Methods for producing immune cells lacking endogenous effector function from pluripotent stem cells

Abstract

The present invention relates to a method for producing immune cells lacking endogenous effector function from pluripotent stem cells. Furthermore, the present invention relates to a method for producing immune cells lacking endogenous effector function but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function; immune cells or immune cell populations obtainable or obtained by the method of the present invention, pharmaceutical compositions comprising the immune cells or immune cell populations, and immune cells or immune cell populations or pharmaceutical compositions obtainable or obtained by the method of the present invention for use in medicine.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to EP patent application No. 23210624.5, filed on November 17, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This invention relates to a method for producing immune cells lacking endogenous effector function from pluripotent stem cells. Furthermore, this invention relates to a method for producing immune cells lacking endogenous effector function but containing one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function; immune cells or immune cell populations obtainable or acquired by the method of this invention; pharmaceutical compositions containing said immune cells or immune cell populations; and immune cells or immune cell populations or pharmaceutical compositions obtainable or acquired by the method of this invention for use in medicine. Background Technology

[0004] Developing immune cell populations, particularly T cell populations, for diagnostic and therapeutic purposes is a current task. Stem cells are an excellent source for providing such immune cell populations, such as differentiated T cell populations. A key characteristic of stem cells is their ability to self-renew indefinitely and differentiate into many different types of cells or tissues. This self-renewal capacity is crucial to their character as a primitive, undifferentiated cell pool. Furthermore, the high “flexibility” and “plasticity” of stem cells are built upon their ability to transdifferentiate into tissues that may differ from their original source tissues. Pluripotent stem cells (PSCs) have attracted considerable attention as a source for T cell production. Currently, the most commonly used pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Specifically, iPSCs, representing non-embryonic cells and isolated from a developed organism, are reprogrammed into pluripotent stem cells. ESCs originate from the embryonic blastocyst and are therefore pluripotent, undifferentiated, and unprogrammed.

[0005] T cells derived from human iPSCs are known in the art. Additional and separately engineered T cells are a powerful tool for targeted therapy of diseases requiring a directed immune response, such as cancer. T cells have been shown to be cultured and proliferated in vitro for adoptive cell immunotherapy or cancer treatment, where their anti-tumor activity has been demonstrated in cancer patients. However, when used for treatment, these differentiated and engineered T cells may produce side effects in such treatments and therapies, making patient safety profiles difficult to predict.

[0006] Therefore, there is a need for an immune cell that can be used for treatment and is safer for patients, thus not causing any side effects.

[0007] Therefore, the object of the present invention is to provide a method for producing immune cells that meet the above-mentioned requirements. This object is achieved by the subject matter of the independent claims. Summary of the Invention

[0008] The inventors have discovered a method for producing immune cells or immune cell populations lacking endogenous effector functions. This means that these immune cells themselves (as defined elsewhere herein, which the inventors can characterize as NK-like precursor cells by the expression of the NK cell phenotypic marker CD56) do not possess any naturally occurring effector functions against any specific target cell and / or other effector cells they interact with (e.g., they do not have any cytolytic function when attacking a specific target cell (e.g., target tumor cells), and / or they do not have any immunosuppressive or immunomodulatory function against another effector cell (e.g., autoreactive T cells).

[0009] The lack of any endogenous effector function may include the absence of expression of any endogenous (naturally occurring) surface molecules on the cell surface of the produced immune cells. Such surface molecules are naturally required to elicit any effector function against the target cells (e.g., tumor target cells). In this regard, the surface molecules required to elicit any effector function may refer to, but are not limited to, T-cell receptors (TCRs); or CD16 receptors, CD94 / NKG2D receptors, or NKp30 receptors (see [link to relevant documentation]). Figure 1 The term may also include situations where the generated immune cells, upon interacting with target cells, fail to produce any effector molecules (such as cytokines) or any regulatory molecules (such as cytokines), such as IL-10 and / or TGF-β cytokines. The term "lack of any endogenous effector function" may not include situations where the generated immune cells lack endogenous MHC class I and / or MHC class II molecules on their cell surface and additionally express recombinant immunomodulatory proteins as defined herein (preferably single-chain fused HLA class I proteins, the effect of which prevents such immune cells from being attacked by recipient T cells—in other words, when immune cells lack endogenous MHC class I and / or MHC class II molecules and additionally express recombinant immunomodulatory proteins as defined herein, the risk of rejection by the recipient's immune system is reduced).

[0010] Similarly, the proliferative potential of immune cells produced in this way is limited (see...). Figure 1 (C and D) This means that such immune cells die off more quickly for their own safety reasons compared to naturally occurring immune cells that still contain their natural effector functions.

[0011] In summary, immune cells produced in this way that lack any endogenous effector function, if used in various treatments and transformed into the specific effector cells required for that treatment as described below, can be considered "neutral cells" that do not provide any side effects (due to the lack of all effector functions naturally inherent in such cells).

[0012] Providing such immune cells is achieved by a first method of the present invention as described herein, the method comprising the steps of: (i) inducing 3D cell aggregate formation, mesodermal differentiation, and hematopoietic differentiation by the following steps: (a) seeding PSCs onto a solid support suitable for 3D cell aggregate formation in a first serum-free medium under suitable conditions, thereby allowing the formation of 3D cell aggregates; (b) collecting the 3D cell aggregates of step (a), resuspending the 3D cell aggregates in a second serum-free medium under suitable conditions, and transferring the resuspended 3D cell aggregates to a third serum-free medium, and culturing them in suspension under suitable conditions for about 1 to about 3 days, thereby allowing mesodermal differentiation; and (c) transferring the cultured 3D cell aggregates of step (b) to a fourth serum-free medium, culturing them in suspension under suitable conditions for about 2 to about 4 days, and further culturing the 3D cell aggregates in suspension under suitable conditions for about 2 to about 6 days in a fifth serum-free medium, thereby allowing hematopoietic differentiation; and (ii) Immune cell differentiation is induced by culturing single cells of the 3D cell aggregates obtained in step (i)(c) in suspension culture under suitable conditions for an appropriate time in a sixth serum-free medium; thereby providing immune cells lacking endogenous effector function.

[0013] Furthermore, the inventors have discovered that such immune cells obtained by the above method can be considered as cellular neutral carriers, used for example to introduce targeted desired molecules, thereby introducing a predetermined effector function required in a specific treatment, and thus obtaining a more effective treatment method. In other words, such immune cells lacking any endogenous effector function can, for example, serve as a basis for introducing exogenous gain-of-function genetic modifications, thereby producing immune cells that, as described above, lack any endogenous effector function but contain one or more exogenous genetic modifications, each of which introduces a predetermined effector function.

[0014] Such immune cells are provided by a second method of the present invention as described herein, the method comprising the steps of: (i) inducing 3D cell aggregate formation, mesodermal differentiation, and hematopoietic differentiation by the following steps: (a) seeding PSCs onto a solid support suitable for 3D cell aggregate formation in a first serum-free medium under suitable conditions, thereby allowing the formation of 3D cell aggregates; (b) collecting the 3D cell aggregates of step (a), resuspending the 3D cell aggregates in a second serum-free medium under suitable conditions, and transferring the resuspended 3D cell aggregates to a third serum-free medium and culturing them in suspension under suitable conditions for about 1 to about 3 days, thereby allowing mesodermal differentiation; and (c) transferring the cultured 3D cell aggregates of step (b) to a fourth serum-free medium and culturing them in suspension under suitable conditions for about 2 to about 4 days, and further culturing the 3D cell aggregates in suspension under suitable conditions for about 2 to about 6 days in a fifth serum-free medium, thereby allowing hematopoietic differentiation; and (ii) Immune cell differentiation is induced by culturing single cells of the 3D cell aggregates obtained in step (i) and (c) in suspension culture under suitable conditions for an appropriate time in a sixth serum-free medium; wherein the method further includes introducing one or more exogenous modifications into genes in PSCs before step (a), during steps (a) to (c) of step (i), or at any time during step (ii), wherein each of the one or more exogenous modifications introduces a predetermined effector function, thereby providing genetically modified immune cells that lack endogenous effector functions but contain one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function.

[0015] In summary, the immune cells or populations of immune cells lacking endogenous effector function, as described herein, provided by the specific steps and sequence of the first method of the present invention, have the advantage that subsequent application of such cells in treatment is safer for patients. Then, by introducing one or more exogenous modifications (i.e., into the genome of the PSC) at the PSC level prior to step (a) of the second method of the present invention, or in any step (a), (b), or (c) of step (i), or in step (ii), immune cells or populations of immune cells lacking endogenous effector function can be generated, which subsequently exhibit the predetermined effector function required for the specific application of the immune cell due to the artificial introduction of modifications into the PSC genome.

[0016] The present invention also relates to immune cells or populations of immune cells lacking endogenous effector function that can be obtained or acquired by the first method of the present invention as defined herein. Furthermore, the present invention also relates to immune cells or populations of immune cells lacking endogenous effector function but containing one or more exogenous modifications that can be obtained or acquired by the second method of the present invention, wherein each of the one or more exogenous modifications introduces a predetermined effector function.

[0017] The present invention also relates to a pharmaceutical composition comprising immune cells or populations of immune cells that are available or obtainable by the methods of the present invention as defined elsewhere herein.

[0018] Furthermore, this invention relates to immune cells or populations of immune cells that can be obtained or acquired by the methods of this invention as defined elsewhere herein, or pharmaceutical compositions as defined elsewhere herein, for use in medicine. Additionally, this invention relates to immune cells or populations of immune cells that can be obtained or acquired by the methods of this invention as defined elsewhere herein, or pharmaceutical compositions as defined elsewhere herein, for use in methods of preventing or treating cancer. Attached Figure Description

[0019] Figure 1 This demonstrates that iPSC-derived cells have limited functional potential. (A) iPSC-differentiated lymphocytes are similar to cells derived from native lymphocyte compartments. (B) iPSC-differentiated lymphocytes (e.g., NK-like progenitor cells) do not express surface molecules (e.g., CD94, CD16, NKG2D, NKp30) required for triggering effector functions. Figure 1 (B) The last inset contains CD3xCD19 staining, demonstrating the absence of TCR / CD3 receptor expression, a prerequisite for T cell recognition and endogenous cytolytic function. The absence of CD3 signaling also rules out its recognition as an NKT cell. (C) and (D) iPSC-differentiated lymphocytes have limited expansion potential in vitro.

[0020] Figure 2 This shows that iPSC-derived cells did not exhibit cytolytic function in vitro. (A) A conventional CAR construct (anti-CD19) was stably knocked into iPSC lines as a control (CAR iPSCdiff.). (B) CD19 + GFP + Co-culture of Raji (target) cells and CAR iPSC diff. (effectons) demonstrated the interaction between the CAR receptor and the CD19 antigen through the disappearance of CD19 signaling. (C) and (D) co-culture of K652 and Raji cells showed that, in contrast to controls (NK cells and CAR T cells, respectively), CAR iPSC diff. lacked both nonspecific and antigen-specific cytolytic functions.

[0021] Figure 3 The study showed that iPSC-derived cells did not exhibit cytolytic function in vivo. Compared to CAR T-cell controls, CAR iPSC diff. did not show tumor control in an in vivo NSG mouse model pre-injected with Raji cells.

[0022] Figure 4 This diagram illustrates how iPSC-derived cells can serve as a basis for introducing gain-of-function genetic modifications. (A) The diagram depicts terminally differentiated lymphocytes (marked with an asterisk) from unmodified iPSCs, effector cells such as T cells or CAR T cells (marked with circles), and targets such as tumor cells (unmarked). (B) The diagram depicts terminally differentiated lymphocytes from gene-edited iPSCs that, upon interaction with tumor target cells (regulated by a target-specific induction system), can exert the effects of effector molecules (such as cytokines). (C) The diagram depicts the enhanced function of terminally differentiated lymphocytes from gene-modified iPSCs (carrying tumor-specific antigens). Interaction with CAR T cells will enhance CAR T cell function to more effectively kill target tumor cells. (D) The diagram depicts the immunomodulatory (immunosuppressive) function of terminally differentiated lymphocytes from gene-modified iPSCs that, upon interaction with effector cells (autoreactive T cells), can exert the effects of regulatory molecules (such as cytokines). In this case, non-malignant target cells will be protected. Detailed Implementation

[0023] The method of the present invention for producing immune cells or populations of immune cells lacking any endogenous effector function and derived from PSCs as defined elsewhere herein particularly includes step (i), which involves the formation of 3D cell aggregates corresponding to step (i)(a), followed by a mesodermal differentiation step corresponding to step (i)(b), and then a hematopoietic differentiation step corresponding to step (i)(c).

[0024] The 3D cell aggregate formation in step (i) (a) involves seeding PSCs onto a solid support suitable for 3D cell aggregate formation, as defined elsewhere herein, in a first serum-free medium under suitable conditions, thereby allowing the formation of such 3D cell aggregates. In this context, "first serum-free medium" refers to a serum-free medium for culturing PSCs, such as iPSC amplification medium, preferably supplemented with GSK-3 inhibitors and ROCK inhibitors as defined elsewhere herein, more preferably at concentrations of about 5 to about 15 μM (see Example 1). "Under suitable conditions" in this context can refer to conditions known to those skilled in the art that allow 3D cell aggregate formation when PSCs are seeded onto a solid support suitable for 3D cell aggregate formation placed in the first serum-free medium described elsewhere herein. Preferably, about 1.5 to about 4.5 × 10⁻⁶ μM of the GSK-3 inhibitor is used. 6 Cells, preferably about 3 × 10⁶ 6 Cells are seeded on a solid support as defined herein. Adjusting the number of PSCs within this specific range can affect the ratio of growth factors in the culture medium and can also affect subsequent differentiation.

[0025] The mesodermal differentiation in step (i)(b) includes collecting the 3D cell aggregates formed in step (i)(a), followed by a resuspension step of the 3D cell aggregates in a second serum-free medium (different from the first serum-free medium used in step (i)(a)) under suitable conditions, and then transferring the resuspension 3D cell aggregates to a third serum-free medium and culturing the aggregates in suspension culture as defined elsewhere herein in the third serum-free medium (different from the first and second serum-free media) under suitable conditions for about 1 to about 3 days, such as about 1 day, about 2 days, about 3 days, preferably about 2 days. In this context, the “second serum-free medium” used for the resuspension step refers to a medium that does not contain any serum and induces mesodermal differentiation, such as a mesodermal induction medium, preferably supplemented with growth factors, such as bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF), more preferably at a concentration of about 10 to about 50 ng / ml (see Example 1). "Third serum-free medium" for culturing 3D cell aggregates in mesodermal differentiation refers to a medium that does not contain any serum and induces final mesodermal differentiation, such as mesodermal induction medium, preferably supplemented with growth factors, such as BMP4, VEGF, FGF, and an ALK receptor inhibitor (see Example 1), more preferably with concentrations of BMP4, VEGF, and FGF of about 10 to about 50 ng / ml, and the ALK receptor inhibitor of about 5 to about 15 μM. Such ALK receptor inhibitors (e.g., SB431542) may refer to TGF-β inhibitors, which are required to achieve more efficient mesodermal differentiation and can prevent differentiation of cell types other than mesodermal cells enhanced by TGF-β. In this context, "under suitable conditions" may refer to conditions known to those skilled in the art that allow the 3D cell aggregates collected after step (i)(a) to undergo mesodermal differentiation in the second and third serum-free media as described elsewhere herein. When subsequently cultured in suspension as defined herein, the 3D cell aggregates already formed from step (i)(a) will not adhere to such culture plates because such suspension culture plates are ultra-low adhesion suspension culture plates. By including the aggregates in ultra-low adhesion suspension culture, the aggregates will not adhere to the culture plate, thereby preventing such cells from differentiating into undesirable cell types.

[0026] The hematopoietic differentiation in step (i) (c) includes transferring the cultured 3D cell aggregates from step (b) to a fourth serum-free medium (different from the first, second, or third serum-free medium) and culturing the aggregates in suspension under suitable conditions in the fourth serum-free medium for about 2 to 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 in suspension under suitable conditions in a fifth serum-free medium (different from the first, second, third, or fourth serum-free medium) for about 2 to 6 days, such as about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days, preferably about 4 days. In this context, the “fourth serum-free medium” used in the first culture step of this hematopoietic differentiation refers to a medium that does not contain any serum and 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 at a concentration of about 10 to about 50 ng / ml (see Example 1). The “fifth serum-free medium” used in the second culture step of hematopoietic differentiation refers to a medium that does not contain any serum and induces final hematopoietic differentiation, such as a hematopoietic induction medium, preferably supplemented with growth factors such as VEGF, FGF, SCF, and FMS-like tyrosine kinase 3 ligand (Flt-3l) and thrombopoietin (TPO), more preferably at concentrations of about 10 to about 50 ng / ml for each of VEGF, FGF, SCF, Flt-3l, and TPO (see Example 1). Final differentiation of PSCs may require Flt-3l and TPO to allow hematopoietic differentiation. In this context, “under suitable conditions” can refer to conditions known to those skilled in the art that allow the 3D cell aggregates obtained after mesodermal differentiation in steps (i) and (b) to undergo hematopoietic differentiation in the fourth and fifth serum-free media as described elsewhere herein. Even though the aggregates are placed in the same type of culture plate, the suspension cultures used in steps (b) and (c) are considered to be different. However, because the aggregates are exposed to different growth factors in the different media used in each step, they are at different stages of development or differentiation.

[0027] In addition to step (i) as defined above, the method of the present invention for producing immune cells or populations of immune cells lacking any endogenous effector function and derived from PSCs as defined elsewhere herein further includes step (ii), which refers to the step of immune cell differentiation. Step (ii) includes culturing single cells of the 3D cell aggregates obtained in step (i), particularly those obtained in step (i)(c), in suspension under suitable conditions in a sixth serum-free medium (different from the first, second, third, fourth, or fifth serum-free media) for an appropriate time. In this context, "sixth serum-free medium" used in immune cell differentiation refers to a medium that does not contain any serum and induces immune cell differentiation, such as an immune cell differentiation medium, preferably supplemented with IL-3, SCF, TPO, Flt3l, IL-7, IL-15, SDF, and SB203580, more preferably with concentrations of IL-3, TPO, IL-15, SDF, SCF, Flt3l, and IL-7 of about 10 to about 50 ng / ml and SB203580 of about 7 to about 23 μM (see Example 1). In this context, "under suitable conditions" can refer to conditions known to those skilled in the art that allow the hematopoietic differentiated 3D cell aggregates in step (i) (c) to undergo immune cell differentiation in a sixth serum-free medium as described elsewhere herein. The suspension culture described here is considered different from the suspension cultures used in steps (b) and (c) because the culture plate used in step (ii) is different from the culture plates used in steps (b) and (c) as further defined herein (e.g., Notch ligand-coated or uncoated culture plates in step (ii)). Also in this step, the aggregates themselves are not cultured in this suspension culture, but rather single cells of the 3D cell aggregates are cultured after the aggregates have been dissociated into single cells. In this context, “suitable time” can refer to a specific time range known to those skilled in the art, within which, under the suspension culture conditions defined herein for step (ii), PSC single cells from the 3D cell aggregates transform into the immune cells of the present invention, preferably in less than 50 days, more preferably less than 25 days, and most preferably less than 21 days.

[0028] Additionally, the method of the present invention as defined elsewhere herein may further include, prior to step (a) of the method of the present invention, or in any step (a), (b) or (c) of step (i), or at any time point in step (ii), introducing one or more exogenous modifications into genes in a PSC to provide genetically modified immune cells lacking endogenous effector function but containing one or more exogenous modifications, each of which introduces a predetermined effector function. In other words, at the PSC level of the method of the present invention as defined elsewhere herein, genetic modification of a PSC may be performed prior to step (a), or in any step (a), (b) or (c) of step (i), or in step (ii). This means that the PSC genome can be genetically modified by introducing one or more exogenous (genetic) modifications to subsequently modify later-generated immune cells lacking endogenous effector function derived from the genetically modified PSC. In this context, the term "prior to step (a) of step (i)" may refer to any step that can be performed on a PSC as defined herein, such as, particularly, culturing such PSCs prior to inoculation as described in step (a).

[0029] In the context of “endogenous effector function” as used herein, the term “endogenous” may be replaced by the term “naturally occurring”, meaning any effector function inherent in the immune cell itself as defined herein. The term “lack” as used in the context of this invention means that such immune cells, after being generated by the method of this invention, do not have or contain any or all of the endogenous effector functions as defined herein.

[0030] On the other hand, the term "exogenous" means "artificial" or "recombinant." Therefore, "exogenous modification" can refer to a modification as defined herein introduced into the PSC (PSC genome) by any suitable technique known to those skilled in the art (including, for example, homologous recombination, gene editing methods, or recombinase technology), such that such modification within the PSC genome is artificial / recombinant. The term "one or more (genetic) modifications" as used herein generally refers to one or more alterations to nucleic acids (DNA or RNA), such as those within the PSC genome. For example, gene modification can refer to alterations, insertions (e.g., gene knock-in), and / or deletions (e.g., gene knockout). Preferably, such one or more gene modifications can result in the incorporation and expression of one or more recombinant genes encoding a specific molecule that enables the genetically modified immune cells to produce the desired effector function.

[0031] In each step (a), (b), and / or (c) of step (i) of the method of the present invention, or before step (a) of step (i) or in step (ii), when an exogenous modification is introduced into a gene, one modification may be introduced, but more than one, such as two, three, four, or five modifications, may also be introduced. Preferably, the exogenous (gene) modification is selected from gene knock-in, gene knockout, gene substitution, point mutation, and deletion, insertion, or substitution of genes, gene fragments, or nucleotides, or combinations thereof. As used herein, the term "gene knockout (deletion or inactivation)" refers to a gene modification resulting from the loss of genetic information encoded at a chromosomal locus within the PSC genome, or a gene modification resulting from gene inactivation (alteration of genetic information encoded at a chromosomal locus), thereby affecting gene transcription and / or translation. As used herein, the term "gene knock-in (insertion)" is a gene modification that achieves the insertion of a gene not previously present in the genome into a specific locus within the PSC genome. As used herein, the term "gene substitution" is a genetic modification that achieves the replacement of genetic information encoded at a chromosomal locus within the PSC genome (e.g., from a human) with a corresponding gene from another subject. The term "gene" is used broadly herein to refer to any segment of DNA containing a gene sequence relevant to a biological function. Therefore, a gene includes coding and / or non-coding sequences and / or regulatory and control sequences required for their expression. Functional gene segments may also include the aforementioned sequences for desired expression.

[0032] In one embodiment, the exogenous modification introduced into the PSC is a gene knock-in as defined herein. In another embodiment, the exogenous modification introduced into the PSC is a gene knockout as defined herein. In yet another embodiment, the exogenous modification introduced into the PSC is a gene substitution as defined herein. In still another embodiment, the exogenous modification introduced into the PSC is a point mutation as defined herein. In yet another embodiment, the exogenous modification introduced into the PSC is a deletion of a gene, gene segment, or nucleotide as defined herein. In yet another embodiment, the exogenous modification introduced into the PSC is an insertion of a gene, gene segment, or nucleotide as defined herein. In yet another embodiment, the exogenous modification introduced into the PSC is a substitution of a gene, gene segment, or nucleotide as defined herein. Therefore, this document may also include cases where the exogenous modification introduced into the PSC refers to gene knockout, and another exogenous modification introduced into the PSC (collectively, two modifications) refers to gene knock-in, or where the exogenous modification introduced into the PSC refers to gene knock-in, and another exogenous modification introduced into the PSC (collectively, two modifications) refers to another gene knock-in, and so on. If such modification is for example, a modification of T cells, these modifications can be introduced into the same PSC used in the method of the present invention. If such modification is for example, a modification of T cells and for example, NK cells, these modifications are introduced into different PSCs used in the method of the present invention to provide T cells and NK cells lacking endogenous effector function, both cell types exhibiting their desired effector function due to their respective introduced modifications.

[0033] Each exogenous modification at the gene level, as defined herein, achieves the introduction of a predetermined effector function. In other words, if, for example, two exogenous modifications are introduced into a PSC as defined herein, the first modification achieves, for example, the introduction of a predetermined effector function, and the second modification achieves the introduction of another predetermined effector function. In this context, a “predetermined” effector function is a specific effector function as defined herein, such as a function targeting a specific target cell or another effector cell. Genetically modified immune cells generated by the method of the present invention should contain said specific effector function. Therefore, the introduction of one or more exogenous modifications into the PSC genome is specifically chosen in advance to achieve a specific effector function, or to ensure that genetically modified immune cells lacking any endogenous effector function due to the method of the present invention truly contain the specific effector function as defined herein due to the exogenously introduced modification (see [link to original text]). Figure 4 ).

[0034] Preferably, gene knock-in comprises knocking in at least one gene selected from genes encoding: chimeric antigen receptor (CAR); exogenous T-cell receptor (TCR); exogenous IL-15 receptor; exogenous CD16 receptor; exogenous CD19 antigen; exogenous IL-10 cytokine; exogenous TGF-β cytokine; and exogenous PD ligand 1 (PD-L1) antigen, or combinations thereof. In one embodiment, gene knock-in comprises knocking in a gene encoding a CAR. In another embodiment, gene knock-in comprises knocking in a gene encoding an exogenous TCR that provides cytolytic function against target cells. In yet another embodiment, gene knock-in comprises knocking in a gene encoding an exogenous IL-15 receptor that provides immunomodulatory function (e.g., persistence of immune cells). In still another embodiment, gene knock-in comprises knocking in a gene encoding an exogenous CD16 receptor that provides cytolytic function against target cells. In yet another embodiment, gene knock-in includes knocking in a gene encoding a foreign CD19 antigen that provides immunomodulatory (immunostimulatory) function. In still another embodiment, gene knock-in includes knocking in a gene encoding a foreign IL-10 cytokine that provides immunosuppressive function. In yet another embodiment, gene knock-in includes knocking in a gene encoding a foreign TGF-β cytokine that provides immunosuppressive function. In yet another embodiment, gene knock-in includes knocking in a gene encoding a foreign PD ligand 1 (PD-L1) antigen that provides immunosuppressive function.

[0035] CAR proteins can contain single-chain variable fragments (scFvs) of antibodies capable of binding to specific tumor-associated antigens, linked to intracellular co-stimulatory domains (e.g., CD28, OX40, and CD137) via transmembrane peptides. These peptides are then fused to a signaling domain of the TCRζ chain, which activates CAR T cells when the CAR binds to its epitope on tumor cells. The subsequent release of granzyme and perforin leads to tumor cell lysis (June CH, O'Connor RS, KawalekarOU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science (New York, NY) 2018;359:1361–65).

[0036] Each of the gene knock-ins described above, preferably a CAR or exogenous TCR, can be introduced into an endogenous TCR-α or endogenous TCR-β gene for conditional expression, as defined herein. Introduction can be performed using a constitutive promoter (such as CAG) to express the gene knock-in per cell, or using a conditional promoter from an endogenous gene (such as TCR-α or β or any other gene), with or without gene disruption. Alternatively, such gene knock-ins as defined above can be introduced into a safe harbor locus known to those skilled in the art for continuous expression (to prevent silencing of the knock-in gene during differentiation), preferably using a conditional promoter.

[0037] More preferably, gene knockout comprises the knockout of at least one gene selected from genes encoding endogenous PD-1 cell surface protein; endogenous VEGF receptor; endogenous VCAM-1 cell surface protein; and endogenous ICAM-1 cell surface protein, or combinations thereof. In one embodiment, gene knockout comprises knocking out a gene encoding an endogenous PD-1 cell surface protein that provides immunomodulatory functions (e.g., persistence / efficiency of immune cells). In another embodiment, gene knockout comprises knocking out a gene encoding an endogenous VEGF receptor that provides immunomodulatory functions (e.g., persistence of immune cells). In yet another embodiment, gene knockout comprises knocking out a gene encoding an endogenous VCAM-1 cell surface protein that provides immunomodulatory functions (e.g., persistence of immune cells). In still another embodiment, gene knockout comprises knocking out a gene encoding an endogenous ICAM-1 cell surface protein that provides immunomodulatory functions (e.g., persistence of immune cells).

[0038] Each embodiment of the method of the present invention described herein (for producing immune cells as defined herein) is also applicable to the method of the present invention for producing populations of immune cells as defined herein.

[0039] As used herein, when referring to a time interval or period of time, the term “about” should be understood to include any intervals that deviate from a specific value of the given interval. For example, an interval of about 2 to 4 days (meaning 48 to 96 hours) also includes intervals that deviate from the given interval by 1, 2, 3, or 4 hours, such that the given interval may be shortened or lengthened by 1, 2, 3, or 4 hours at the beginning and / or end of the interval.

[0040] In one embodiment of the method of the present invention, the first serum-free culture medium preferably comprises, in step (i)(a), a GSK-3 inhibitor and a ROCK inhibitor, each at a concentration of about 5 to about 15 μM. Alternatively or additionally, the second serum-free culture medium used in step (i)(b) may comprise BMP4, VEGF, and FGF, each at a concentration of about 10 ng / mL to about 50 ng / mL, and the third serum-free culture medium used in step (i)(b) may comprise BMP4, VEGF, and FGF, each at a concentration of about 10 ng / mL to about 50 ng / mL, and additionally comprise an ALK receptor inhibitor at a concentration of about 5 μM to about 15 μM. Alternatively or alternatively, the fourth serum-free culture medium used in step (i) (c) may contain VEGF, FGF and SCF at a concentration of about 10 ng / mL to about 50 ng / mL, and the fifth serum-free culture medium used in step (i) (c) may contain VEGF, FGF and SCF at a concentration of about 10 ng / mL to about 50 ng / mL, and additionally contain about 10 ng / mL to about 50 ng / mL Flt-3l and about 10 ng / mL to about 50 ng / mL TPO. Alternatively or alternatively, the sixth serum-free culture medium used in step (ii) may contain about 7 to about 23% of BIT 9500 serum substitute, about 25 mM to about 75 mM of β-mercaptoethanol, about 10 ng / ml to about 50 ng / ml of IL-3, about 10 ng / ml to about 50 ng / ml of TPO, about 10 ng / ml to about 50 ng / ml of IL-15, about 10 ng / ml to about 50 ng / ml of SDF, about 7 µM to about 23 µM of SB203580, about 10 ng / ml to about 50 ng / ml of SCF, about 10 ng / ml to about 50 ng / ml of Flt-3l, about 10 ng / ml to about 50 ng / ml of IL-7, and about 15 mM to about 45 mM of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.

[0041] As used herein, the term “about” in relation to any concentration value or range must be understood to also include any concentration or range that deviates from the specific value of a given concentration or range. For example, a concentration of about 50 ng / mL also includes lower concentrations of 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 ng / mL, or higher concentrations of 51, 52, 53, 54, 55, 56, 57, 58, or 59 ng / mL.

[0042] In a preferred embodiment of the invention, the ROCK inhibitor is Y-27632 dihydrochloride, the ALK receptor inhibitor is SB43152, and the GSK-3 inhibitor is CHIR99021. The use of the ROCK inhibitor provides the advantage of limiting / avoiding apoptosis and dedifferentiation. Therefore, the use of the ROCK inhibitor and the ALK receptor inhibitor avoids cell loss due to apoptosis, meaning that the method of the invention provides a precise quantity of cells. Furthermore, because the cells retain the desired differentiation characteristics, differentiation loss is reduced. This means that the method of the invention provides cells of a defined quality. More CD34-positive PSCs are generated after step (i)(c) by adding a GSK-3 inhibitor that activates the WNT pathway, such as CHIR99021, to the first serum-free medium in step (i)(a).

[0043] The PSCs used in this invention can be pluripotent stem cells that lack endogenous major histocompatibility complex (MHC) class I molecules present on the surface of PSC cells and contain immunomodulatory proteins on their surface.

[0044] Cells or pluripotent stem cells that "lack endogenous MHC class I molecules present on their cell surface" are those lacking functional MHC class I molecules on their surface. Such defective cells / pluripotent stem cells also do not contain functional MHC class I molecules in their cell membranes. In this context, the term "endogenous" refers to any MHC class I protein that is naturally present in the cell or pluripotent stem cell, rather than being artificially introduced. The presence of endogenous MHC class I molecules increases the risk of rejection by the recipient's immune system, but the absence of MHC class I molecules on the cell surface may be recognized by the immune system as a "lack of self" signal. Therefore, the characteristic of cells or pluripotent stem cells lacking MHC class I molecules on their surface does not apply to any immunomodulatory proteins and / or recombinant immunomodulatory proteins that can be introduced into the pluripotent stem cell. In one embodiment, the absence of MHC class I molecules on the cell surface can be achieved by disrupting all copies of the β2-microglobulin gene in the pluripotent stem cell. The MHC complex is a heterodimer of α-microglobulin and β2-microglobulin. Therefore, if β2-microglobulin is absent, the functional MHC class I complex cannot be assembled, and MHC class I molecules are consequently absent from the cell membrane and / or cell surface. Those skilled in the art know many feasible ways to modify the genome of pluripotent stem cells to lack MHC class I molecules and contain immunomodulatory proteins. It should be noted that the pluripotent stem cells of the present invention lacking MHC class I molecules can express immunomodulatory proteins, even if they are MHC class I molecules such as HLA-E as described herein. Therefore, the term "lacking MHC class I molecules" refers to endogenous MHC class I molecules and does not exclude the presence of (recombinant) immunomodulatory proteins.

[0045] In another preferred embodiment of the invention, the immunomodulatory protein is a single-chain fusion HLA class I protein.

[0046] Preferably, the single-chain fusion HLA class I protein may include at least a portion of B2M covalently linked to at least a portion of the HLA class I α chain, said HLA class I α chain being selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, preferably HLA-E. Optionally, the introduction of such recombinant immunomodulatory proteins (preferably single-chain fusion HLA class I proteins, more preferably HLA-E) into PSCs can protect them from NK cell-mediated host rejection, thereby producing non-immunogenic PSCs as defined elsewhere herein.

[0047] In a preferred embodiment of the invention, the PSC further expresses a target peptide antigen, which is presented by a single-chain fused HLA class I protein on the surface of the pluripotent cell. The target peptide antigen is preferably covalently linked to the single-chain fused HLA class I protein. In an even more preferred embodiment, the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO: 1).

[0048] Preferably, in the PSC, substantially all copies of the β-microglobulin 2 gene are disrupted. In the PSC of the present invention, the B2M gene can be disrupted such that no functional endogenous B2M protein is produced from the disrupted locus. In some embodiments, the disruption results in the expression of non-functional B2M protein, including but not limited to truncated, deleted, point-mutated, and inserted forms. In other embodiments, the disruption results in no protein expression of the B2M gene.

[0049] In another preferred embodiment, the PSC as defined herein is additionally or optionally lacking endogenous MHC class II. Preferably, the PSC lacks endogenous MHC class II by disrupting the C2TA gene (an MHC class II transactivator).

[0050] Through the above-described gene modifications, non-immunogenic immune cells can be generated that are not attacked by the recipient's immune cells (such as T and NK cells). When one or more exogenous modifications are introduced into the genes of the PSC to introduce a predetermined effector function as defined herein, the above-described gene modifications may not be included in the one or more exogenous modifications introduced before step (a) of the method of the present invention, in (a), (b) or (c) of step (i), or at any time during step (ii).

[0051] In a preferred embodiment, the PSC is selected from embryonic stem cells, induced pluripotent stem cells, and parthenogenetic stem cells.

[0052] As used herein, the term "pluripotent stem cell" (PSC) refers to any such stem cell type capable of differentiating into immune cells. In the context of this invention, it is preferred not to use methods involving alteration of human germline genetic identity or methods involving the use of human embryos for industrial or commercial purposes to produce these PSCs. The PSCs used in the methods of this invention may refer to wild-type PSCs (without any non-immunogenic modifications as defined herein) or non-immunogenic PSCs as defined herein. Therefore, immune cells derived from such PSCs, lacking any endogenous effector function due to the methods of this invention, may additionally be non-immunogenic immune cells as defined elsewhere herein. Preferably, the PSCs are primate-derived, more preferably human-derived. In another preferred embodiment of the invention, the PSCs are human induced pluripotent stem (iPS) cells, preferably CD34-positive cells isolated from umbilical cord blood, and most preferably the human iPS cell line TC-1133, which is also the cell line ND-50039 from the NINDS Human Cell and Database (see Example 1). The TC-1133 cell line has been reprogrammed under c-GMP conditions and is available from Lonza. Other suitable PSCs, including induced PSCs, can be obtained, for example, from the NIH Human Embryonic Stem Cell Registry, the European Induced Pluripotent Stem Cell Bank (EBiSC), the stem cell bank of the German Center for Cardiovascular Research (DZHK), or the ATCC, to name just a few. PSCs are also commercially available, for example, from the NINDS Human Sequence and Cell Bank (https: / / stemcells.nindsgenetics.org), operated by the National Institute for Neurological Disorders and Stroke (NINDS) and which widely distributes human cell resources to academic and industry researchers.

[0053] Other exemplary iPSC cell lines that can be used in this invention include, but are not limited to, Gibco. TMThe Episomal iPSC cell line (catalog 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 obtained from ATTC. Alternatively, any person skilled in the art of reprogramming can readily generate suitable iPSC cell lines using known methods, such as those described by Okita et al., “A more efficient method to generate integration-free human iPS cells”, Nature Methods, Vol.8 No.5, May 2011, pages 409-411, or 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 pluripotentstem cells”, Biomaterials 35 (2014) 2816e2826. The (induced) pluripotent stem cells used in this invention can be derived from any suitable cell type (e.g., stem cells derived from mesenchymal stem cells or epithelial stem cells, or differentiated cells such as fibroblasts) and from any suitable source (body fluid or tissue). Examples of such sources (body fluid or tissue) include umbilical cord blood, skin, gums, urine, blood, bone marrow, any compartment of the umbilical cord (e.g., umbilical cord amnion or Wharton's jelly), cord-placental junction, placenta, or adipose tissue, to name just a few. In one illustrative example, CD34-positive cells isolated from umbilical cord blood are selected, for example, using magnetic cell fractionation with a specific antibody against CD34, and subsequently reprogrammed as described by Chou et al., (2011), Cell Research, 21:518-529. Baghbaderani et al. (2015), Stem Cell Reports, 5(4):647-659, demonstrate that the iPSC generation process can comply with Good Manufacturing Practices (GMP) for pharmaceuticals to produce cell line ND-50039. Therefore, preferably, the PSC meets the requirements of Good Manufacturing Practice (GMP) for pharmaceuticals.

[0054] The PSCs described herein can be cultured first on a solid support containing at least one extracellular matrix protein, and then seeded onto another solid support as described in step (i)(a) of this invention. This step (as defined below, referred to as step (a')) refers to the PSC culture step prior to step (a) of the method of the present invention. The aforementioned at least one extracellular matrix protein can be selected from: fibronectin, laminin, collagen, fibronectin, elastin, matrigol, peptides containing the amino acid sequence RGD, proteins containing the amino acid sequence RGD, and combinations thereof. Those skilled in the art can empirically select the corresponding optimal extracellular matrix protein or optimal combination of extracellular matrix proteins. Therefore, in this step, two, three, four, or more extracellular matrix proteins can also be combined for PSC culture. Preferably, prior to step (i)(a), the PSCs described herein can be cultured on a matrigol-coated container (e.g., a matrigol-coated T-flask). Preferably, PSCs are cultured on such matrix-coated containers in iPSC amplification media such as Miltenyi StemMACS iPS-Brew XF so that these PSCs remain pluripotent and undifferentiated. This document also covers the method of the invention further comprising step (a') prior to step (a), which includes culturing PSCs in a serum-free medium under suitable conditions on a solid support containing at least one extracellular matrix protein. In this context, "serum-free medium" means a medium without serum for culturing PSCs so that they remain pluripotent and undifferentiated, such as iPSC amplification medium, preferably supplemented with the ROCK inhibitor Y-27632. "Under suitable conditions" in this context may refer to conditions known to those skilled in the art that allow PSCs to be cultured on coated solid supports in serum-free media as described elsewhere herein, such that the PSCs remain pluripotent and undifferentiated.

[0055] Preferably, the PSCs are primate-derived pluripotent stem cells, more preferably human pluripotent stem cells. In a preferred embodiment, the PSCs are generated from CD34-positive cells isolated from umbilical cord blood. In an even more preferred embodiment, the PSCs used in this invention are ND-50039 cells from the NINDS Human Cell and Cell Database.

[0056] Preferably, in step (i) (a) of the method of the present invention, the solid support comprises one or more pores, wherein each pore comprises a V-shaped or conical cavity. In a preferred embodiment, the solid support is a microwell culture plate. Any suitable microwell culture plate can be used, as long as the plate allows for the generation of 3D cell aggregates. Examples of suitable microwell culture plates include, but are not limited to, AggreWell. TMBoard (available from StemCell Technologies), BIOFLOAT TM 96-well plates (available from facellitate) or Nunclon Sphera 3D culture plates (available from ThermoFisher). Such supports used in step (i)(a) (preferably if AggreWell) TM When preparing the substrate (for 3D cell aggregates), pretreatment with the anti-adhesion solution described above is preferred. This anti-adhesion solution allows for reduced surface tension and prevents cells from adhering to the support, allowing only cell-to-cell adhesion. This provides a low-adhesion surface conducive to the formation of 3D cell aggregates. The formation of 3D cell aggregates can be carried out in a very simple and robust manner, which also allows for scale-up.

[0057] Preferably, the suspension culture used in steps (i), (b), and (c) of the method of the present invention is a dynamic suspension culture, which means culture under agitation, for example, by culturing on a shaking device. Any suitable shaking device / shaker can be used, such as a stationary shaker, a horizontal shaker, or a linear shaker. The culture can be moved / agitated under any suitable conditions that can be determined experimentally by those skilled in the art. For example, a shaker (e.g., a stationary shaker) can be operated at a rate of about 60 to about 80 revolutions per minute (rpm). Unlike adherent cell culture, the suspension cell culture used herein offers several advantages. Adherent cell culture requires more space and is, for example, more cumbersome for cell passage operations. Suspension culture is a form of cell culture that provides a scalable method of cell culture, which is useful for providing a greater number of cells. This greater number of cells is particularly needed for the clinical application of cell products. Dynamic suspension culture is also easier to transfer to bioreactor conditions compared to adherent cell culture, such as 2D cell culture or static cell culture, because the characteristics of dynamic cell culture are very similar to those of culture in a bioreactor. Furthermore, in dynamic cell culture, the growth factors present in the culture medium can circulate. This allows each cell to have sufficient contact with these growth factors. This can positively influence the culture process, especially cell differentiation, and contribute to obtaining a more homogeneous and well-defined cell population.

[0058] Preferably, the method of the present invention further includes dissociating the 3D cell aggregates into single cells after steps (i) and (c) and before step (ii), and then optionally sorting CD34-positive PSCs. This dissociation step can be performed by applying type II collagenase. The sorting of CD34-positive PSCs can be performed by MACS or FACS known to those skilled in the art.

[0059] Induction of immune cell differentiation can be performed at any suitable time, preferably less than 50 days, more preferably less than 25 days, and most preferably less than 21 days. The suitable time can be determined experimentally, for example by collecting cell samples over a certain period and determining the nature / differentiation state of these cells, for example, by analyzing cell surface markers (see Experimental Section, where CD45 markers of cells are first screened to scan immune cells (leukocytes)). Figure 1 A). Then, the CD56 of the immune cells thus generated can be further characterized. 高 Phenotype (referring to NK-like precursor cells) (see also...) Figure 1 B). In a preferred embodiment of the invention, step (ii) of inducing immune cell differentiation involves culturing single cells of the 3D cell aggregates obtained from step (i) and (c) in suspension culture under suitable conditions 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 about 14 days. The suspension culture used in step (ii) of the method of the present invention is not a dynamic suspension culture, while the suspension cultures in steps (i), (b), and (c) of the method of the present invention can be dynamic suspension cultures. Such a suspension culture in step (ii) is preferably a Notch ligand-coated suspension culture plate. Immune cell differentiation can also be induced when a Notch ligand-coated suspension culture plate is not used, i.e., an uncoated suspension culture plate. The Notch ligand can be Notch ligand Delta-like ligand 4 (DLL4) or Notch ligand Delta-like ligand 1 (DLL1). Notch signaling is reportedly important for immune cell differentiation.

[0060] The immune cells generated by the method of the present invention (excluding any modification steps at the PSC level) are preferably NK-like progenitor cells lacking endogenous effector function as described elsewhere herein. This document also includes a method of the present invention as defined herein, which generates a population of immune cells that may comprise NK-like progenitor cells lacking any endogenous effector function. For the purposes of this method, "immune cell population" can be understood as a single homogeneous population of immune cells, such as a pure or homogeneous population of NK-like progenitor cells, all lacking endogenous effector function as defined herein, which can be characterized by those skilled in the art using methods known in the art (e.g., flow cytometry). In cases where PSCs are genetically modified as defined herein to obtain genetically modified immune cells, the immune cells generated by this method are preferably T-cell-like cells or NK-cell-like cells lacking endogenous effector function, comprising one or more exogenous modifications, each of which introduces a predetermined effector function. In this context, "T-cell-like cells" means that the resulting immune cells are not naturally occurring T cells because, based on methods including the introduction of exogenous modifications, genetically modified T cells are produced. These T cells lack any endogenous effector functions, but if an exogenous modification leading to the predetermined effector function has been introduced into the PSCs that subsequently develop into such modified T cells, then these T cells, for example, contain only one predetermined effector function. This also applies, with appropriate adjustments, to "NK-cell-like cells," "B-cell-like cells," etc. As used herein, the term "immune cell population" used in methods including modification steps includes a mixture of (different) immune cells contained within that immune cell population. Therefore, in this respect, "immune cell population" can be understood as a single homogeneous immune cell population, such as a population of pure or homogeneous T-cell-like cells or NK-cell-like cells, etc. (where homogeneity can be assessed by the percentage of cells expressing or lacking a specific marker protein). The term "immune cell population" can also be understood as a heterogeneous immune cell population, which includes any type of immune cell, but different immune cells or different types of immune cells (e.g., T-cell-like cells and NK-cell-like cells) lack endogenous effector functions, but include predetermined effector functions due to exogenously introduced genetic modifications dependent on the intended application. Those skilled in the art can distinguish and subsequently separate these cells from each other using methods known in the art, such as flow cytometry. This means that, for example, if an exogenous TCR is introduced into the genome of a PSC and an exogenous CD16 receptor is introduced into the genome of a PSC, PSCs modified with the exogenous TCR gene produce T-cell-like cells, while PSCs modified with the exogenous CD16 receptor gene produce NK-cell-like cells, thereby obtaining a population of different immune cells as defined herein.Immune cells include neutrophils, eosinophils, basophils, lymphocytes, and monocytes, as well as lymphocytes such as B cells, T cells, and natural killer (NK) cells. The immune cell population may also include cells representing precursor cells of T-cell-like cells or NK-cell-like cells, and cells representing mature T-cell-like cells or mature NK-cell-like cells.

[0061] The method of the present invention can also be adapted to produce a high cell population of immune cells by using bioreactor conditions in any or all of steps (i)(a), (i)(b), (i)(c), and / or (ii), or even in the PSC culture step prior to step (i)(a). The use of a bioreactor offers the advantage of achieving higher cell yields due to the use of a large-scale bioreactor. The use of a bioreactor allows for optimization of the concentrations of different factors while producing higher cell numbers. Therefore, the use of a bioreactor enables a scaled-up increase in the cell number of the immune cell population provided by the method of the present invention. This achieves the cell number of the immune cell population required for clinical applications. Furthermore, this provides a highly standardized and cost-effective method. In addition, the use of a bioreactor simplifies the operational process, as it provides a less labor-intensive and easier-to-implement method.

[0062] All definitions provided herein for the methods of this invention can be applied to immune cells or immune cell populations that lack endogenous effector function and are obtainable or acquireable by the methods of this invention. All definitions provided herein for the methods of this invention, with appropriate adjustments, can also be applied to immune cells or immune cell populations that lack endogenous effector function but contain one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function that is obtainable or acquireable by the methods of this invention.

[0063] In a preferred embodiment, this document provides immune cells or populations of immune cells that can be obtained or acquired by the specific methods of the present invention, preferably NK-like precursor cells that express CD45 but do not express endogenous CD94 receptor, endogenous CD16 receptor, endogenous NKG2D receptor, endogenous NKp30 receptor, endogenous TCR and endogenous CD19 antigen.

[0064] In a preferred embodiment, this document provides immune cells or populations of immune cells obtainable or acquireable by specific methods of the present invention, lacking endogenous effector functions as defined herein, but containing one or more exogenous modifications, each of which introduces a predetermined effector function, wherein the one or more exogenous modifications are gene knock-ins comprising knock-ins of at least one gene selected from genes encoding: CAR, exogenous TCR, exogenous IL-15 receptor, exogenous CD16 receptor, exogenous CD19 antigen, exogenous IL-10 cytokine, exogenous TGF-β cytokine, and exogenous PD ligand 1 (PD-L1) antigen. In other words, this document also includes immune cells or populations of immune cells obtainable or acquireable by specific methods as defined herein, preferably T cell-like cells or populations of T cell-like cells, lacking endogenous effector functions, but expressing at least one of CAR, exogenous TCR, exogenous IL-10 cytokine, exogenous TGF-β cytokine, or exogenous PD ligand 1 (PD-L1) antigen. This document also includes immune cells or populations of immune cells, preferably NK cell-like cells or populations, that are available or obtainable by the specific methods defined herein, lacking endogenous effector function but expressing at least one of exogenous IL-15 receptor or exogenous CD16 receptor. This document also includes immune cells or populations of immune cells, preferably B cell-like cells or populations, that are available or obtainable by the specific methods defined herein, lacking endogenous effector function but expressing exogenous CD19 antigen and / or exogenous PD ligand 1 (PD-L1) antigen.

[0065] The present invention also includes a method for converting immune cells lacking endogenous effector function, which are obtainable or acquired by the methods of the present invention as defined herein, into immune cells lacking endogenous effector function but containing 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 hematopoietic differentiation by the following steps: (a) seeding PSCs on a solid support suitable for 3D cell aggregate formation in a first serum-free medium under suitable conditions, thereby allowing the formation of 3D cell aggregates; (b) collecting the 3D cell aggregates of step (a), resuspending the 3D cell aggregates in a second serum-free medium under suitable conditions, transferring the resuspended 3D cell aggregates to a third serum-free medium, and culturing them in suspension under suitable conditions for about 1 to about 3 days, thereby allowing mesodermal differentiation; and (c) The cultured 3D cell aggregates from step (b) are transferred to a fourth serum-free medium and cultured in suspension under suitable conditions for approximately 2 to 4 days, and further cultured in a fifth serum-free medium in suspension under suitable conditions for approximately 2 to 6 days, thereby allowing hematopoietic differentiation; subsequently (ii) immune cell differentiation is induced by culturing single cells of the 3D cell aggregates obtained in steps (i) and (c) in suspension under suitable conditions for an appropriate time; wherein the method further includes introducing one or more exogenous modifications into genes in PSCs before step (a), during steps (a) to (c) of step (i), or at any time during step (ii), wherein each of the one or more exogenous modifications introduces a predetermined effector function, thereby providing genetically modified immune cells that lack endogenous effector functions but contain one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function. Where necessary, all definitions provided herein may also be applied to this transformation method.

[0066] Additionally, the present invention includes a pharmaceutical composition comprising immune cells or populations of immune cells obtained / available by the methods of the present invention as defined herein. Such pharmaceutical compositions may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes any carrier that itself does not cause adverse reactions harmful to a subject receiving the pharmaceutical composition. Therefore, the pharmaceutical composition is intended for therapeutic purposes. According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a subject, preferably a human, as defined herein. Pharmaceutical compositions or formulations are generally in a form that allows the active ingredient to exert its biological activity and is therefore applicable to a subject for therapeutic use as described herein. Suitable carriers are typically large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Carriers used in combination with the pharmaceutical compositions of the present invention may be water-based and form aqueous solutions or oil-based carrier solutions.

[0067] This document also includes a kit containing a pharmaceutical composition as defined herein. Such kits may additionally include an instruction manual containing instructions for administering the pharmaceutical composition to a subject in need.

[0068] Furthermore, this invention relates to immune cells or populations of immune cells as defined herein, or pharmaceutical compositions as defined herein, for use in medicine. This invention also includes immune cells or populations of immune cells of this invention, or pharmaceutical compositions of this invention, for use in methods of preventing or treating cancer.

[0069] As used herein, the term "treatment" ("treat", "treating", or "treatment") means to reduce (slow down), stabilize, or inhibit, or at least partially alleviate or eliminate the progression of symptoms associated with a corresponding disease. Therefore, this term includes the administration of cells (preferably in the form of a drug or pharmaceutical composition) as defined herein to a subject as defined elsewhere herein. Those subjects requiring treatment include those who already have a disease (here, cancer). Preferably, treatment reduces (slow down), stabilizes, or inhibits, or at least partially alleviates or eliminates the progression of symptoms associated with the presence and / or progression of a disease or pathological condition. Therefore, "treatment" refers to therapeutic treatment. Specifically, in the context of this invention, treatment or treatment means improvement of symptoms associated with cancer as defined elsewhere herein. When used herein, the term "subject" includes mammalian subjects. Preferably, subjects of this invention are mammals including humans. In some embodiments, the mammal is a mouse. Subjects also include human and veterinary patients. When a subject is a living human who requires treatment for the disease or condition described herein, they are also referred to as a "patient". Patients requiring treatment include those who already have the disease. In this case, the subject may have a disease (in this case, cancer) or symptoms of a corresponding disease (in this case, cancer).

[0070] The term "prevention" ("preventing" or "prevention") refers to preventive or preventative measures that prevent the disease from even occurring or the symptoms associated with the corresponding disease from appearing, i.e., preventing the onset or recurrence of a disease (in this case, cancer). Therefore, this term also includes administering cells (preferably in the form of a drug or pharmaceutical composition) as defined herein to a subject as defined elsewhere herein. The definitions of "subject" provided above also apply here. In this case, the subject may have a susceptibility to a specific disease (in this case, cancer).

[0071] In this context, the invention also includes the immune cells or populations of immune cells of the invention, or the pharmaceutical compositions of the invention, used in methods of preventing or treating cancer as defined herein, wherein the cancer is selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, lymphatic system cancers including lymphoma and multiple myeloma, leukemia, bone or soft tissue sarcoma, cervical cancer, and vulvar cancer.

[0072] Another aspect of the invention relates to the immune cells or populations of immune cells of the invention, or the pharmaceutical compositions of the invention, for use in cell-based therapies. The pharmaceutical use includes administering the immune cells or populations of immune cells of the invention, or cell populations or pharmaceutical compositions derived from the immune cell populations of the invention, to a subject in need. The subject is typically a mammal, such as a human. Where necessary, the definitions above may also apply to this paragraph.

[0073] Therefore, the immune cells or immune cell populations or pharmaceutical compositions of the present invention, as defined elsewhere herein, are typically administered to subjects for cancer treatment or immunotherapy. The immune cells or immune cell populations or pharmaceutical compositions of the present invention, as defined elsewhere elsewhere, are used in adoptive T-cell transfer or similar treatment regimens. Among various types of immunocellular therapies (such as T-cell immunotherapy), adoptive cell therapy has received considerable attention and interest in recent years. Adoptive cell therapy is a personalized therapy in which the patient's own immune cells are removed, modified immune cells are generated using the methods of the present invention, expanded to a large quantity in vitro, and then infused back into the patient to eliminate the tumor. Guedan et al., Rev Immunol. 2019 Apr 26; 37: 145–171. doi:10.1146 / annurev-immunol-042718-041407 provides an overview of the development of adoptive cell therapy.

[0074] ***

[0075] Unless otherwise stated, the following terms as used herein (including the specification and claims) have the definitions given below.

[0076] Those skilled in the art can recognize or determine many equivalent embodiments of the present invention described herein using only conventional experiments. This invention is intended to cover such equivalent embodiments.

[0077] It is important to note that, as used herein, the singular forms “a,” “an,” and “the” include plural 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 equivalent steps and methods known to those skilled in the art that can modify or replace the methods described herein.

[0078] Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in that series. Those skilled in the art can recognize or determine numerous equivalents of the specific embodiments of the invention described herein using only conventional experiments. This invention is intended to cover such equivalents.

[0079] The term “and / or” as used anywhere in this document includes the meanings of “and,” “or,” and “all or any other combination of elements connected by the term.”

[0080] The terms “about” or “approximately” refer to an acceptable range of error for a particular value as determined by those skilled in the art, which will depend in part on how the value was measured or determined, i.e., on the limitations of the measurement system. For example, “about” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” may represent a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Or, as used herein, “about” means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, the term also includes specific numerical values; for example, about 20 includes 20. Or, particularly for biological systems or processes, the term may refer to an order of magnitude of a value, preferably within 5 times, more preferably within 2 times. In cases where a particular value is described in this application and claims, unless otherwise stated, it should be assumed that the term “about” means within an acceptable range of error for the particular value.

[0081] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” shall be understood to mean comprising the said integer or step or group of integers or steps, but not excluding any other integer or step or group of integers or steps. When used herein, the term “containing” may be replaced by the terms “comprising” or “including”, or sometimes by the term “having”.

[0082] When used herein, "consisting of..." excludes any element, step, or ingredient not specified in the elements of the claim. When used herein, "consisting substantially of..." does not exclude materials or steps that do not substantially affect the essential and novel features of the claim.

[0083] In each case herein, any one of the terms “containing,” “consistently composed of,” and “composed of” may be replaced by any of the other two terms.

[0084] It should be understood that the present invention is not limited to the specific methods, schemes, materials, reagents, and substances described herein, as these can be modified. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0085] All publications cited in this specification (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.) are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to any prior art based on such disclosure. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, this specification supersedes any such material.

[0086] Embodiments of the present invention

[0087] The following embodiments are used to illustrate the present invention. These embodiments should not be construed as limiting the scope of the invention. They are included for illustrative purposes only, and the invention is defined solely by the claims.

[0088] Example 1: Production of immune cell populations from pluripotent stem cells (PSCs).

[0089] Human induced pluripotent stem (iPS) cell line TC-1133 was purchased from Lonza and reprogrammed under c-GMP conditions. This cell line also refers to ND-50039 cells from the NINDS Human Cell and Database. Human iPS cells were cultured and maintained in matrix gel-coated T-flasks in iPS amplification medium (Miltenyi StemMACS iPS-Brew XF) as defined elsewhere in this document.

[0090] 3D-aggregate formation and mesodermal induction (days 1 to 3 of differentiation):

[0091] 1. Use 2 mL of anti-adhesion rinsing solution (Stem Cell Technologies, 07010) per well to rinse AggreWell. TM Pretreatment was performed using 4006-well plates (Stemcell Technologies, 34425).

[0092] 2. Draw out the anti-adhesion rinsing fluid from the hole.

[0093] 3. Rinse each well with warm Dulbecco's phosphate-buffered saline (DPBS, Gibco 14190136) and keep at room temperature until used.

[0094] 4. Wash the human iPS cells in the T-flask with 5 mL of DPBS (Gibco 14190136).

[0095] 5. Add 3 mL of Accutase (Gibco A1110501) and incubate the cells at 37°C for 4 minutes.

[0096] 6. Gently tap the culture flask to detach the cells, then rinse the cells with 5 mL of DPBS.

[0097] 7. Transfer the cells to Falcon and centrifuge at 300g for 4 minutes.

[0098] 8. Discard the supernatant, resuspend the cells in 1 mL of iPSC amplification medium (Miltenyi StemMACSiPS-Brew XF) and count them.

[0099] 9. 3.0 × 10 6 AggreWell cells were seeded in iPSC amplification medium supplemented with CHIR99021 (10 μM), BMP4 (50 ng / mL), VEGF (50 ng / mL), FGF (50 ng / mL), and ROCK inhibitor Y-27632 dihydrochloride (10 μM) in AggreWell medium. TM In each hole of a 400 6-well plate.

[0100] 10. Centrifuge the plate at 100g for 3 minutes to capture cells in the microwells.

[0101] 11. Incubate the plate at 37°C, 5% CO2 and 95% humidity for 24 hours.

[0102] 12. On day 1, aggregate formation can be observed under a microscope.

[0103] 13. To collect 3D aggregates from the plate, gently tap the plate to dislodge the aggregates, rotate the plate to gather the aggregates in the center of the well, collect the aggregates from the plate using a serum pipette, and transfer them to Falcon.

[0104] 14. Once all the aggregates have settled to the bottom of the Falcon, the culture medium can be carefully discarded without disturbing the aggregates.

[0105] 15. The aggregates were resuspended in mesotherapy induction medium and transferred to an ultra-low adhesion suspension culture plate, wherein the mesotherapy induction medium consisted of StemPro34-SFM medium (Gibco 10639011) supplemented with BMP4 (50 ng / ml), VEGF (50 ng / ml) and FGF (50 ng / ml).

[0106] 16. Place the plate on a fixed-track shaker and maintain the aggregates in dynamic suspension culture by rotating the shaker at 80 rpm.

[0107] 17. On day 2, the culture medium was replaced with mesotherapy induction medium, which consisted of StemPro34-SFM medium supplemented with BMP4 (50 ng / mL), VEGF (50 ng / mL), FGF (50 ng / mL) and SB431542 (10 μM).

[0108] Hematopoietic induction (differentiation days 4 to 11):

[0109] 1. On day 4, the culture medium in the plates was replaced with hematopoietic induction medium, which consisted of StemPro34-SFM medium supplemented with VEGF (50 ng / ml), FGF (50 ng / ml) and SCF (50 ng / ml).

[0110] 2. On days 7 and 9, the culture medium was replaced again with hematopoietic induction medium, which consisted 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).

[0111] 3. On day 11, aggregates were collected from the wells and dissociated into single cells using type II collagenase.

[0112] 4. Then sort CD34 positive cells using MACS or FACS.

[0113] Immune cell differentiation (days 11 to 25 of differentiation):

[0114] From day 11 to day 25 of differentiation, aggregates (iPSC-derived CD34+ cells) were cultured in suspension on Notch-ligand-coated or uncoated cell culture plates in immunocellular differentiation medium consisting of IMDM (Thermo Fischer Sci, 31980030) supplemented with 15% BIT 9500 serum substitute (Stemcell Technologies, 09500), β-mercaptoethanol (50 mM), IL-3 (10 ng / mL, added only from day 11 to day 18), TPO (50 ng / mL), IL-15 (50 ng / mL), SDF (10 ng / mL), SB203580 (15 μM), 50 ng / mL SCF, 50 ng / mL Flt-3L, 50 ng / mL IL-7, and 30 mM L-ascorbic acid 2-phosphate sesquimagnesium salt salt hydrate and 1% penicillin / streptomycin.

[0115] In the following examples, iPSC-derived immune cells as described in the differentiation protocol of Example 1 were used. Specifically, wild-type (WT) PSCs (not the non-immunogenic variant as defined herein) and CD19-CAR-expressing cells were used. The CD19-CAR construct was stably knocked into the safe harbor locus at the iPSC level as defined herein. Downstream experiments employed multiple clonal populations.

[0116] Example 2: Phenotypic characterization of iPSC-derived immune cells - These immune cells have no functional potential in vitro.

[0117] For phenotypic characterization, iPSC-derived immune cells were analyzed using flow cytometry and the following antibodies: anti-CD45 (AF700), anti-CD94 (B525), anti-CD16 (V450), anti-NKG2D (V610), NKp30 (V763), anti-CD3 (FITC), and anti-CD19 (PB) (all from BioLegend). (See also: [link to relevant documentation]) Figure 1 CAR expression was monitored using an anti-idiotype antibody specific to CD19-directed CARs (Miltaneyi Biotec). Antibody staining was performed according to the manufacturer's instructions. Cell-related fluorescence was analyzed by flow cytometry using a CytoFLEX LX flow cytometer (Beckman Coulter). For all scatter plots shown, pre-gating was performed for live cells and single cells.

[0118] For cell growth and expansion folds, live nucleated cells were counted using a NucelloCounter NC-3000 (Chemotec) according to the manufacturer’s instructions.

[0119] Example 3: Phenotypic characterization of iPSC-derived immune cells - These immune cells do not have cell-lysing function in vitro.

[0120] To test in vitro cell lysis function, iPSC-derived cells (effective cells) were mixed with CD19 at a specified cell E:T ratio. + GFP + Raji cell lines (target cells) were co-cultured for 0, 24, and 48 hours. Changes in CD19 antigen expression and cell percentage were assessed using flow cytometry, as described above. Primary human T and NK cells isolated from PBMC fractions using a positive selection kit (Miltenyi Biotec kit) served as positive controls (see [link to relevant documentation]). Figure 2 ).

[0121] The following control group was transfected with primary human T cells for CAR expression. The purified T cells were activated for 48 hours and then injected with 300 IU / ml... -1Recombinant human IL-2, 5 ng / ml -1 Recombinant human IL-7 (Peprotech) and 5 ng / ml -1 Cultured with IL-15. Then, using a 4DNucleofector X unit (Lonza, pulse code EH100), electroporated 1 × 10⁻⁶ cells in 20 μl of Nucleofector solution P3 (Lonza) with Cas9 ribonucleoprotein and CD19-CAR DNA template. 6 Cells. After electroporation, in a solution containing 180 IU / ml -1 Cells were cultured in serum-free IL-2 medium until co-culture experiments were conducted.

[0122] Example 4: Phenotypic characterization of iPSC-derived immune cells - These immune cells do not have cytolytic function in vivo.

[0123] To evaluate the in vivo function of CAR T cells, male NSGS mice aged 6-8 weeks were selected. First, 5 × 10⁶ T cells were used... 5 CD19 + Raji / ffluc cells were used to inject tumor cells via the tail vein. Seven days later, mice were additionally injected intravenously with PBS (control) or 0.75 × 10⁻⁶ mcg of PBS. 6 CAR selection + Cells. For bioluminescence imaging, mice were intraperitoneally injected with luciferin substrate (XenoLight d-luciferin, Perkin Elmer) resuspended in PBS (15 μg / g body weight), anesthetized with isoflurane, and imaged 10 minutes post-luciferin injection using an IVIS Lumina imaging system (Perkin Elmer) (in small pixel binning mode, acquisition time up to 1 minute to obtain unsaturated images). Luciferase activity was analyzed using Living Image software (Perkin Elmer) (see [link to documentation]). Figure 3 ).

[0124] project

[0125] 1. A method for producing immune cells lacking endogenous effector function from pluripotent stem cells (PSCs), the method comprising the following steps:

[0126] (i) Inducing 3D cell aggregate formation, mesodermal differentiation, and hematopoietic differentiation through the following steps:

[0127] (a) In a first serum-free medium, under suitable conditions, PSCs are seeded on a solid support suitable for the formation of 3D cell aggregates, thereby allowing the formation of 3D cell aggregates;

[0128] (b) Collect the 3D cell aggregates from step (a), resuspend the 3D cell aggregates in a second serum-free medium under suitable conditions, transfer the resuspended 3D cell aggregates to a third serum-free medium, and culture in suspension under suitable conditions for approximately 1 to 3 days to allow mesodermal differentiation; and

[0129] (c) The cultured 3D cell aggregates from step (b) are transferred to a fourth serum-free medium and cultured in suspension under suitable conditions for approximately 2 to 4 days, and further cultured in suspension under suitable conditions in a fifth serum-free medium for approximately 2 to 6 days, thereby allowing hematopoietic differentiation; and

[0130] (ii) Inducing immune cell differentiation by culturing single cells of the 3D cell aggregates obtained in step (i)(c) in suspension culture under suitable conditions for an appropriate time in a sixth serum-free medium;

[0131] This provides immune cells that lack endogenous effector functions.

[0132] 2. A method for producing genetically modified immune cells from pluripotent stem cells (PSCs), said immune cells lacking endogenous effector function but containing one or more exogenous modifications, wherein each of said one or more exogenous modifications introduces a predetermined effector function, said method comprising the steps of:

[0133] (i) Inducing 3D cell aggregate formation, mesodermal differentiation, and hematopoietic differentiation through the following steps:

[0134] (a) In a first serum-free medium, under suitable conditions, PSCs are seeded on a solid support suitable for the formation of 3D cell aggregates, thereby allowing the formation of 3D cell aggregates;

[0135] (b) Collect the 3D cell aggregates from step (a), resuspend the 3D cell aggregates in a second serum-free medium under suitable conditions, transfer the resuspended 3D cell aggregates to a third serum-free medium, and culture in suspension under suitable conditions for approximately 1 to 3 days to allow mesodermal differentiation; and

[0136] (c) The cultured 3D cell aggregates from step (b) are transferred to a fourth serum-free medium and cultured in suspension under suitable conditions for approximately 2 to 4 days, and further cultured in suspension under suitable conditions in a fifth serum-free medium for approximately 2 to 6 days, thereby allowing hematopoietic differentiation; and

[0137] (ii) Inducing immune cell differentiation by culturing single cells of the 3D cell aggregates obtained in step (i)(c) in suspension culture under suitable conditions for an appropriate time in a sixth serum-free medium;

[0138] The method further includes introducing one or more exogenous modifications into a gene in the PSC before step (a), during steps (a) to (c) of step (i), or at any time during step (ii), wherein each of the one or more exogenous modifications introduces a predetermined effector function.

[0139] This provides genetically modified immune cells that lack endogenous effector functions but contain one or more exogenous modifications, each of which introduces a predetermined effector function.

[0140] 3. The method according to Project 2, wherein the exogenous modification is selected from gene knock-in, gene knockout, gene substitution, point mutation, and deletion, insertion or substitution of genes, gene fragments or nucleotides, or combinations thereof.

[0141] 4. The method according to Project 3, wherein the gene knock-in comprises knocking in at least one gene selected from genes encoding the following substances:

[0142] i) Chimeric antigen receptor (CAR);

[0143] ii) Exogenous T cell receptor (TCR);

[0144] iii) Exogenous IL-15 receptor;

[0145] iv) Exogenous CD16 receptor;

[0146] v) Exogenous CD19 antigen;

[0147] vi) Exogenous IL-10 cytokine;

[0148] vii) Exogenous TGF-β cytokines; and

[0149] viii) Exogenous PD-1 ligand 1 antigen, or a combination thereof, and / or

[0150] The gene knockout mentioned above includes the knockout of at least one gene selected from genes encoding the following substances:

[0151] ix) Endogenous PD-1 cell surface protein;

[0152] x) Endogenous VEGF receptors;

[0153] xi) Endogenous VCAM-1 cell surface protein; and

[0154] xii) Endogenous ICAM-1 cell surface proteins, or combinations thereof.

[0155] 5. The method according to any one of the foregoing items, wherein:

[0156] In step (i)(a), the first serum-free culture medium contains a GSK-3 inhibitor and a ROCK inhibitor, each at a concentration of about 5 µM to about 15 µM.

[0157] In step (i) (b), the second serum-free culture medium contains bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF), each at a concentration of approximately 10 ng / mL to approximately 50 ng / mL, and

[0158] The third serum-free culture medium contains BMP4, VEGF, and FGF, each at a concentration of approximately 10 ng / mL to approximately 50 ng / mL, and additionally contains an ALK receptor inhibitor at a concentration of approximately 5 µM to approximately 15 µM; and / or

[0159] In step (i) (c), the fourth serum-free culture medium contains VEGF, FGF, and stem cell factor (SCF) at concentrations of approximately 10 ng / mL to approximately 50 ng / mL, and

[0160] The fifth serum-free culture medium contains VEGF, FGF, and SCF at concentrations of approximately 10 ng / mL to approximately 50 ng / mL, and additionally contains approximately 10 ng / mL to approximately 50 ng / mL of FMS-like tyrosine kinase 3 ligand (Flt-3l) and approximately 10 ng / mL to approximately 50 ng / mL of thrombopoietin (TPO); and / or

[0161] In step (ii), the sixth serum-free culture medium comprises about 7% to about 23% of BIT 9500 serum substitute, about 25 mM to about 75 mM of β-mercaptoethanol, about 10 ng / ml to about 50 ng / ml of IL-3, about 10 ng / ml to about 50 ng / ml of TPO, about 10 ng / ml to about 50 ng / ml of IL-15, about 10 ng / ml to about 50 ng / ml of SDF, about 7 µM to about 23 µM of SB203580, about 10 ng / ml to about 50 ng / ml of SCF, about 10 ng / ml to about 50 ng / ml of Flt-3l, about 10 ng / ml to about 50 ng / ml of IL-7, and about 15 mM to about 45 mM of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.

[0162] 6. The method according to any one of the foregoing items, wherein the ROCK inhibitor is Y-27632 dihydrochloride, the ALK receptor inhibitor is SB43152, and the GSK-3 inhibitor is CHIR99021.

[0163] 7. The method according to any one of the preceding items, wherein the PSC is a pluripotent stem cell lacking endogenous MHC class I molecules presented on its cell surface but containing immunomodulatory proteins on its surface.

[0164] 8. The method according to Project 7, wherein the immunomodulatory protein is a single-chain fusion HLA class I protein.

[0165] 9. The method according to Item 8, wherein the single-chain fusion HLA class I protein comprises at least a portion of β-2 microglobulin (B2M) covalently linked to at least a portion of an HLA class I α chain, wherein the HLA class I α chain is selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0166] 10. The method according to any one of items 8 to 9, wherein the PSC further expresses a target peptide antigen, the target peptide antigen being presented by the single-chain fusion HLA class I protein on the surface of the pluripotent cell.

[0167] 11. The method according to Item 10, wherein the target peptide antigen is covalently linked to the single-chain fusion HLA class I protein.

[0168] 12. The method according to item 10 or 11, wherein the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO: 1).

[0169] 13. The method according to any one of items 7 to 12, wherein in the PSC, substantially all copies of the B2M gene are destroyed.

[0170] 14. The method according to any one of the preceding items, wherein the PSC is a pluripotent stem cell lacking endogenous MHC class II.

[0171] 15. The method according to Project 14, wherein the PSC is deprived of endogenous MHC class II by disrupting the C2TA gene-MHC class II transactivator.

[0172] 16. The method according to any one of the preceding items, wherein the PSC is selected from embryonic stem cells, induced pluripotent stem cells, and parthenogenetic stem cells.

[0173] 17. The method according to any one of the preceding items, wherein the PSC is a primate-derived pluripotent stem cell, preferably a human pluripotent stem cell.

[0174] 18. The method according to any one of the preceding items, wherein the PSC is generated from CD34-positive cells isolated from umbilical cord blood.

[0175] 19. The method according to any one of the preceding items, wherein the PSC is ND-50039 cells from the NINDS Human Cell and Database.

[0176] 20. The method according to any one of the preceding items, wherein in step (i)(a), the solid support comprises one or more holes, wherein each hole comprises a V-shaped or conical cavity.

[0177] 21. The method according to item 20, wherein the solid support is a microwell culture plate.

[0178] 22. The method according to item 21, wherein the microplate is AggreWell. TM Board, BIOFLOAT TM Either a 96-well plate or Nunclon Sphera 3D culture medium.

[0179] 23. The method according to any one of the foregoing items, wherein the suspension culture in steps (i), (b) and (c) is a dynamic suspension culture, preferably achieved by culturing on a shaker at a rotation speed of about 60 to about 80 rpm.

[0180] 24. The method according to any one of the preceding items further includes dissociating the 3D cell aggregates into single cells after steps (i) and (c) and sorting them for CD34-positive PSCs.

[0181] 25. The method according to any one of the foregoing items, wherein step (ii) is performed for about 7 days to about 21 days.

[0182] 26. The method according to any one of the preceding items, wherein in step (ii), the suspension culture is carried out via a Notch ligand-coated suspension culture plate.

[0183] 27. The method according to any one of the preceding items, wherein the immune cell is an NK-like precursor cell lacking endogenous effector function.

[0184] 28. An immune cell or population of immune cells lacking endogenous effector function, which can be obtained by any one of items 1, 3 to 27.

[0185] 29. An immune cell or population of immune cells lacking endogenous effector function, obtained by any one of items 1, 3 to 27.

[0186] 30. An immune cell or immune cell population lacking endogenous effector function but comprising one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function, said immune cell or immune cell population being obtained by any one of items 2, 3 to 27.

[0187] 31. An immune cell or immune cell population lacking endogenous effector function but containing one or more exogenous modifications, wherein each of the one or more exogenous modifications introduces a predetermined effector function, said immune cell or immune cell population being obtained by any one of items 2, 3 to 27.

[0188] 32. A pharmaceutical composition comprising any one of items 28 to 31, the immune cells or populations of immune cells.

[0189] 33. The immune cells or immune cell populations according to any one of items 28 to 31, or the pharmaceutical composition according to item 32, used in medicine.

[0190] 34. The immune cells or immune cell populations according to any one of items 28 to 31, or the pharmaceutical composition according to item 32, in a method of preventing or treating cancer.

[0191] 35. Immune cells or immune cell populations, or pharmaceutical compositions, for the purposes described in item 34, wherein the cancer is selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, lymphatic system cancers including lymphoma and multiple myeloma, leukemia, bone or soft tissue sarcoma, cervical cancer, and vulvar cancer.

Claims

1. A method for producing immune cells lacking endogenous effector function from pluripotent stem cells (PSCs), the method comprising the following steps: (i) Inducing 3D cell aggregate formation, mesodermal differentiation, and hematopoietic differentiation through the following steps: (a) In a first serum-free medium, under suitable conditions, PSCs are seeded on a solid support suitable for the formation of 3D cell aggregates, thereby allowing the formation of 3D cell aggregates; (b) Collect the 3D cell aggregates from step (a), resuspend the 3D cell aggregates in a second serum-free medium under suitable conditions, transfer the resuspended 3D cell aggregates to a third serum-free medium, and culture them in suspension under suitable conditions for about 1 to about 3 days to allow mesodermal differentiation. and (c) The cultured 3D cell aggregates from step (b) are transferred to a fourth serum-free medium and cultured in suspension under suitable conditions for approximately 2 to 4 days, and further cultured in suspension under suitable conditions in a fifth serum-free medium for approximately 2 to 6 days, thereby allowing hematopoietic differentiation; and (ii) Inducing immune cell differentiation by culturing single cells of the 3D cell aggregates obtained in step (i)(c) in suspension culture under suitable conditions for an appropriate time in a sixth serum-free medium; This provides immune cells that lack endogenous effector functions.

2. A method for producing genetically modified immune cells from pluripotent stem cells (PSCs), said immune cells lacking endogenous effector function but containing one or more exogenous modifications, wherein each of said one or more exogenous modifications introduces a predetermined effector function, said method comprising the steps of: (i) Inducing 3D cell aggregate formation, mesodermal differentiation, and hematopoietic differentiation through the following steps: (a) In a first serum-free medium, under suitable conditions, PSCs are seeded on a solid support suitable for the formation of 3D cell aggregates, thereby allowing the formation of 3D cell aggregates; (b) Collect the 3D cell aggregates from step (a), resuspend the 3D cell aggregates in a second serum-free medium under suitable conditions, transfer the resuspended 3D cell aggregates to a third serum-free medium, and culture them in suspension under suitable conditions for about 1 to about 3 days to allow mesodermal differentiation. and (c) The cultured 3D cell aggregates from step (b) are transferred to a fourth serum-free medium and cultured in suspension under suitable conditions for approximately 2 to 4 days, and further cultured in suspension under suitable conditions in a fifth serum-free medium for approximately 2 to 6 days, thereby allowing hematopoietic differentiation; and (ii) Inducing immune cell differentiation by culturing single cells of the 3D cell aggregates obtained in step (i)(c) in suspension culture under suitable conditions for an appropriate time in a sixth serum-free medium; The method further includes introducing one or more exogenous modifications into a gene in the PSC before step (a), during steps (a) to (c) of step (i), or at any time during step (ii), wherein each of the one or more exogenous modifications introduces a predetermined effector function. This provides genetically modified immune cells that lack endogenous effector functions but contain one or more exogenous modifications, each of which introduces a predetermined effector function.

3. The method according to claim 2, wherein the exogenous modification is selected from gene knock-in, gene knockout, gene substitution, point mutation, and deletion, insertion or substitution of genes, gene fragments or nucleotides, or combinations thereof.

4. The method of claim 3, wherein the gene knock-in comprises knocking in at least one gene selected from genes encoding: i) Chimeric antigen receptor (CAR); ii) Exogenous T cell receptor (TCR); iii) Exogenous IL-15 receptor; iv) Exogenous CD16 receptor; v) Exogenous CD19 antigen; vi) Exogenous IL-10 cytokine; vii) Exogenous TGF-β cytokines; and viii) Exogenous PD-1 ligand 1 antigen, or a combination thereof, and / or The gene knockout mentioned above includes the knockout of at least one gene selected from genes encoding the following substances: ix) Endogenous PD-1 cell surface protein; x) Endogenous VEGF receptors; xi) Endogenous VCAM-1 cell surface protein; and xii) Endogenous ICAM-1 cell surface proteins, or combinations thereof.

5. The method according to any one of the preceding claims, wherein: In step (i)(a), the first serum-free culture medium contains a GSK-3 inhibitor and a ROCK inhibitor, each at a concentration of about 5 µM to about 15 µM. In steps (i) and (b), the second serum-free culture medium contains bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF), each at a concentration of approximately 10 ng / mL to approximately 50 ng / mL. The third serum-free culture medium contains BMP4, VEGF, and FGF, each at a concentration of approximately 10 ng / mL to approximately 50 ng / mL, and additionally contains an ALK receptor inhibitor at a concentration of approximately 5 µM to approximately 15 µM; and / or In step (i)(c), the fourth serum-free culture medium contains VEGF, FGF, and stem cell factor (SCF), each at a concentration of approximately 10 ng / mL to approximately 50 ng / mL, and The fifth serum-free culture medium contains VEGF, FGF, and SCF at concentrations of approximately 10 ng / mL to approximately 50 ng / mL, and additionally contains approximately 10 ng / mL to approximately 50 ng / mL of FMS-like tyrosine kinase 3 ligand (Flt-3l) and approximately 10 ng / mL to approximately 50 ng / mL of thrombopoietin (TPO); and / or In step (ii), the sixth serum-free culture medium comprises about 7% to about 23% of BIT 9500 serum substitute, about 25 mM to about 75 mM of β-mercaptoethanol, about 10 ng / ml to about 50 ng / ml of IL-3, about 10 ng / ml to about 50 ng / ml of TPO, about 10 ng / ml to about 50 ng / ml of IL-15, about 10 ng / ml to about 50 ng / ml of SDF, about 7 µM to about 23 µM of SB203580, about 10 ng / ml to about 50 ng / ml of SCF, about 10 ng / ml to about 50 ng / ml of Flt-3l, about 10 ng / ml to about 50 ng / ml of IL-7, and about 15 mM to about 45 mM of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.

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

7. The method according to any one of the preceding claims, wherein the PSC is a pluripotent stem cell lacking endogenous MHC class I molecules presented on its cell surface but containing immunomodulatory proteins on its surface.

8. The method according to claim 7, wherein the immunomodulatory protein is a single-chain fusion HLA class I protein.

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

10. The method according to any one of claims 8 to 9, wherein the PSC further expresses a target peptide antigen, the target peptide antigen being presented by the single-chain fused HLA class I protein on the surface of the pluripotent cell.

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

12. The method according to claim 10 or 11, wherein the target peptide antigen comprises the sequence VMAPRTLFL (SEQ ID NO: 1).

13. The method according to any one of claims 7 to 12, wherein in the PSC, substantially all copies of the B2M gene are destroyed.

14. The method according to any one of the preceding claims, wherein the PSC is a pluripotent stem cell lacking endogenous MHC class II.

15. The method of claim 14, wherein the PSC is deprived of endogenous MHC class II by disrupting the C2TA gene-MHC class II transactivator.

16. The method according to any one of the preceding claims, wherein the PSC is selected from embryonic stem cells, induced pluripotent stem cells, and parthenogenetic stem cells.

17. The method according to any one of the preceding claims, wherein the PSC is a primate-derived pluripotent stem cell, preferably a human pluripotent stem cell.

18. The method according to any one of the preceding claims, wherein the PSC is generated from CD34-positive cells isolated from umbilical cord blood.

19. The method according to any one of the preceding claims, wherein the PSC is ND-50039 cells from the NINDS Human Cell and Database.

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

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

22. The method of claim 21, wherein the microwell culture plate is AggreWell. TM Board, BIOFLOAT TM Either a 96-well plate or Nunclon Sphera 3D culture medium.

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

24. The method according to any one of the preceding claims further includes dissociating the 3D cell aggregates into single cells after steps (i) and (c) and sorting them for CD34-positive PSCs.

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

26. The method according to any one of the preceding claims, wherein in step (ii), the suspension culture is carried out via a Notch ligand-coated suspension culture plate.

27. The method according to any one of the preceding claims, wherein the immune cell is an NK-like progenitor cell lacking endogenous effector function.

28. An immune cell or population of immune cells lacking endogenous effector function, which can be obtained or acquired by the method of any one of claims 1, 3 to 27.

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

30. A pharmaceutical composition comprising immune cells or populations of immune cells as described in any one of claims 28 to 29.

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

32. The immune cells or immune cell populations according to any one of claims 28 to 29, or the pharmaceutical composition according to claim 30, in a method of preventing or treating cancer.

33. The immune cells or immune cell populations, or pharmaceutical compositions for the use described in claim 32, wherein the cancer is selected from lung cancer, prostate cancer, ovarian cancer, testicular cancer, brain cancer, skin cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, breast cancer, lymphatic system cancers including lymphoma and multiple myeloma, leukemia, bone or soft tissue sarcoma, cervical cancer, and vulvar cancer.

Images