Enhanced immune effector cells and use thereof

Abstract

To provide a functionally enhanced induced effector cell that is obtained by designated differentiation of a genome-engineered iPSC, a method for obtaining the cell, and a composition.SOLUTION: Provided is a cell or a population thereof, where (i) the cell is (a) an induced pluripotent cell (iPSC), a clone iPSC, or a cell from an iPS cell line, or (b) an induced cell obtained by differentiating the cell of (a), and (ii) the cell includes a polynucleotide encoding a IL15 / IL15Rα fusion protein (IL15Δ) which does not have CD38 knock-out or intracellular domain. The provided induced cell is equipped with stable and functional genome editing which brings about improved or enhanced therapeutic effect. Also provided are a therapeutic composition which includes only a functionally enhanced induced effector cell, or includes combination therapy with an antibody or a checkpoint inhibitor, and use thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 609,827, filed December 22, 2017; U.S. Provisional Patent Application No. 62 / 649,781, filed March 29, 2018; and U.S. Provisional Patent Application No. 62 / 774,052, filed November 30, 2018, the disclosures of which are incorporated herein by reference in their entirety.

[0002] The present disclosure broadly relates to the field of off-the-shelf immune cell products. More particularly, the present disclosure relates to strategies for developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. The cell products developed under the present disclosure address significant limitations of patient-derived cell therapies.

Background Art

[0003] The field of adoptive cell therapy is currently focused on using patient-derived and donor-derived cells, making it particularly difficult to achieve consistent manufacturing of cancer immunotherapies and to provide treatment to all patients who may benefit. There is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. Lymphocytes such as T cells and natural killer (NK) cells are powerful anti-tumor effectors that play important roles in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains difficult, and there are unmet needs for improvement. Thus, there remain important opportunities to maximize the potential of T cells, NK cells, or other lymphocytes in adoptive immunotherapy.

Summary of the Invention

[0004] Functionally improved effector cells are needed to address various issues such as response rate, cell depletion, loss of transfused cells (survival rate and / or persistence), tumor escape due to loss of target or lineage conversion, accuracy of tumor targeting, off-target toxicity, off-tumor effects, effectiveness against solid tumors, i.e., tumor microenvironment and associated immunosuppression, mobilization, transport, and infiltration.

[0005] The object of the present invention is to provide a method and composition for generating induced non-pluripotent cells differentiated from a single-cell-derived iPSC (induced pluripotent stem cell) clone strain, wherein this iPSC strain contains one or several gene modifications in its genome. The aforementioned one or several gene modifications include DNA insertions, deletions, and substitutions, and these modifications are retained and continue to function in the subsequent induced cells after differentiation, proliferation, passage, and / or transplantation.

[0006] The non-pluripotent cells derived from iPSCs of the present application include, but are not limited to, CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, and B cells. The non-pluripotent cells derived from iPSCs of the present application contain one or several gene modifications in their genomes through differentiation from iPSCs containing the same gene modification. In the engineered clone iPSC differentiation strategy for obtaining genetically engineered induced cells, it is also necessary that the likelihood of iPSC generation in the indicated differentiation is not adversely affected by the engineered modality of the iPSC, and that the engineered modality functions as intended in the induced cells. Furthermore, this strategy overcomes the current barriers when manipulating primary lymphocytes such as T cells or NK cells obtained from peripheral blood, i.e., such cells often result in cells with insufficient cell persistence lacking reproducibility and uniformity, accompanied by high cell death and low cell proliferation, making it difficult to manipulate such cells. Additionally, this strategy avoids the generation of heterogeneous effector cell populations obtained by other methods using a primary cell source that is initially heterogeneous.

[0007] Some aspects of the present invention provide genomically engineered iPSCs obtained using methods that include (I), (II), or (III), respectively, following, simultaneously with, and prior to a reprogramming process, each reflecting a strategy of genomic manipulation:

[0008] (I): Genetically manipulate the iPSC with either or both of (i) and (ii) in any order: (i) introduce one or more constructs into the iPSC to enable targeted integration at a selected site; (ii) (a) introduce one or more double-strand breaks into the iPSC at the selected site using one or more endonucleases capable of recognizing the selected site; (b) culture the iPSC of step (I)(ii)(a) to allow endogenous DNA repair to generate targeted indels at the selected site; thereby obtaining genomically engineered iPSCs capable of differentiating into partially or fully differentiated cells.

[0009] (II): Genetically manipulate the reprogrammed non-pluripotent cells to obtain genomically engineered iPSCs: (i) contact the non-pluripotent cells with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells; (ii) introduce either or both of (a) and (b) in any order into the reprogrammed non-pluripotent cells contacted in step (II): (a) one or more constructs enabling targeted integration at a selected site; (b) after introducing one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, culture the cells of step (II)(ii)(b) for endogenous DNA repair to allow generation of targeted indels at the selected site; thus, the obtained genomically engineered iPSCs contain at least one functional targeted genomic edit, and the aforementioned genomically engineered iPSCs can differentiate into partially or fully differentiated cells.

[0010] (III): Genetically engineering non-pluripotent cells for reprogramming to obtain genomically engineered iPSCs comprising (i) and (ii): (i) Introducing into the non-pluripotent cells either or both of (a) and (b) in any order: (a) One or more constructs enabling targeted integration at a selected site; (b) Performing one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, where the cells of step (III)(i)(b) are cultured to perform endogenous DNA repair to enable the generation of indels targeted at the selected site; (ii) Contacting the cells of step (III)(i) with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to obtain genomically engineered iPSCs comprising at least one functionally targeted genomic edit at the selected site, and the aforementioned genomically engineered iPSCs can differentiate into partially differentiated cells or fully differentiated cells.

[0011] In one embodiment of the above method, at least one targeted genomic editing at one or more selected sites comprises inserting one or more exogenous polynucleotides encoding a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival rate of genomically engineered iPSCs or their derived cells. In some embodiments, the exogenous polynucleotide for insertion is (1) one or more exogenous promoters including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, transient, tissue-specific, or cell-type specific promoters; or (2) operably linked to one or more endogenous promoters contained at a selected site including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors. In some embodiments, the genomically engineered iPSCs generated using the above method contain one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, wherein when the genomically engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into various safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotide encodes a partial or complete peptide of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors. In some embodiments, the partial or complete peptide of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors encoded by the exogenous polynucleotide is in the form of a fusion protein.

[0012] In some other embodiments, the genome-edited iPSCs generated using the methods provided herein include indels in one or more endogenous genes related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins that suppress engraftment, transport, homing, viability, self-renewal, persistence, and / or survival rate of iPSCs or their derived cells. In some embodiments, the endogenous genes for disruption include at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.

[0013] In still some other embodiments, the genome-edited iPSCs generated using the methods provided herein include caspase encoding an exogenous polynucleotide at the AAVS1 locus and thymidine kinase encoding an exogenous polynucleotide at the H11 locus.

[0014] In still some other embodiments, approaches (I), (II) and / or (III) further include contacting the genome-edited iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain the pluripotency of the genome-edited iPSCs. In one embodiment, the obtained genome-edited iPSCs comprising at least one targeted genome editing are functional and have the ability to differentiate, and can differentiate into non-pluripotent cells comprising the same functional genome editing.

[0015] The present invention also provides the following.

[0016] One aspect of the present application is a cell or a population thereof, wherein the cell is an induced pluripotent stem cell (iPSC), a cloned iPSC, or an iPSC line cell, or an induced cell obtained by differentiating any of the above iPSCs; any of the above cells comprises at least one CD38 knockout or a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) having no intracellular domain. In some embodiments of the induced cells obtained from iPSC differentiation, the induced cells are hematopoietic cells including, but not limited to, CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells; these hematopoietic cells (i.e., induced CD34 cells, induced hematopoietic endothelial cells, induced hematopoietic stem and progenitor cells, induced hematopoietic multipotent progenitor cells, induced T cell progenitors, induced NK cell progenitors, induced T cells, induced NKT cells, induced NK cells, or induced B cells) contain longer telomeres compared to their natural counterpart cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue.

[0017] In some embodiments of the iPSCs and their derived cells comprising a polynucleotide encoding CD38 knockout or an IL15 / IL15Rα fusion protein (IL15Δ) without an intracellular domain, the cells further comprise one or more of the following genome edits: (i) B2M null or low; (ii) CIITA null or low; (iii) introduction of HLA-G or non-cleavable HLA-G expression; (iv) high-affinity non-cleavable CD16 (hnCD16) or a variant thereof; (v) chimeric antigen receptor (CAR); (vi) partial or complete peptide of an exogenous cytokine or its receptor for cell surface expression; (vii) at least one of the genotypes listed in Table 1; (viii) deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region; and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptor, engager, and introduced or increased expression in at least one of surface trigger receptors for binding to bispecific or multispecific or universal engagers.

[0018] A polynucleotide encoding at least a CD38 knockout or an IL15 / IL15Rα fusion protein without an intracellular domain (IL15Δ), and in some embodiments of the iPSCs and their derived cells including the above and additional genome editing described throughout this application, the cells may comprise: (i) one or more exogenous polynucleotides integrated into one safe harbor locus, (ii) or three or more exogenous polynucleotides integrated into different safe harbor loci, or (iii) a polynucleotide encoding IL15Δ comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 17, 19, or 21. In some embodiments, the safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1. In a specific embodiment, the safe harbor locus TCR is the constant region of TCR alpha.

[0019] In some embodiments of a cell or a population thereof, the cell comprising at least a CD38 knockout or IL15Δ and one or more of the above-described additional genome editing is an induced NK or induced T cell, and the induced NK or induced T cell comprises at least one of the following characteristics, but is not limited thereto: (i) improved persistence and / or survival rate when compared to its natural counterpart NK or T cell obtained from peripheral blood, umbilical cord blood, or any other donor tissue, (ii) increased resistance to natural immune cells, (iii) increased cytotoxicity, (iv) improved tumor penetration, (v) enhanced or acquired ADCC, (vi) enhanced ability to migrate, activate or mobilize bystander immune cells to the tumor site, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability to rescue tumor antigen escape, reduced fratricide.

[0020] In one embodiment of a cell or population thereof, the cell comprising a CD38 knockout or IL15Δ further comprises a high-affinity non-cleavable CD16 (hnCD16) or a variant thereof. Some embodiments of the high-affinity non-cleavable CD16 (hnCD16) or a variant thereof include at least any one of the following: (a) F176V and S197P in the extracellular domain of CD16, (b) a complete or partial extracellular domain derived from CD64, (c) a non-natural (or non-CD16) transmembrane domain, (d) a non-natural (or non-CD16) intracellular domain, (e) a non-natural (or non-CD16) signaling domain, (f) a non-natural stimulatory domain, and (g) transmembrane, signaling, and stimulatory domains not derived from CD16 and derived from the same or different polypeptides. In some embodiments, the non-natural transmembrane domain is derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor (TCR) polypeptide. In some embodiments, the non-natural stimulatory domain is derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some other embodiments, the non-natural signaling domain is derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In some specific embodiments of the hnCD16 variant, the non-natural transmembrane domain is derived from NKG2D, the non-natural stimulatory domain is derived from 2B4, and the non-natural signaling domain is derived from CD3ζ.

[0021] In one embodiment of the cell or population thereof, the cell comprising CD38 knockout or IL15Δ further comprises a chimeric antigen receptor (CAR), and the CAR can be any one or more of the following: (i) T cell-specific or NK cell-specific; (ii) bispecific antigen-binding CAR; (iii) switchable CAR; (iv) dimerized CAR; (v) split CAR; (vi) multichain CAR; (vii) inducible CAR; (viii) co-expression with another CAR; (ix) co-expressed with a partial or complete peptide of an exogenous cytokine or its receptor for cell surface expression, optionally in a separate construct or in a bicistronic construct; (xi) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct; (xii) specific for CD19 or BCMA; and / or (xiii) ADGRE2, carbonic anhydrase IX (CAlX), CCRI, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9) Specific for any of Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, cancer fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigens.

[0022] In some embodiments where the checkpoint inhibitor is co-expressed with the CAR, the checkpoint inhibitor is an antagonist to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. The checkpoint inhibitor co-expressed with the CAR can be an antibody specific for any of the above-described checkpoint molecules, or a humanized or Fc-modified variant or fragment thereof, as well as their functional equivalents and biosimilars. In some embodiments, any one of (i)-(ix) CARs can be inserted into the TRAC locus. In some embodiments, any one of (i)-(ix) CARs inserted into the TRAC locus can be driven by the endogenous promoter of the TCR. In some embodiments, the insertion of any one of (i)-(ix) CARs at the TRAC locus results in a TCR knockout.

[0023] In one embodiment of the cell or population thereof, the cell comprising a CD38 knockout further comprises a partial or complete peptide of an exogenous cytokine or its receptor on cell surface expression, and the exogenous cytokine or its receptor may comprise at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (i) co-expression of IL15 and IL15Rα using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein with the intracellular domain of IL15Rα cleaved, (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and the common receptor γC, where the common receptor γC is natural or modified, (vii) may comprise at least one of homodimers of IL15Rβ, wherein any one of (i)-(vii) may be co-expressed with the CAR in a separate construct or a bicistronic construct. In some embodiments, the partial or complete peptide of the exogenous cytokine or receptor on the cell surface is transiently expressed in the cells provided herein.

[0024] In another embodiment of the cell or population thereof, the cell comprises a partial or complete peptide of an exogenous cytokine or receptor expressed on the cell surface, and the cytokine may comprise at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors. In an embodiment of a cell or population thereof comprising an IL15 cytokine or receptor, the cell is (i) co-expressing IL15 and IL15Rα using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein with the intracellular domain of IL15Rα cleaved, (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and the common receptor γC, where the common receptor γC is natural or modified, (vii) may comprise at least one of homodimers of IL15Rβ, where any one of (i)-(vii) can be co-expressed with the CAR in a separate construct or in a bicistronic construct. In some embodiments, the partial or complete peptide of the exogenous cytokine or receptor on the cell surface is transiently expressed in the cells provided herein. In one embodiment, the cell or population thereof comprises a polynucleotide encoding an IL15Δ comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 17, 19 or 21.In one embodiment of a cell or a population thereof, a cell comprising IL15Δ may further comprise one or more of B2M null or low, CIITA null or low, introduced expression of HLA-G or non-cleavable HLA-G, high affinity non-cleavable CD16 (hnCD16) or a variant thereof, a chimeric antigen receptor (CAR), a partial or complete peptide of an exogenous cytokine or its receptor with cell surface expression (the cytokine is not IL15), at least one of the genotypes listed in Table 1, a deletion or decreased expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region, and introduced expression or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptor, an engager, and a surface trigger receptor for binding to a bispecific or multispecific or universal engager. In an embodiment of a cell or a population thereof comprising both IL15Δ and a CAR, IL15Δ may be co-expressed with the CAR in a separate construct or a bicistronic construct.

[0025] In one embodiment of the cell or population thereof, the cell comprising CD38 knockout or IL15Δ is an induced NK cell or an induced T cell, and the induced NK cell can recruit and / or migrate T cells to the tumor site, where the induced NK cell or the induced T cell can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. In some embodiments, the checkpoint inhibitor is an antagonist to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some other embodiments, the checkpoint inhibitor comprises (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0026] Another aspect of the present application provides a composition comprising any cell or population thereof described above and throughout the present application. In some embodiments, the iPSC or iPSC-derived cells (induced cells) can include any one of the genotypes listed in Table 1 of the present application. In some embodiments, the iPSC or induced cells therefrom include CD38 knockout (CD38− / −). In some embodiments, the iPSC or induced cells therefrom include IL15Δ. In some embodiments, the iPSC or induced cells therefrom include hnCD16 and CD38 knockout. In some embodiments, the iPSC or induced cells therefrom include hnCD16 and IL15Δ. In some embodiments, the iPSC or induced cells therefrom include hnCD16, CD38 knockout, and IL15Δ. In some embodiments, the iPSC or induced cells therefrom include hnCD16, CD38− / −, and CAR. In some embodiments, the iPSC or induced cells therefrom include hnCD16, IL15Δ, and CAR. In some embodiments, the iPSC or induced cells therefrom include hnCD16, IL15Δ, CD38− / −, and CAR. In some embodiments, the iPSC or induced cells therefrom include hnCD16, CD38− / −, CAR, and a partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface described above and throughout the present application. In some embodiments of cells comprising hnCD16, CD38− / −, and CAR, the CAR is specific for CD19. In some embodiments of cells comprising hnCD16, IL15Δ, and CAR, the CAR is specific for CD19. In some embodiments of cells comprising hnCD16, IL15Δ, CD38− / −, and CAR, the CAR is specific for CD19. In other embodiments of cells comprising hnCD16, CD38− / −, and CAR, the CAR is specific for CD269 (BCMA). In other embodiments of cells comprising hnCD16, IL15Δ, and CAR, the CAR is specific for CD269 (BCMA).In some other embodiments of the cells comprising hnCD16, IL15Δ, CD38− / −, and CAR, the CAR is specific for CD269 (BCMA). In some further other embodiments, the CAR is specific for any of ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD8, CLEC12A, an antigen of cytomegalovirus (CMV)-infected cells (e.g., a cell surface antigen), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin β7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCS1, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and any of various pathogen antigens known in the art.

[0027] Accordingly, a further aspect of the present application provides a composition for therapeutic use comprising one or more therapeutic agents in addition to any of the induced cells provided herein. In some embodiments of the composition for therapeutic use, the therapeutic agent comprises a peptide, cytokine, checkpoint inhibitor, mitogen, growth factor, small molecule RNA, dsRNA (double-stranded RNA), mononuclear cells, feeder cells, feeder cell components or their replenishing factors, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In some embodiments of the composition for therapeutic use, the checkpoint inhibitor used with the provided cells comprises an antagonist to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some embodiments of the composition for therapeutic use, the checkpoint inhibitor used with the provided cells comprises one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof. In some other embodiments of the composition for therapeutic use, the checkpoint inhibitor used with the provided cells comprises at least one of atezolizumab, nivolumab, and pembrolizumab. In some embodiments of the composition for therapeutic use, the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide.

[0028] In some embodiments of the composition for therapeutic use, the antibody used with the provided cells comprises any one of anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies. In some embodiments of the composition for therapeutic use, the antibody used with the provided cells comprises one or more of rituximab, belzutifan, ofatumumab, ublituximab, ocrelizumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and their humanized or Fc-modified variants or fragments, as well as their functional equivalents and biosimilars. In yet some other embodiments of the composition for therapeutic use, the antibody used with the provided cells comprises daratumumab.

[0029] This application also provides for the therapeutic use of the cells or therapeutic composition described herein by introducing the composition into a subject suitable for adoptive cell therapy. In some embodiments, the subject suitable for and in need of adoptive cell therapy has an autoimmune disorder, a hematologic malignancy, a solid tumor; cancer, or a viral infection.

[0030] A further aspect of the present application provides a method for producing the induced cells described herein, the method comprising differentiating iPSCs comprising CD38 knockout or IL15Δ, and optionally one or more of the following: (i) B2M null or low, (ii) CIITA null or low, (iii) introduction of HLA-G or non-cleavable HLA-G expression, (iv) high-affinity non-cleavable CD16 (hnCD16) or a variant thereof, (v) chimeric antigen receptor (CAR), (vi) partial or complete peptide of an exogenous cytokine or its receptor for cell surface expression, (vii) at least one of the genotypes listed in Table 1, (viii) deletion or decreased expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosomal 6p21 region, and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptor, engager, and introduced or increased expression in at least one of surface trigger receptors for binding to bispecific or multispecific or universal engagers.

[0031] In some embodiments of the manufacturing method, the method further comprises genomically engineering the cloned iPSCs to knockout CD38 or knockin IL15Δ, and optionally knockout B2M and CIITA, or introduce the expression of HLA-G or non-cleavable HLA-G, high-affinity non-cleavable CD16 or variants thereof, CAR, and / or a partial or full peptide of an exogenous cytokine or its receptor for cell surface expression, wherein the CAR and the partial or full peptide of the exogenous cytokine or its receptor for cell surface expression are co-expressed in separate constructs or a bicistronic construct. In some embodiments of the manufacturing method, the genomic engineering of the iPSCs comprises targeted editing. In some embodiments, the targeted editing comprises deletions, insertions, or indels. In some embodiments, the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0032] This application further provides for CRISPR-mediated editing of cloned iPSCs, thereby generating edited cloned iPSCs that contain a CD38 knockout, an IL15Δ knock-in, or at least one of the genotypes listed in Table 1. In some embodiments of the CRISPR-mediated editing, the resulting CD38 knockout is homozygous. In some embodiments of the CRISPR-mediated editing, the CD38 knockout is a nucleic acid cleavage between a first and a second target sequence, wherein the targeting sequences comprise SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In some embodiments of the CRISPR-mediated editing, the resulting IL15Δ knock-in comprises a polynucleotide encoding an IL15Δ having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21. In some embodiments of the CRISPR-mediated editing described above, the editing further comprises insertion of a CAR at the TRAC locus, and / or the CAR is driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by the CAR insertion.

[0033] Additional aspects of the present application provide a method of improving anti-CD38 antibody therapy, including administering effector cells without CD38 expression to a subject under treatment. In some embodiments of the anti-CD38 antibody therapy, the anti-CD38 antibody can be daratumumab, isatuximab, or MOR202, or humanized or Fc-modified variants or fragments, functional equivalents, and biosimilars thereof. In some embodiments, the effector cells provided for the method of improving anti-CD38 antibody therapy include induced hematopoietic cells including induced NK cells or induced T cells, and the induced NK cells or induced T cells include CD38 knockout, high-affinity non-cleavable CD16 or variants thereof, and optionally (i) B2M and CIITA knockout, (ii) HLA-G or non-cleavable HLA-G, CAR, and / or expression with introduction of a partial or complete peptide of an exogenous cytokine or its receptor for cell surface expression, wherein the CAR and the partial or complete peptide of the exogenous cytokine or its receptor for cell surface expression are co-expressed in separate constructs or a bicistronic construct, and / or (iii) at least one of the genotypes listed in Table 1. In some embodiments of the method of improving anti-CD38 antibody therapy, the method reduces the reduction of anti-CD38 antibody-induced effector cells in a subject under such treatment.

[0034] Yet another aspect of the present application is a method of reducing or preventing allogeneic rejection of allogeneic effector cells by using a CD38-specific antagonist, wherein the allogeneic effector cells comprise a CD38 knockout and the CD38-specific antagonist is capable of suppressing activated T cells and B cells in the recipient of the allogeneic effector cells. In some embodiments, the CD38-specific antagonist is an anti-CD38 antibody, a CD38-specific engager, or a CD38 chimeric antigen receptor (CAR). In some other embodiments, the anti-CD38 antibody is daratumumab, isatuximab, or MOR202, or a humanized or Fc-modified variant or fragment, functional equivalent, and biosimilar thereof. In yet another embodiment, the anti-CD38 antibody is daratumumab, and the method provided herein provides a novel use of daratumumab.

[0035] The various objectives and advantages of the compositions and methods provided herein will become apparent from the following description in conjunction with the accompanying drawings, which illustrate specific embodiments of the invention by way of example and illustration.

Brief Description of the Drawings

[0036]

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Mode for Carrying Out the Invention

[0037] Genome modification of iPSCs (induced pluripotent stem cells) includes insertion, deletion, and substitution of polynucleotides. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or reduced gene expression in the dedifferentiated cell types from cells derived from the genome-engineered iPSCs after long-term clonal expansion of the original genome-engineered iPSCs, after cell differentiation. On the other hand, it is difficult to directly manipulate primary immune cells such as T cells or NK cells, which poses an obstacle to the preparation and delivery of engineered immune cells for adoptive cell therapy. The present invention provides an efficient and reliable targeted approach for stably integrating one or more exogenous genes including suicide genes and other functional modalities, which confers improved therapeutic properties related to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, lifespan, self-renewal, persistence, and / or survival rate to iPSC-derived cells including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells.

[0038] Definitions Unless otherwise specifically defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context specifically requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0039] It is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein and may therefore vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined only by the claims.

[0040] As used herein, the articles "a," "an," and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0041] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0042] The term "and / or" should be understood to mean either or both of the alternatives.

[0043] As used herein, the terms "about" or "approximately" refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by an amount of 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to the referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms "about" or "approximately" refer to a level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is approximately, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0044] As used herein, the terms "substantially" or "essentially" refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more, as compared to a referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms "substantially the same" or "essentially the same" refer to a range of an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is approximately the same as a referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0045] As used herein, the terms "substantially free of" and "essentially free of" are used interchangeably and, when used to describe a composition such as a cell population or a culture medium, refer to a composition that is, for example, 95% free of, 96% free of, 97% free of, 98% free of, 99% free of, etc., of a particular substance or source thereof, or undetectable by conventional means of measurement. The terms "free of" or "essentially free of" a particular component or substance in a composition also mean that such a component or substance is (1) not present in the composition at any concentration or (2) present in the composition at a low density and is functionally inert. A similar meaning may apply to the term "absent," which refers to the absence of a particular substance or source thereof in a composition.

[0046] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to mean including the stated step, or element, or group of steps or elements but not to mean excluding any other step, or element, or group of steps or elements. In particular embodiments, the terms "include," "have," "contain," and "contain" are used synonymously.

[0047] "Consisting of" means including and being limited to what follows the phrase "consisting of". Accordingly, the phrase "consisting of" indicates that the recited elements are necessary or essential and that no other elements can exist.

[0048] "Consisting essentially of" means including any elements recited after the phrase, and being limited to other elements that do not interfere with or contribute to the activity or operation specified by the disclosure of the recited elements. Accordingly, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that other elements are not necessarily required and may or may not be present depending on whether they affect the activity or operation of the recited elements.

[0049] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "a particular embodiment", "additional embodiments", or "further embodiments" or combinations thereof mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0050] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements conducted within or on biological tissue in an artificial environment outside of the body, preferably with minimal changes to natural conditions. In certain embodiments, an "ex vivo" procedure includes living cells or tissue that have been removed from a living organism and cultured in an experimental apparatus, typically under aseptic conditions, for a period of several hours or up to about 24 hours, but in some circumstances up to 48 hours or 72 hours or more. In certain embodiments, such tissue or cells can be collected and frozen and later thawed for ex vivo processing. Tissue culture experiments or procedures that continue for longer than a few days using viable cells or tissue are typically considered to be "in vitro", but in certain embodiments, the term can be used interchangeably with ex vivo.

[0051] The term "in vivo" generally refers to activities that occur within a living organism.

[0052] As used herein, the terms "reprogramming" or "dedifferentiation" or "increased differentiation potential" or "increased developmental potential" refer to methods of increasing the differentiation potential of a cell, or methods of dedifferentiating a cell to a less differentiated state. For example, increased differentiation potential means having more developmental plasticity (i.e., being able to differentiate into more cell types) compared to the same cell in an un-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in an un-reprogrammed state.

[0053] As used herein, the term "differentiation" is the process by which unspecialized ("uncommitted") or less-specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or differentiation-induced cells are cells that assume a more specialized ("committed") position within the cell lineage. The term "committed", when applied to the process of differentiation, refers to cells that have progressed along the differentiation pathway to a position where they will continue to differentiate into a particular cell type or subset of cell types under normal circumstances and, under normal circumstances, cannot differentiate into different cell types or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell (i.e., the embryo itself) to form all lineages of the organism or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers, the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential that ranges from less primitive and less pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can generate a complete organism.

[0054] As used herein, the term "induced pluripotent stem cell" or "iPSC" means that a stem cell has been reprogrammed from a differentiated adult, neonatal, or fetal cell that has been induced or altered, i.e., to a cell that can differentiate into all tissues of the three germ layers or the dermal layer: mesoderm, endoderm, and ectoderm. The generated iPSCs do not refer to naturally occurring cells.

[0055] As used herein, the term "embryonic stem cell" refers to the naturally occurring pluripotent stem cells of the inner cell mass of the blastocyst. Embryonic stem cells are pluripotent and give rise to derivatives of all three major germ layers, the ectoderm, endoderm, and mesoderm. They do not contribute to the extraembryonic membranes or placenta, i.e., they are not totipotent.

[0056] As used herein, the term "pluripotent stem cell" refers to a cell that is capable of differentiating into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Thus, pluripotent cells can also be referred to as "partially differentiated cells." Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells such as, for example, hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell can form many types of cells of a particular lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to the state of a cell having a lower degree of developmental potential than totipotency and pluripotency.

[0057] Pluripotency can be determined in part by assessing the pluripotent characteristics of the cell. Characteristics of pluripotency include, but are not limited to, (i) the morphology of pluripotent stem cells, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers such as SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) the formation of teratomas consisting of all three somatic cell lineages, and (vi) the formation of embryoid bodies consisting of cells of the three somatic lineages.

[0058] Two types of pluripotency have been described: “primed” or “quasi-stable” pluripotency similar to epiblast stem cells (EpiSCs) of late blastocysts, and “naïve” or “ground” pluripotency similar to the inner cell mass of early / pre-implantation blastocysts. Both pluripotent states exhibit the characteristics as described above, but the naïve or ground state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and viability in single-cell culture, (iii) an overall decrease in DNA methylation, (iv) a decrease in the deposition of the H3K27me3 repressive chromatin mark on developmental regulatory gene promoters, and (v) decreased expression of differentiation markers compared to primed-state pluripotent cells. The standard methodology of cell reprogramming in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or removed from the resulting pluripotent cells is generally considered to have the characteristics of the primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the primed state and the characteristics of the ground state are observed unless exogenous transgene expression is maintained.

[0059] As used herein, the term “morphology of pluripotent stem cells” refers to the classical morphological characteristics of embryonic stem cells. The morphology of normal embryonic stem cells is characterized by a high nucleus-to-cytoplasm ratio, prominent nucleoli, and a typical intercellular spacing, with a round and small shape.

[0060] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0061] “Pluripotency factor” or “reprogramming factor” refers to an agent that can increase the developmental potential of a cell, either alone or in combination with other agents. Pluripotency factors include, without limitation, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0062] "Culture" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (in both cases the singular "medium"), "supplemental component" and "medium supplemental component" refer to the nutrient composition for culturing cell cultures.

[0063] "To culture" or "to maintain" refers to, for example, maintaining, growing (proliferating), and / or differentiating tissue or cells outside the body in a sterile plastic (or coated plastic) cell culture dish or flask. "To culture" or "to maintain" can utilize a medium as a source of nutrients, hormones, and / or other factors useful for cell growth and / or maintenance.

[0064] As used herein, the term "mesoderm" refers to one of three germ layers that appear during early embryonic development and give rise to various specialized cell types including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0065] As used herein, the term "secondary hematopoietic endothelium" (HE) or "induced hematopoietic endothelium from pluripotent stem cells" (iHE) refers to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial to hematopoietic transition. Hematopoietic cell development in the embryo proceeds sequentially from the lateral plate mesoderm through angioblasts to secondary hematopoietic endothelial cells and hematopoietic progenitor cells.

[0066] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursors" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation and include pluripotent hematopoietic stem cells (blood cells), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphocyte progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid (T cells, B cells, NK cells) lineages. As used herein, the term "secondary hematopoietic stem cells" refers to CD34+ hematopoietic cells that are capable of giving rise to both mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0067] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cells that have completed maturation in the thymus and have various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and inactivation of other immune cells. T cells can be any T cell, e.g., cultured T cells, such as primary T cells, or T cell lines derived from cultured T cells, such as Jurkat, SupT1, etc., or T cells obtained from mammals. T cells can be CD3+ cells. T cells can be CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma-delta T cells (γδT cells), etc., including but not limited to any type of T cell, and can be at any stage of development. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

[0068] "CD4+ T cells" refers to a subset of T cells that express CD4 on their surface and are involved in the cellular immune response. They are characterized by their secretion profile after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein originally defined as a differentiation antigen of T lymphocytes, but is also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as an associated recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, it defines the helper / inducer subset.

[0069] "CD8+ T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is an associated recognition element in major histocompatibility complex class I-restricted interactions.

[0070] As used herein, the terms "NK cells" or "natural killer cells" refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and phenotypically are CD3- and CD56+, express at least one of NKG2C and CD57, and optionally CD16, but lack the expression of one or more of PLZF, SYK, FceRγ, and EAT-2, and refer to a subset of NK cells. In some embodiments, an isolated subpopulation of CD56+ NK cells includes the expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. CD56+ can be either dim or bright expression.

[0071] As used herein, the term "NKT cell" or "natural killer T cell" refers to CD1d-restricted T cells that express a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a canonical α-chain (Vα24-Jα18 in humans) associated with a very limited TCR repertoire - a restricted range of β-chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, exhibits a more heterogeneous use of TCRαβ. Type I NKT cells are thought to be suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one or more of the markers TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0072] As used herein, terms such as "isolated" refer to a cell or population of cells that has been separated from its original environment, i.e., the environment of an isolated cell is substantially free of at least one component found in the environment where "unseparated" reference cells are present. This term includes, for example, cells that have been isolated from a tissue or biopsy sample and removed from some or all of the components as seen in their natural environment. This term also includes cells that have been removed from at least one, some, or all components because the cells are seen in an unnatural environment, such as an environment isolated from a cell culture or cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells, or cell populations, whether seen in its natural state or growing, stored, or persisting in an unnatural environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a medium not found in nature. Isolated cells can be obtained by separating a desired cell or population of cells from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0073] As used herein, terms such as "purify" refer to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0074] As used herein, the term "encoding" refers to the property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that functions as a template for the synthesis of a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and other polymers and macromolecules in a biological process resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the nucleotide sequence that is identical to the mRNA sequence and which is usually the coding strand as set forth in the Sequence Listing, and the non-coding strand that is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0075] "Construct" refers to a macromolecular or molecular complex that includes a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of inducing delivery or introduction of foreign genetic material into a target cell and of replicating and / or expressing therein. As used herein, the term "vector" includes the construct being delivered. A vector can be a linear or circular molecule. A vector can be either integrated or not integrated. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0076] "Integration" means that one or more nucleotides of a construct are stably inserted into the cellular genome, i.e., covalently bound to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted into the chromosomal or mitochondrial DNA of the cell at a preselected site or "integration site". As used herein, the term "integration" further refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of endogenous sequences or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" may further include substitution of the deleted nucleotides with endogenous sequences or one or more inserted nucleotides.

[0077] As used herein, the term "exogenous" is intended to mean that the reference molecule or activity is introduced into the host cell or is non-native to the host cell. A molecule can be introduced, for example, by introducing the coding nucleic acid into the genetic material of the host, such as by integration into the host's chromosome or by introduction as non-chromosomal genetic material, such as a plasmid. Thus, the term used with respect to the expression of a coding nucleic acid refers to introducing the coding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a reference molecule or activity present in the host cell. Similarly, when this term is used with respect to the expression of a coding nucleic acid, it refers to the expression of a coding nucleic acid contained within the cell and not exogenously introduced.

[0078] As used herein, an "objective gene" or "objective polynucleotide sequence" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo. The objective gene or polynucleotide can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, the objective gene can encode miRNA, shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, a variant polypeptide (i.e., a variant of a natural polypeptide having less than 100% sequence identity to the natural polypeptide) or a fragment thereof, a modified polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0079] As used herein, the term "polynucleotide" refers to a nucleotide in polymeric form of any length, which can be either deoxyribonucleotide or ribonucleotide, or any analog thereof. The sequence of a polynucleotide consists of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, when the polynucleotide is RNA, uracil (U) instead of thymine. Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotide also refers to both double-stranded and single-stranded molecules.

[0080] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, which are generally also referred to in the art as peptides, oligopeptides, and oligomers, and long chains, which are generally referred to in the art as polypeptides or proteins. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0081] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it can affect the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either the sense or antisense direction.

[0082] As used herein, the term "gene imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic attributes of a source cell or iPSC and can be retained in iPSCs derived from the source cell and / or hematopoietic lineage cells derived from the iPSC. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and iPSCs derived from the source cell can further differentiate into specific cell types, including any hematopoietic lineage cells. iPSCs derived from the source cell, and cells differentiated therefrom, may collectively be referred to as "induced" cells or "derived" cells, depending on the context. For example, induced effector cells, or induced NK cells or induced T cells, as used throughout this application, are cells differentiated from iPSCs when compared to their primary counterparts obtained from natural / natural sources such as peripheral blood, cord blood, or other donor tissues. As used herein, a gene imprint conferring preferential therapeutic attributes is incorporated into iPSCs by reprogramming selected source cells that are specific to a donor, disease, or treatment response, or by introducing gene recombination modalities into iPSCs using genome editing. In aspects of source cells obtained from specific selected donors, diseases, or treatment situations, gene imprints contributing to preferential therapeutic attributes may include context-specific genetic or epigenetic, heritable phenotypes, i.e., representing preferential therapeutic attributes, that are passed on to induced cells of the selected source cell, whether or not underlying molecular events have been identified. Source cells specific to a donor, disease, or treatment response can contain gene imprints that can be retained in iPSCs and derived hematopoietic lineage cells, and these gene imprints include, but are not limited to, pre-arranged single-specificity TCRs from virus-specific T cells or invariant natural killer T (iNKT) cells, traceable desirable genetic polymorphisms, e.g., homozygosity for a point mutation encoding a high-affinity CD16 receptor of a selected donor, and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting an increased haplotype in the population.As used herein, preferred therapeutic attributes include improved engraftment, transport, homing, viability, self-renewal, persistence, regulation and modulation of the immune response, survival rate, and cytotoxicity of the induced cells. Preferred therapeutic attributes may also be related to the expression of antigen-targeting receptors, HLA presentation or its absence, resistance to the tumor microenvironment, induction and immune modulation of bystander immune cells, improved target specificity with reduced off-tumor effects, and resistance to treatments such as chemotherapy.

[0083] As used herein, the term "enhanced therapeutic properties" refers to the enhanced therapeutic properties of cells compared to typical immune cells of the same general cell type. For example, NK cells having "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties compared to typical unmodified and / or naturally occurring NK cells. Therapeutic properties of immune cells include, but are not limited to, cell engraftment, transport, homing, viability, self-renewal, persistence, regulation and modulation of the immune response, survival rate, and cytotoxicity. Therapeutic properties of immune cells are also manifested by the expression of antigen-targeting receptors, HLA presentation or its absence, resistance to the tumor microenvironment, induction and immune modulation of bystander immune cells, improved target specificity with reduced off-tumor effects, and resistance to treatments such as chemotherapy.

[0084] As used herein, the term "engager" refers to a molecule, such as a fusion polypeptide, that can form a linkage between an immune cell, such as a T cell, NK cell, NKT cell, B cell, macrophage, neutrophil, and a tumor cell and can activate the immune cell. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTE), bispecific killer cell engagers (BiKE), trispecific killer cell engagers, or multispecific killer cell engagers, or universal engagers that can be compatible with multiple immune cell types.

[0085] As used herein, the term "surface trigger receptor" refers to a receptor that can induce or initiate an immune response, such as a cytotoxic response. The surface trigger receptor can be engineered and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, neutrophils. In some embodiments, the surface trigger receptor facilitates the binding of a bispecific or multispecific antibody between an effector cell and a specific target cell, such as a tumor cell, independent of the natural receptor and cell type of the effector cell. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and differentiated into populations of various effector cell types expressing such universal surface trigger receptors. "Universal" means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind or link to an engager having the same epitope recognized by the surface trigger receptor regardless of the tumor-binding specificity of the engager. In some embodiments, an engager having the same tumor targeting specificity is used to bind to the universal surface trigger receptor. In some embodiments, an engager having different tumor targeting specificities is used to bind to the universal surface trigger receptor. Thus, one or more effector cell types may be used to kill specific types of tumor cells or two or more types of tumors. The surface trigger receptor generally includes a co-stimulatory domain for activation of the effector cell and an anti-epitope specific for the epitope of the engager. The bispecific engager is specific for the anti-epitope of the surface trigger receptor at one end and specific for the tumor antigen at the other end.

[0086] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of cell therapy. In some examples, the expression of the safety switch protein is conditionally controlled to address concerns about the safety of transplanted engineered cells that have permanently integrated into the genome the gene encoding the safety switch protein. This conditional regulation can vary and may include post-translational activation via small molecules and control by tissue-specific and / or transient transcriptional regulation. The safety switch may be capable of mediating induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation and / or antibody-mediated depletion. In some examples, the safety switch protein is activated by an exogenous molecule, such as a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, the prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product to kill the transduced cells.

[0087] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide that can achieve biological and / or pharmaceutical effects on an organism. A pharmaceutically active protein has curative or palliative properties against a disease and can be administered to improve, reduce, alleviate, reverse, or mitigate the severity of the disease. A pharmaceutically active protein also has preventive properties and is used to prevent the onset of a disease or, if such a disease has occurred, to reduce the severity of such disease or condition. Pharmaceutically active proteins include the whole protein or peptide or a pharmaceutically active fragment thereof. It also includes pharmaceutically active analogs of the protein or peptide or analogs of fragments of the protein or peptide. The term "pharmaceutically active protein" also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0088] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or increases cell signaling. Cell signaling refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along a pathway that ultimately causes biochemical events within a cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Toll-like receptor signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0089] As used herein, the term "targeting modality" refers to a molecule, e.g., a polypeptide, that is genetically incorporated into a cell and promotes the specificity of an antigen and / or epitope, including, but not limited to, antigen specificity when associated with an endogenous chimeric antigen receptor (CAR) or T cell receptor (TCR), engager specificity when associated with a monoclonal antibody or bispecific engager, targeting of transformed cells, targeting of cancer stem cells, and other targeting strategies in the absence of a specific antigen or surface molecule.

[0090] As used herein, the terms "specific" or "specificity" can be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as contrasted with non-specific or non-selective binding.

[0091] As used herein, the term "adoptive cell therapy" refers to a cell-based immunotherapy used herein that is related to the transfusion of autologous or allogeneic lymphocytes identified as T or B cells, which are expanded ex vivo prior to transfusion, and which may be genetically modified or unmodified.

[0092] As used herein, "a therapeutically sufficient amount" includes, within the meaning thereof, a non-toxic but sufficient and / or effective amount of the particular treatment and / or pharmaceutical composition to which it refers, and provides the desired therapeutic effect. The exact amount required varies from subject to subject depending on factors such as the overall health of the patient, the age of the patient, and the disease and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0093] The differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium and changes in the physical state of the cells. The most common strategy is to utilize the formation of embryoid bodies (EBs) as a common and important intermediate for initiating lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryonic development and generates multiple lineages within a three-dimensional region. Through a differentiation process that typically takes several hours to several days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and grow into cystic EBs, which typically takes several days to several weeks. At this time, they are further processed to continue differentiating. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other within a three-dimensional multi-layer cluster of cells, which is typically achieved by one of several methods, including sedimenting pluripotent cells as droplets, sedimenting the cells into a "U" bottom well plate, or by mechanical agitation. Aggregates maintained in a pluripotent culture maintenance medium do not form appropriate EBs, so in order to promote EB growth, aggregates of pluripotent stem cells require further cues for differentiation. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cues for induction into the selected lineage. EB-based culture of pluripotent stem cells typically generates a moderately proliferating differentiated cell population (ectodermal, mesodermal, and endodermal germ layers) within the EB cell cluster. EBs have been shown to promote cell differentiation, but due to the lack of consistency in the exposure of three-dimensional structured cells to differentiation cues from the environment, they generate heterogeneous cells in various differentiation states. Furthermore, EBs are cumbersome to create and maintain. Additionally, cell differentiation by EBs is accompanied by moderate cell proliferation, which also leads to a decrease in differentiation efficiency.

[0094] In contrast, "aggregate formation", unlike "EB formation", can be used to grow a population of pluripotent stem cell-derived cells. For example, during the growth of pluripotent stem cells based on aggregates, the culture medium is selected to maintain growth and pluripotency. Cell growth generally increases the size of the aggregates that form larger aggregates, and these aggregates can be routinely dissociated mechanically or enzymatically into smaller aggregates to maintain cell growth in culture and increase the number of cells. Unlike EB culture, cells cultured in aggregates in maintenance culture maintain pluripotency markers. Pluripotent stem cell aggregates require additional cues for differentiation to induce differentiation.

[0095] As used herein, "monolayer differentiation" is a term that refers to a differentiation method different from differentiation by three-dimensional multi-layer clusters of cells, i.e., "EB formation". Among other advantages disclosed herein, monolayer differentiation avoids the need for EB formation for initiation of differentiation. Since monolayer culture does not mimic embryogenesis such as EB formation, differentiation into a specific lineage is considered minimal compared to differentiation of all three germ layers of the EB.

[0096] As used herein, "dissociated" cells refer to cells that are substantially separated or purified from other cells or from a surface (e.g., the surface of a culture plate). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be dissociated from each other, for example, by enzymatic or mechanical dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from the culture plate or other surface. Thus, dissociation involves disrupting cell interactions with the extracellular matrix (ECM) and substrate (such as the culture surface), or disrupting the ECM between cells.

[0097] As used herein, "feeder cell" or "feeder" refers to a type of cell that, when co-cultured with a second type of cell, provides an environment in which the second type of cell can proliferate, expand, or differentiate, and the feeder cells provide stimulation, growth factors, nutrients, and support for the second cell type. Feeder cells may be derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the proliferation and maturation of natural killer cells. Feeder cells can typically be inactivated by treatment with an anti-mitotic agent such as irradiation or mitomycin when co-cultured with other cells to prevent them from growing more than the cells they support. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one specific type of feeder cell can be a human feeder such as human skin fibroblasts. Another type of feeder cell can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation into specific lineages, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells.

[0098] As used herein, a "feeder-free" (FF) environment refers to an environment such as a culture condition, cell culture, or culture medium that is essentially free of feeder or stromal cells and / or has not been pretreated by culturing feeder cells. A "pretreated" medium refers to a medium taken after feeder cells have been cultured in the medium for a period such as at least one day. The pretreated medium contains many mediator substances, including growth factors and cytokines secreted from the feeder cells cultured in the medium. In some embodiments, the feeder-free environment contains neither feeder cells nor stromal cells and has not been pretreated by culturing feeder cells.

[0099] "Functional," as used in the context of genomic editing or modification of iPSCs and their derived non-pluripotent cells, or genomic editing or modification of non-pluripotent cells and their derived iPSCs reprogrammed therefrom, means (1) successful knock-in, knock-out, knockdown of gene expression, transgenic or regulated gene expression, such as inducible or transient expression at a desired cell developmental stage, achieved by direct genomic editing or modification or by "passage" through differentiation or reprogramming from an initially genomically engineered starting cell, or (2) (i) modification of gene expression obtained in such cells by direct genomic editing, (ii) modification of gene expression maintained in such cells by "passage" through differentiation or reprogramming from an initially genomically engineered starting cell, (iii) downstream gene regulation in such cells as a result of modification of gene expression that appears only at the early developmental stage of such cells or only in the starting cells that give rise to such cells through differentiation or reprogramming, or (iv) removal, addition, or alteration of cell functions / characteristics at the cell level by enhanced or newly achieved cell functions or attributes presented in a mature cell product that are initially derived from genomic editing or modification performed on iPSCs, progenitor cells, or dedifferentiated cells of origin.

[0100] "HLA deficiency," including HLA class I deficiency, or HLA class II deficiency, or both, refers to a cell in which the level of surface expression of the complete MHC complex containing the HLA class I protein heterodimer and / or the HLA class II heterodimer is insufficient, or no longer maintained, or is lower, decreased, or reduced to a level lower than that naturally detectable by other cells or synthetic methods.

[0101] As used herein, "modified HLA-deficient iPSC" refers to HLA-deficient iPSCs that are further modified by introducing genes that express proteins related to, but not limited to, improved differentiation ability, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3z, 41BBL, CD47, CD113, and PDL1. Cells with "modified HLA deficiency" include cells other than iPSCs.

[0102] "Fc receptor" is abbreviated as FcR and is classified based on the type of antibody it recognizes. For example, those that bind to the most common class of antibody IgG are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). The classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signal transduction properties of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and thus different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b).

[0103] The term "chimeric Fc receptor", abbreviated as CFcR, is used to describe an engineered Fc receptor in which the native transmembrane and / or intracellular signaling domain has been modified or replaced with a non-native transmembrane and / or intracellular signaling domain. In some embodiments of the chimeric Fc receptor, in addition to one or both of the transmembrane and signaling domains being non-native, one or more stimulatory domains are introduced into the intracellular portion of the engineered Fc receptor to enhance cell activation, proliferation, and function upon receptor engagement. Unlike chimeric antigen receptors (CARs) that contain an antigen-binding domain for a target antigen, chimeric Fc receptors bind to the Fc fragment, or the Fc region of an antibody, or the Fc region contained in an engager or binding molecule, and can activate cellular functions by binding to the molecule, with or without bringing the target cell into proximity. For example, Fcγ receptors can be engineered to contain a selected transmembrane domain, stimulatory domain, and / or signaling domain in the intracellular region that generates CFcR in response to IgG binding in the extracellular domain. In one example, CFcR is generated by engineering CD16, an Fcγ receptor, by replacing its transmembrane domain and / or intracellular domain. To further improve the binding affinity of the CD16-based CFcR, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcR containing the high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or replaced such that the extracellular domain of the receptor is non-cleavable, i.e., not shed, thereby yielding an hnCD16-based CFcR.

[0104] The Fcγ receptor CD16 has been identified to have two isoforms, the Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate the NK cells and promote antibody-dependent cell cytotoxicity (ADCC). As used herein, "high-affinity CD16", "non-cleavable CD16", or "high-affinity non-cleavable CD16 (hnCD16)" refers to natural or non-natural variants of CD16. Wild-type CD16 has low affinity and is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes when the NK cells are activated. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane-proximal region (positions 189-212) is altered or deleted do not shed. The cleavage site and the membrane-proximal region are described in detail in WO2015148926, the complete disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. A CD16 variant containing both F158V and S197P has high affinity and is non-cleavable. Another exemplary high-affinity non-cleavable CD16 (hnCD16) variant is an engineered CD16 that contains an extracellular domain derived from one or more of the three exons of the CD64 extracellular domain.

[0105] I. Cells and Compositions Useful for Adoptive Cell Therapies with Enhanced Properties Provided herein is a strategy for systematically manipulating the regulatory circuitry of clonal iPSCs while enhancing the therapeutic properties of the derived cells without affecting the differentiation potential of the iPSCs and the cell developmental biology of the iPSCs and their derived cells. The derived cells are functionally improved and a combination of select modalities is suitable for adoptive cell therapy after being introduced into the cells at the iPSC level through genome engineering. Prior to the present invention, it was unclear whether modified iPSCs containing one or more provided gene edits retained the ability to enter cell development while maintaining the modified activity and / or the ability to mature into functionally differentiated cells. Unexpected failures during directed cell differentiation from iPSCs are due to aspects including, but not limited to, the absence or presence of specific gene expression at the developmental stage, requirements for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure differentiation protocols that allow for changes in cell phenotype and / or function. This application shows that one or more selected genome modifications provided herein do not adversely affect iPSC differentiation potential and that functional effector cells derived from the engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from the individual or combined genome modifications that are retained in the effector cells after iPSC differentiation.

[0106] 1. CD38 Knockout The cell surface molecule CD38 is highly upregulated in multiple hematological malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated depletion of cancer cells is typically due to a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cell cytotoxicity). In addition to ADCC, immune effector mechanisms associated with therapeutic antibodies can also include phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0107] In addition to being highly expressed in malignant cells, CD38 is also expressed in plasma cells as well as NK cells, activated T cells, and B cells. During hematopoiesis, CD38 is expressed in CD34 + stem cells, progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, and at the final stage of maturation that continues to the plasma cell stage. CD38, a type II transmembrane glycoprotein, functions in cell function as both a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 catalyzes the hydrolysis of the reaction from synthesis and NAD + to ADP-ribose, thereby generating the second messengers CADPR and NAADP, which are calcium-dependent and important for the process of cell adhesion and stimulate the release of calcium from the endoplasmic reticulum and lysosomes. CD38 recognizes CD31 as a receptor and regulates cytokine release and cytotoxicity of activated NK cells. CD38 has also been reported to associate with cell surface proteins in lipid rafts, regulate cytoplasmic Ca 2+ flux, and mediate signaling in lymphoid and myeloid cells.

[0108] In the treatment of malignant tumors, when T cells transduced with a CD38 antigen-binding receptor are used systemically, the CD38+ fractions of CD34+ hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells are lysed, and the recipient's immune effector cell function is impaired, resulting in an incomplete treatment response and reduced or eliminated efficacy. Furthermore, in multiple myeloma patients treated with daratumumab, a CD38-specific antibody, a decrease in NK cells was observed in both the bone marrow and peripheral blood, while other immune cell types such as T cells and B cells were not affected despite CD38 expression (Casneuf et al., Blood Advances. 2017;1(23):2105-2114). Without being bound by theory, the present application provides a strategy to maximize the potential of CD38-targeted cancer therapy by overcoming the depletion or reduction of effector cells through fratricide induced by CD38-specific antibodies and / or CD38 antigen-binding domains. Furthermore, since CD38 is upregulated in activated lymphocytes such as T cells and B cells, the use of a CD38-specific antibody such as daratumumab in recipients of allogeneic effector cells suppresses the activation of these lymphocytes, thereby reducing and / or preventing allogeneic rejection against these effector cells and increasing the survival rate and persistence of effector cells. Thus, the present application also provides a strategy to enhance the persistence and / or survival rate of effector cells through the reduction or prevention of allogeneic rejection by using a CD38-specific antibody, a secreted CD38-specific engager, or a CD38 CAR (chimeric antigen receptor) against the activation of recipient T cells and B cells. Specifically, the provided strategy includes the generation of CD38 knockout iPSC lines and the acquisition of CD38 null (CD38 - / - ) induced effector cells by inducing the differentiation of the engineered iPSC lines. Prior to the present application, considering that CD38 plays many important roles in cell developmental biology and cell function as described above, it was unclear whether disrupting CD38 in iPSCs would disrupt aspects including iPSC differentiation, induced cell phenotype, and effector cell function.

[0109] In one embodiment provided herein, the CD38 knockout in the iPSC line is a biallelic knockout. As disclosed herein, the provided CD38 null iPSC line differentiates as directed to generate mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, including but not limited to functional induced hematopoietic cells. In some embodiments, when using an anti-CD38 antibody to induce ADCC or an anti-CD38 CAR for targeted cell killing, CD38 - / - iPSCs and / or their induced effector cells are not eliminated by anti-CD38 antibodies or anti-CD38 CARs, thereby increasing the persistence and / or viability of iPSCs and their effector cells in the presence and / or after exposure to such therapeutic agents. In some embodiments, the effector cells have increased persistence and / or viability in vivo in the presence of such therapeutic agents and / or after exposure. In some embodiments, the CD38 null effector cells are NK cells derived from iPSCs. In some embodiments, the CD38 null effector cells are T cells derived from iPSCs. In some embodiments, the CD38 null iPSCs and induced cells include one or more additional genome edits described herein, including but not limited to hnCD16 expression, CAR expression, cytokine / cytokine receptor expression, HLA I and / or HLAII knockout, and additional modalities provided.

[0110] 2. hnCD16 knock-in CD16 has been identified as two isoforms, the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high-affinity CD16", "non-cleavable CD16", or "high-affinity non-cleavable CD16" refers to various CD16 variants. Wild-type CD16 has low affinity and, upon activation of NK cells, is subject to an ectodomain shedding proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V (also called F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is an exemplary genetically engineered non-cleavable version of CD16. Engineered CD16 variants containing both F176V and S197P have high affinity and are non-cleavable, which is described in more detail in WO2015 / 148926, the complete disclosure of which is incorporated herein by reference. Furthermore, a chimeric CD16 receptor in which the ectodomain of CD16 is essentially replaced by at least a portion of the ectodomain of CD64 can also achieve the desirable high affinity and non-cleavable function of a CD16 receptor capable of performing ADCC. In some embodiments, the substituted ectodomain of the chimeric CD16 comprises one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or an isoform or polymorphic variant thereof).

[0111] Thus, in some embodiments, the high-affinity non-cleavable CD16 receptor (hnCD16) comprises both F176V and S197P, and in some embodiments, comprises F176V and has the cleavage region removed. In some other embodiments, hnCD16 comprises a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between identity when compared to any of SEQ ID NOs: 7, 8, and 9, which are exemplary sequences each comprising at least a portion of the CD64 extracellular domain. SEQ ID NOs: 7, 8, and 9 are each encoded by way of exemplifying SEQ ID NOs: 10 - 12. As used throughout this specification and this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be performed using mathematical algorithms recognized in the art. TIFF2025084845000002.tif44161 TIFF2025084845000003.tif49160 TIFF2025084845000004.tif45159 TIFF2025084845000005.tif88157 TIFF2025084845000006.tif87158 TIFF2025084845000007.tif87157

[0112] Accordingly, provided herein are cloned iPSCs genetically engineered to contain, among other edits contemplated and described herein, a high-affinity non-cleavable CD16 receptor (hnCD16), and the genetically engineered iPSCs can differentiate into effector cells containing the hnCD16 introduced into the iPSCs. In some embodiments, the induced effector cells containing hnCD16 are NK cells. In some embodiments, the induced effector cells containing hnCD16 are T cells. The exogenous hnCD16 expressed in the iPSCs or their induced cells exhibits high affinity not only for ADCC antibodies or fragments thereof, but also for binding to bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domains of CD16 or CD64 of the hnCD16. Bispecific, trispecific, or multispecific engagers or binders are further described below in this application (see Section I.7). Thus, this application provides induced effector cells or a population thereof pre-loaded with one or more pre-selected ADCC antibodies via high-affinity binding to the extracellular domain of hnCD16 expressed on the induced effector cells, in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed in Section V below, wherein the hnCD16 comprises the extracellular binding domain of CD64 or CD16 having F176V and S197P.

[0113] In some other embodiments, the native CD16 transmembrane domain and / or intracellular domain of hnCD16 are further modified or replaced such that the chimeric Fc receptor (CFcR) is generated to include a non-native transmembrane domain, a non-native stimulatory domain, and / or a non-native signaling domain. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor other than the receptor that provides the extracellular domain. In the exemplification herein, the CFcR based on CD16 or its variant does not have a transmembrane domain, stimulatory domain, or signaling domain derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native transmembrane domain derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T cell receptor polypeptide. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native stimulatory / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one aspect of hnCD16, the chimeric receptor provided includes a transmembrane domain and a signaling domain both derived from any of IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides.One specific embodiment of the hnCD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P. Another embodiment of the hnCD16-based chimeric Fc receptor comprises the transmembrane domain and the signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P.

[0114] Various embodiments of the hnCD16-based chimeric Fc receptors as described above can bind with high affinity to the Fc region of an antibody or its fragment, or to the Fc region of a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory domain and / or signaling domain of the chimeric receptor enables effector cell activation and cytokine secretion, killing the tumor cells targeted by the antibody, or the bispecific, trispecific, or multispecific engager or binder having the tumor antigen-binding component and Fc region as described above. Without being limited by theory, through the non-native transmembrane, stimulatory and / or signaling domains of the hnCD16-based chimeric Fc receptor, or through the binding of the engager to the external domain, the CFcR contributes to the killing ability of effector cells and increases the proliferation and / or the potential for proliferation of effector cells. The antibody and the engager can bring into proximity the tumor cells expressing the antigen and the effector cells expressing the CFcR, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding effector cells expressing the hnCD16-based CFcR when attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0115] Unlike the endogenous CD16 receptor expressed by primary NK cells that is cleaved from the cell surface following NK cell activation, the various non-cleavable versions of CD16 in induced NK avoid CD16 shedding and maintain a constant expression. In induced NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, which are indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cell cytotoxicity (ADCC) and the binding of bispecific, trispecific, or multispecific engagers. ADCC is the mechanism of NK cell-mediated lysis via the binding of CD16 to antibody-coated target cells. The additional high-affinity properties of hnCD16 introduced into induced NK cells enable in vitro loading of ADCC antibodies onto NK cells via hnCD16 before administering the cells to a subject in need of cell therapy. As presented, hnCD16 can include F176V and S197P in some embodiments, or can include a full or partial extracellular domain derived from CD64 as exemplified by SEQ ID NO: 7, 8, or 9, or can further include at least one of a non-native transmembrane domain, stimulatory domain, and signaling domain. As disclosed, this application also provides induced NK cells or a population thereof pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as described in detail in Section V below. In some embodiments, NK cells comprising hnCD16 further comprise a CD38 knockout. In some embodiments, induced NK cells comprising hnCD16 and a CD38 knockout are pre-loaded with an anti-CD38 antibody. In some embodiments, the pre-loaded anti-CD38 antibody is daratumumab.

[0116] Unlike primary NK cells, mature T cells derived from a primary source (i.e., natural / natural sources such as peripheral blood, cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing exogenous non-cleavable CD16 that is expressed could differentiate into functional T cells that not only express exogenous CD16 but also execute functions through the acquired ADCC mechanism without impairing the developmental biology of T cells. This ADCC acquired in induced T cells can be further used as an approach to rescue antigen escape, which often occurs in bispecific targeting and / or CAR-T cell therapy, where tumors relapse with reduced or absent CAR-T target antigen expression or mutant antigens that avoid recognition by the CAR (chimeric antigen receptor). When the induced T cells contain ADCC acquired through exogenous CD16 expression and the antibody targets a tumor antigen different from the tumor antigen targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent the recurrence or relapse of the target tumor, which is often seen in CAR-T therapy. Such a strategy of reducing and / or preventing antigen escape while achieving bispecific targeting can be similarly applied to NK cells expressing one or more CARs. Various CARs that can be used in this antigen escape reduction and prevention strategy are described in detail below.

[0117] Thus, the present invention provides induced T cells comprising exogenous CD16. In further provided embodiments, the induced T cells obtained herein comprise CD38 knockout in addition to the expression of hnCD16. In some embodiments, the hnCD16 comprised in the induced T cells comprises F176V and S197P. In some other embodiments, the hnCD16 comprised in the induced T cells comprises a complete or partial extracellular domain derived from CD64 as exemplified by SEQ ID NO: 7, 8, or 9, or may further comprise at least one of a non-natural transmembrane domain, a stimulatory domain, and a signaling domain. As described, such induced T cells have an acquired mechanism of targeting tumors with monoclonal antibodies mediated by ADCC to enhance the therapeutic effect of the antibodies. As disclosed, the present application also provides induced T cells or a cell population thereof pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as described in detail in Section V below. In some other embodiments, the induced T cells expressing hnCD16 are also CD38 null, such that the cells can be avoided from being eliminated when in the presence of a therapeutic agent targeting the tumor antigen CD38. In one embodiment, the therapeutic agent targeting the tumor antigen CD38 is an anti-CD38 antibody. In another embodiment, the therapeutic agent targeting the tumor antigen CD38 is a CAR comprising a CD38 binding region, such as an anti-CD38 scFV.

[0118] 3. Expression of CAR What is applicable to the genetically engineered iPSCs and their induced effector cells can be any CAR design known in the art. A CAR, that is, a chimeric antigen receptor, is generally a fusion protein that includes an extracellular domain containing an antigen recognition region, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain can further include a signal peptide or leader sequence and / or a spacer. In some embodiments, the intracellular domain can further include a signaling peptide that activates the effector cells expressing the CAR. In some embodiments, the antigen recognition domain can specifically bind to an antigen. In some embodiments, the antigen recognition domain can specifically bind to an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, and the tumor can be a liquid tumor or a solid tumor. In some embodiments, the CAR is suitable for activating either T cells or NK cells expressing the above CAR. In some embodiments, the CAR is NK cell-specific and includes an NK-specific signaling component. In certain embodiments, the above T cells are derived from CAR-expressing iPSCs, and the induced T cells can include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof. In certain embodiments, the above NK cells are derived from CAR-expressing iPSCs.

[0119] In certain embodiments, the above antigen recognition region includes a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy-chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab)’2, F(ab)’3, Fv, single-chain antigen-binding fragment (scFv), (scFv) 2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody are included. Non-limiting examples of antigens that can be targeted by CARs include ADGRE2, carbonic anhydrase IX (CAlX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD269 (BCMA), CDS, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCS1, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, cancer embryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various pathogen antigens known in the art are included.Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that have the potential to cause disease.

[0120] In some embodiments, the transmembrane domain of the CAR comprises the full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor polypeptide.

[0121] In some embodiments, the signaling peptide of the internal domain (or intracellular domain) comprises the full length or at least a portion of the polypeptide of CD28, CD3ε, CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the signaling peptide of the CAR comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to at least one ITAM (immunoreceptor tyrosine-based activation motif) of CD3ζ.

[0122] In certain embodiments, the internal domain further comprises at least one co-stimulatory signaling region. The co-stimulatory signaling region can comprise the full length or at least a portion of the polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.

[0123] In one embodiment, the CAR applicable to the cells provided in the present application includes a co-stimulatory domain derived from CD28 and a signaling domain including a natural or modified ITAM1 of CD3ζ represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 13. In a further embodiment, the CAR including the co-stimulatory domain derived from CD28 and the natural or modified ITAM1 of CD3ζ also includes a hinge domain and a transmembrane domain derived from CD28, the scFv can be connected to the transmembrane domain via the hinge, and the CAR includes an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 14. TIFF2025084845000008.tif33148 TIFF2025084845000009.tif32159

[0124] In another embodiment, the CAR applicable to the cells provided in the present application includes a transmembrane domain derived from NKG2D, a co-stimulatory domain derived from 2B4, and a signaling domain including a natural or modified CD3ζ represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 15. The above CAR including the transmembrane domain derived from NKG2D, the co-stimulatory domain derived from 2B4, and the signaling domain including the natural or modified CD3ζ may further include a CD8 hinge, and the amino acid sequence of such a structure has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 16. TIFF2025084845000010.tif39145 TIFF2025084845000011.tif45146

[0125] Non-limiting CAR strategies further include conditional activation CARs dimerizing intracellular domain pairs to form heterodimers (see, e.g., U.S. Patent No. 9,587,020), split CARs that are antigen-binding, hinge, and internal domain homologous recombination to generate CARs (see, e.g., U.S. Patent Publication No. 2017 / 0183407), multi-chain CARs that enable non-covalent linkages between two transmembrane domains each connected to an antigen-binding domain and a signaling domain (see, e.g., U.S. Patent Publication No. 2014 / 0134142), CARs having bispecific antigen-binding domains (see, e.g., U.S. Patent No. 9,447,194), or pairs of antigen-binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Patent No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Invest. 2016;126(8):3036-3052); inducible CARs (see, e.g., U.S. Patent Publications Nos. 2016 / 0046700, 2016 / 0058857, 2017 / 0166877), switchable CARs (see, e.g., U.S. Patent Publication No. 2014 / 0219975), and other designs known in the art.

[0126] Accordingly, provided herein are induced cells obtained from the differentiation of genomically engineered iPSCs, wherein both the iPSCs and the induced cells comprise one or more CARs along with additional engineered modalities including, but not limited to, CD38 knockout and / or hnCD16. In certain embodiments, the iPSCs and their induced cells comprise CD38 knockout, hnCD16, and a CAR targeting a selected tumor or viral antigen, wherein the induced cells are NK or T cells, and the induced cells are used with one or more of an ADCC antibody or a bispecific, trispecific, multispecific engager that targets a tumor antigen different from that targeted by the CAR to avoid or reduce tumor antigen escape while achieving dual targeting of the same tumor via hnCD16 binding. In further embodiments, the iPSCs and their induced T cells comprising a CAR have the CAR inserted into the TCR constant region, resulting in TCR knockout and placing CAR expression under the control of the endogenous TCR promoter. In some embodiments, the induced TCR null CAR-T cells derived from the engineered iPSCs further comprise hnCD16 having an extracellular domain native to CD16 (F176V and / or S197P) or derived from CD64, and native or non-native transmembrane, stimulatory, and signaling domains. In another embodiment, the iPSCs and their induced NK cells comprising a CAR have the CAR inserted into the NKG2A locus or the NKG2D locus, resulting in NKG2A or NKG2D knockout and placing CAR expression under the control of the endogenous NKG2A or NKG2D promoter.

[0127] 4. Exogenously Introduced Cytokines and / or Cytokine Signaling By avoiding systemic high-dose administration of clinically appropriate cytokines, the risk of dose-limiting toxicity due to such actions is reduced and cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, one or more partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors are introduced into the cells to enable cytokine signaling, with or without the expression of the cytokine itself, thereby reducing the risk of cytokine toxicity and maintaining or improving cell proliferation, proliferation, proliferation, and / or effector function. In some embodiments, the introduced cytokine and / or its respective native or modified receptor for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary.

[0128] Figure 1 shows several construct designs using IL15 as an example. Any transmembrane (TM) domain of the designs in Figure 1 can be native to the IL15 receptor or modified or replaced with the transmembrane domain of another membrane-bound protein.

[0129] Design 1: IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics trans-presentation of IL15 without eliminating cis-presentation of IL15.

[0130] Design 2: IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without eliminating cis-presentation of IL15 and ensuring membrane binding of IL15.

[0131] Design 3: IL15Rα with a truncated intracellular domain is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation of IL15, maintaining membrane binding of IL15, and eliminating cis-presentation and / or other potential signaling pathways mediated by normal IL15R via its intracellular domain. The intracellular domain of IL15Rα is thought to be important for the receptor to be expressed in IL15-responsive cells and for the responsive cells to proliferate and function. Such a truncated construct comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 17, which may be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 18. In one embodiment of the truncated IL15 / IL15Rα, the construct does not contain the last 4 amino acids "KSRQ" of SEQ ID NO: 17 and comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 21. TIFF2025084845000012.tif51161 TIFF2025084845000013.tif109159 TIFF2025084845000014.tif50160

[0132] One of ordinary skill in the art will understand that the signal peptides and linker sequences described above are exemplary and do not in any way limit the variations suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those of ordinary skill in the art. One of ordinary skill in the art will understand that the signal peptide and / or linker sequence can be replaced with another sequence without altering the activity of the functional peptide directed by the signal peptide or linked by the linker.

[0133] Design 4: Since the constructs of Design 3 have been shown to function in promoting the survival and proliferation of effector cells, the cytoplasmic domain of IL15Rα has been shown to be dispensable in such designs without adversely affecting the autonomous function of effector cells armed with IL15. Design 4 is a construct that provides an alternative functional version of Design 3, in which essentially the entire IL15Rα is deleted except for the Sushi domain, and IL15 is fused to a transmembrane domain at one end (mb-Sushi), and optionally includes a linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of the membrane-bound protein. In constructs such as Design 4, unwanted signaling via IL15Rα, including cis-presentation, is eliminated when only the desired trans-presentation of IL15 is retained. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 19, which may be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 20. TIFF2025084845000015.tif39157 TIFF2025084845000016.tif83159

[0134] One of ordinary skill in the art will understand that the signal peptides and linker sequences described above are exemplary and in no way limit those variations suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to one of ordinary skill in the art. One of ordinary skill in the art will understand that the signal peptide and / or linker sequence can be replaced with another sequence without altering the activity of the functional peptide directed by the signal peptide or linked by the linker.

[0135] Design 5: Native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to enable constitutive signaling and maintain IL15 membrane binding and trans-presentation.

[0136] Design 6: Native or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling and membrane-bound trans-presentation of the cytokine. The common receptor γC, also called the common gamma chain or CD132, is also known as interleukin 2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukins, including but not limited to the IL2, IL4, IL7, IL9, IL15, and IL21 receptors.

[0137] Design 7: Engineered IL15Rβ that forms homodimers in the absence of IL15 is useful for generating constitutive signaling of the cytokine.

[0138] In some embodiments, one or more of the cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or one or more of their receptors can be introduced into iPSCs and their induced cells upon iPSC differentiation using one or more of the designs of FIG. 1. In some embodiments, cell surface expression and signaling of IL2 or IL15 are via constructs shown in any one of Designs 1-7. In some embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 are via constructs shown in Design 5, 6, or 7 by using either a common receptor or a cytokine-specific receptor. In some embodiments, cell surface expression and signaling of IL7 are via constructs shown in Design 5, 6, or 7 by using either a common receptor or a cytokine-specific receptor such as the IL4 receptor. The transmembrane (TM) domain of any of the designs of FIG. 1 can be native to the corresponding cytokine receptor or modified or replaced with the transmembrane domain of another membrane-bound protein.

[0139] In iPSCs and derived cells that include both a CAR and exogenous cytokine and / or cytokine receptor signaling, the CAR and IL are expressed in separate constructs or co-expressed in a bicistronic construct that includes both the CAR and IL. In some further embodiments, IL15 in the form represented by any of the construct designs of FIG. 1 can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence exemplified, for example, as CAR-2A-IL15 or IL15-2A-CAR. Thus, IL15 and the CAR are in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct includes the IL15 of Design 3 of FIG. 1. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct includes the IL15 of Design 3 of FIG. 1. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct includes the IL15 of Design 7 of FIG. 1. When CAR-2A-IL15 or IL15-2A-CAR is expressed, the self-cleaving 2A peptide causes the expressed CAR and IL15 to dissociate, enabling the dissociated IL15 to be presented on the cell surface. The bicistronic design of CAR-2A-IL15 or IL15-2A-CAR enables coordinated expression under the same control mechanism that can be selected for incorporation, such as an inducible promoter for expression of a single ORF, in both timing and quantity. Self-cleaving peptides are found in members of the Picornaviridae family, including Foot-and-Mouth Disease Virus (FMDV), Equine Rhinitis A Virus (ERAV), Thosea asigna Virus (TaV), and Porcine Teschovirus 1 (PTV-1) (Donnelly, ML, et al, J. Gen. Virol, 82, 1027-101 (2001)), as well as cardioviruses such as Theiler's virus (e.g., Theiler's murine encephalomyelitis virus) and Encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes referred to as "F2A", "E2A", "P2A", and "T2A", respectively.

[0140] Regarding IL15, the bicistronic CAR-2A-IL15 or IL15-2A-CAR embodiments disclosed herein contemplate the expression of any other cytokine provided herein, such as IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, cell surface expression and signaling of IL2 are via constructs shown in any of Designs 1-7. In some other embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 are via constructs shown in Designs 5, 6, or 7 that use either a common receptor and / or a cytokine-specific receptor.

[0141] 5. Deficiency of HLA-I- and HLA-II- To avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins need to match for the histocompatibility of allogeneic recipients. Provided herein are iPSC cell lines in which the expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or deletion or reduced expression levels of HLA class I-related genes including, but not limited to, the beta2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers. B2M null cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II-related genes including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through activation of the transcription factor RFX5 required for expression of class II proteins. CIITA null cells are HLA-II deficient. Provided herein are iPSC lines and their derived cells in which both B2M and CIITA are knocked out, and the resulting induced effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoiding recognition and killing by host (allogeneic) T cells.

[0142] In some cell types, lack of class I expression causes lysis by NK cells. To overcome this “missing self” response, HLA-G can be knocked in as needed to avoid recognition and killing of NK cells by HLA-I and HLA-II deficient effector cells derived from engineered iPSCs. In one embodiment, HLA-I and HLA-II deficient iPSCs and their derived cells further comprise CD38 knockout, and optionally one or more of hnCD16, CAR, and IL, without adversely affecting the differentiation ability of the iPSCs and the function of the induced effector cells including induced T cells and induced NK cells.

[0143] 6. Genetically engineered iPSC lines and induced cells provided herein In light of the above, this application is directed to CD38 - / - (also referred to herein as "CD38 null" or CD38 knockout) iPSCs, cell line cells, or a population thereof, and CD38 - / - induced functional induced cells comprising CD38 knockout obtained from the differentiation of iPSCs are provided. In some embodiments, the functional induced cells are hematopoietic cells, including but not limited to mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, the functionally induced hematopoietic cells include effector cells such as T cells, NK cells, and regulatory cells.

[0144] Also provided herein are iPSCs comprising a CD38 knockout and a polynucleotide encoding high-affinity non-cleavable CD16 (hnCD16), which can produce functional hematopoietic cells by directed differentiation. In some embodiments, when using an anti-CD38 antibody to induce hnCD16-mediated enhanced ADCC, the iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing elimination of the effector cells, i.e., a decrease or depletion of CD38-expressing effector cells, thereby increasing the persistence and / or survival rate of the iPSCs and their effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or survival rate in the presence of an anti-CD38 antibody or an anti-CD38 therapeutic agent that can be a CAR-binding CD38. In some embodiments, the effector cells include T cells. T cells derived from iPSCs comprising CD38 null and hnCD16 experience a decrease in cell depletion in the presence of an anti-CD38 antibody or anti-CD38 CAR, acquire ADCC, and result in additional mechanisms of tumor killing mediated by T cells. In some embodiments, the effector cells include NK cells. NK cells derived from iPSCs comprising CD38 null and hnCD16 have enhanced cytotoxicity and a reduced fratricide of NK cells in the presence of an anti-CD38 antibody or anti-CD38 CAR.

[0145] iPSCs comprising a CD38 knockout and a polynucleotide encoding a target-specific chimeric antigen receptor (CAR) are provided herein, and the iPSCs are capable of producing functionally induced effector cells by directed differentiation. In one embodiment, the CAR comprised in the iPSC and its derivative effector cells comprising the CD38 knockout targets the tumor cell surface protein CD38, but the CD38-CAR does not result in elimination of the iPSC and / or its derivative effector cells having the CD38 knockout. In some embodiments, the CAR comprised in the iPSC and its derivative effector cells comprising the CD38 knockout does not target CD38. In some embodiments, CAR-expressing, CD38 null induced effector cells are used with anti-CD38 antibody to induce ADCC without causing elimination of the effector cells, thereby increasing the persistence and / or viability of the iPSC and its effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or viability in combination therapies.

[0146] Furthermore, it includes CD38 knockout and a polynucleotide encoding at least one exogenous cytokine and / or its receptor (IL) that enables cytokine signaling contributing to cell survival, continuous production, and / or proliferation, and the iPSC line can be directed to differentiate to produce functionally induced hematopoietic cells with improved survival, persistence, proliferation, and effector cell function. Exogenously introduced cytokine signaling includes the signaling of any one or two or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or complete peptides of the cytokine and / or its respective receptor for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary. In some embodiments, the transient / temporary expression of cell surface cytokine / cytokine receptor is via RNA including retrovirus, Sendai virus, adenovirus, episome, minicircle, or mRNA. In some embodiments, CD38 - / - The exogenous cell surface cytokine and / or receptor contained in the iPSC or its induced cells enables IL7 signaling. In some embodiments, CD38 - / - The exogenous cell surface cytokine and / or receptor contained in the iPSC or its induced cells enables IL10 signaling. In some embodiments, CD38 - / - The exogenous cell surface cytokine and / or receptor contained in the iPSC or its induced cells enables IL15 signaling. The above CD38 - / - In some embodiments of the IL iPSC, the IL15 expression is by construct 3 in FIG. 1. The above CD38 - / -In some embodiments of the IL iPSCs, IL15 expression is by construct 4 of FIG. 1. The above CD38 of the above-described embodiments - / - IL iPSCs and their differentiated cells can autonomously maintain or improve cell proliferation, expansion, and / or effector function without contact with soluble cytokines additionally supplied in vitro or in vivo. In some embodiments, CD38 - / - IL iPSCs and their differentiated effector cells are used with anti-CD38 antibodies to induce ADCC without causing elimination of the effector cells, thereby synergistically increasing the persistence and / or survival rate of the iPSCs and their effector cells.

[0147] iPSCs comprising CD38 knockout, B2M knockout, and CIITA knockout, and optionally a polynucleotide encoding HLA-G are also provided, and the iPSCs can be directed to differentiate to produce functional induced hematopoietic cells. The above CD38 - / - B2M - / - CIITA - / - The iPSCs and their differentiated effector cells lack both HLA-I and HLA-II and are used with anti-CD38 antibodies to induce ADCC without causing elimination of the effector cells, thereby synergistically increasing the persistence and / or survival rate of the iPSCs and their effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or survival rate.

[0148] From the above perspective, provided herein are iPSCs comprising CD38 knockout and, optionally, one, two, three, or all four of hnCD16, CAR, exogenous cytokine / receptor, and B2M / CIITA knockout, wherein when B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced, and the iPSCs are capable of being directed to differentiate to produce functional induced hematopoietic cells. Also included in the present application are functional iPSC-derived hematopoietic cells comprising CD38 knockout and, optionally, one, two, three, or all four of hnCD16, B2M / CIITA knockout, CAR, and exogenous cytokine / receptor, wherein when B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced, and the induced hematopoietic cells include, but are not limited to, mesodermal cells having definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic system multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0149] Another aspect provided herein includes iPSCs or iPSC-derived cells comprising a shortened fusion protein of IL15 and IL15Rα, wherein the fusion protein does not include an intracellular domain. Although shown as "IL15Rα(ΔICD) fusion" and "IL5 / mb-Sushi" in Figure 1, these embodiments are further abbreviated as IL15Δ throughout Table 1 and the present application. In some embodiments, the shortened IL15 / IL15Rα fusion protein lacking an intracellular domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21. In some embodiments, the shortened IL15 / IL15Rα fusion protein lacking an intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the shortened IL15 / IL15Rα fusion protein lacking an intracellular domain comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the shortened IL15 / IL15Rα fusion protein lacking an intracellular domain comprises the amino acid sequence of SEQ ID NO: 21. In some further embodiments, the iPSCs or iPSC-derived cells comprising the shortened IL15 / IL15Rα fusion protein (IL15Δ) lacking an intracellular domain further comprise one or more of CD38 knockout, hnCD16, CAR, exogenous cytokine / receptor, and B2M / CIITA knockout, wherein when B2M is knocked out, a polynucleotide encoding HLA-G is introduced as needed, and the iPSCs can differentiate as directed to produce functional induced hematopoietic cells, and the induced hematopoietic cells include, but are not limited to, mesodermal cells having definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic system multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0150] Accordingly, the present application provides iPSCs and their functionally induced hematopoietic cells comprising any one of the following genotypes in Table 1. Unless designated as IL15Δ, this is a truncated fusion protein of IL15 and IL15Rα but is described in detail as having no intracellular domain. The "IL" provided in Table 1 represents one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which specific cytokine / receptor expression is selected. Further, when the iPSCs and their functionally induced hematopoietic cells have a genotype containing both CAR and IL, CAR and IL are included in a bicistronic expression cassette containing a 2A sequence. In contrast, in some other embodiments, CAR and IL are in separate expression cassettes contained in the iPSCs and their functionally induced hematopoietic cells. In certain embodiments, the IL15 in construct 3 or 4 of FIG. 1 is included in the iPSCs and their functionally induced effector cells that express both CAR and IL, and the IL15 construct is included in an expression cassette with or separate from CAR.

Table 1

[0151] 7. Further modifications In some embodiments, the iPSCs and their induced effector cells comprising any one of the genotypes in Table 1 may further comprise a deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosomal 6p21 region, or an introduced or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A RR, TCR, Fc receptor, engager, and surface trigger receptor for binding to bispecific, multispecific or universal engager.

[0152] Bispecific or multispecific engagers are fusion proteins consisting of two or more single-chain variable fragments (scFvs) of different antibodies, where at least one scFv binds to an effector cell surface molecule and at least one other binds to tumor cells via a tumor-specific surface molecule. Exemplary effector cell surface molecules or surface trigger receptors that can be used for bispecific or multispecific engager recognition or coupling include, but are not limited to, CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, and the chimeric Fc receptors disclosed herein. In some embodiments, CD16 expressed on the surface of effector cells for engager recognition is hnCD16 as described in Section I.2, which includes the extracellular domain of CD16 (including F176V and optionally S197P) or CD64, and a native or non-native transmembrane, stimulatory, and / or signaling domain. In some embodiments, CD16 expressed on the surface of effector cells for engager recognition is an hnCD16-based chimeric Fc receptor (CFcR). In some embodiments, the hnCD16-based CFcR includes the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, and the extracellular domain of hnCD16 is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 includes F176V and optionally S197P. Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, ROR1. In one embodiment, the bispecific antibody is CD3-CD19.In another embodiment, the bispecific antibody is CD16 - CD30 or CD64 - CD30. In another embodiment, the bispecific antibody is CD16 - BCMA or CD64 - BCMA. In yet another embodiment, the bispecific antibody is CD3 - CD33. In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and the tumor cell antigen binding domain, for example, modified IL15 as a linker for effector NK cells that promotes effector cell proliferation (in some publications, called TriKE, or trispecific killer engager). In one embodiment, the TriKE is CD16 - IL15 - EPCAM or CD64 - IL15 - EPCAM. In another embodiment, the TriKE is CD16 - IL15 - CD33 or CD64 - IL15 - CD33. In yet another embodiment, the TriKE is NKG2C - IL15 - CD33.

[0153] In some embodiments, the surface trigger receptor of the bispecific or multispecific conjugate can sometimes be endogenous to the effector cell, depending on the cell type. In some other embodiments, the methods and compositions provided herein are used, that is, iPSCs comprising the genotypes listed in Table 1 are further engineered to direct the differentiation of iPSCs into T cells, NK cells, or other effector cells comprising the same genotype and surface trigger receptor as the source iPSC, and one or more exogenous surface trigger receptors can be introduced into the effector cells.

[0154] 8. Antibodies for Immunotherapy In some embodiments, in addition to the genomically engineered effector cells provided herein, additional therapeutic agents comprising an antibody or antibody fragment that targets an antigen associated with a condition, disease, or indicator can be used in combination therapy with these effector cells. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, as an additional therapeutic agent for the administered iPSC-derived effector cells, antibodies suitable for combination therapy include anti-CD20 (rituximab, belimumab, ofatumumab, ublituximab, ocrelizumab, obinutuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (cetuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab); and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars, but are not limited thereto. In some embodiments, the iPSC-derived effector cells comprise hematopoietic lineage cells comprising the genotypes listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise NK cells comprising the genotypes listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise T cells comprising the genotypes listed in Table 1. In some embodiments of combinations useful for the treatment of liquid or solid tumors, the combination comprises iPSC-derived NK cells or T cells comprising at least CD38 null and an anti-CD38 antibody.In one embodiment, the combination comprises iPSC-derived NK cells comprising CD38 null and hnCD16 and one of daratumumab, isatuximab, MOR202, which are anti-CD38 antibodies. In one embodiment, the combination comprises iPSC-derived NK cells comprising CD38 null and hnCD16 and daratumumab. In some further embodiments, the iPSC-derived NK cells included in the combination with daratumumab comprise a CAR that targets at least one of CD38 null, hnCD16, IL15, and CD38 or CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, and PDL1, wherein IL15 is co-expressed or expressed separately from the CAR, and IL15 is in any one of the forms presented in constructs 1-7 of FIG. 1. In some specific embodiments, IL15 is in the form of construct 3, 4, or 7 when it is expressed with or separately from the CAR.

[0155] 9. Checkpoint inhibitor Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate the immune response when not inhibited. Tumors have been shown to select specific immune checkpoint pathways as a major mechanism of immune resistance against T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can block inhibitory checkpoints and restore immune system function by reducing the expression of checkpoint genes or gene products or by decreasing the activity of checkpoint molecules. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has changed the landscape of oncology, and these agents have brought about long-term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a major concern. One aspect of the present application provides a therapeutic approach for overcoming CI resistance by including functionally induced cells that are genomically engineered to be provided in combination therapy with a CI. In one embodiment of the combination therapy, the induced cells are NK cells. In another embodiment of the combination therapy, the induced cells are T cells. In addition to demonstrating direct antitumor capabilities, the induced NK cells provided herein are resistant to PDL1-PD1-mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and have been shown to enhance T cell activation at the tumor site. Thus, tumor infiltration of T cells promoted by functionally potent genomically engineered induced NK cells has been shown to synergize with T cell-targeted immunotherapy including checkpoint inhibitors to alleviate local immunosuppression and reduce tumor burden.

[0156] In one embodiment, the induced NK cells for the combination therapy of checkpoint inhibitors include CD38 knockout, and hnCD16 expression, B2M / CIITA knockout, CAR expression, and one, two, three, or all four of exogenous cell surface cytokine and / or receptor expression, wherein when B2M is knocked out, a polynucleotide encoding HLA-G is optionally included. In some embodiments, the induced NK cells include any one of the genotypes listed in Table 1. In some embodiments, the above-described induced NK cells further include a deletion or decreased expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptor, engager, and introduced expression or increased expression in at least one of surface trigger receptors for binding to bispecific, multispecific or universal engagers.

[0157] In another embodiment, the induced T cells for the combination therapy of checkpoint inhibitors include CD38 knockout, and hnCD16 expression, B2M / CIITA knockout, CAR expression, and one, two, three, or all four of exogenous cell surface cytokine and / or receptor expression, wherein when B2M is knocked out, a polynucleotide encoding HLA-G is optionally included. In some embodiments, the induced T cells include any one of the genotypes listed in Table 1. In some embodiments, the above-described induced T cells further include a deletion or decreased expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2A It further includes introduced expression or increased expression in at least one of R, CAR, TCR, Fc receptor, an engager, and a surface trigger receptor for binding to a bispecific, multispecific or universal engager.

[0158] The induced NK cells or induced T cells are obtained by differentiating an iPSC clone strain including CD38 knockout and, optionally, one, two, three, or all four of hnCD16 expression, B2M / CIITA knockout, CAR expression, and exogenous cell surface cytokine expression, where when B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced. In some embodiments, the iPSC clone strain further includes a deletion or decreased expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosomal 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A It further includes introduced expression or increased expression in at least one of R, CAR, TCR, Fc receptor, an engager, and a surface trigger receptor for binding to a bispecific, multispecific or universal engager.

[0159] Checkpoint inhibitors suitable for combination therapy with the induced NK cells or induced T cells provided in this specification include, but are not limited to, PD-1 (Pdcd1, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpd1), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and antagonists of inhibitory KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, 3DL2).

[0160] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which can be more cost-effective to manufacture, easier to use, or more sensitive than the whole antibody. In some embodiments, one or two or three or more checkpoint inhibitors include at least one of atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR), IPH43 (anti-MICA), IPH33 (anti-TLR3), lirilumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0161] In some embodiments, since many miRNAs are found as regulators that control the expression of immune checkpoints, antagonists that inhibit any of the above checkpoint molecules are microRNA-based (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint-antagonistic miRNAs include, but are not limited to, miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197, miR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c.

[0162] Some embodiments of the provided combination therapies with induced NK cells or induced T cells include at least one checkpoint inhibitor that targets at least one checkpoint molecule, and the induced cells have the genotypes listed in Table 1. Some other embodiments of the provided combination therapies with induced NK cells or induced T cells include two, three, or more checkpoint inhibitors such that two, three, or more checkpoint molecules are targeted. In some embodiments of the combination therapy comprising at least one checkpoint inhibitor and induced cells having the genotypes listed in Table 1, the checkpoint inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the checkpoint inhibitor is produced by the induced cells by expressing an exogenous polynucleotide sequence encoding the antibody, or a fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the antibody, or a fragment or variant thereof that inhibits the checkpoint, is co-expressed with the CAR either in a separate construct or in a bicistronic construct that includes both the CAR and the sequence encoding the antibody or a fragment thereof. In some further embodiments, the sequence encoding the antibody or a fragment thereof can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence exemplified, for example, as CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences of the checkpoint inhibitor and the CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered and expressed and secreted as a payload by induced effector cells that can infiltrate the tumor microenvironment (TME), it neutralizes inhibitory checkpoint molecules upon binding to the TME and activates effector cells by activating modalities such as the CAR or by activating receptor activation.In some embodiments, the checkpoint inhibitor co-expressed with the CAR inhibits at least one of checkpoint molecules, PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some embodiments, the checkpoint inhibitor co-expressed with the CAR in induced cells having the genotypes listed in Table 1 is selected from the group consisting of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and their humanized, or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is atezolizumab, or its humanized, or Fc-modified variant, fragment or their functional equivalents or biosimilars. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is nivolumab, or its humanized, or Fc-modified variant, fragment or their functional equivalents or biosimilars. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab, or its humanized, or Fc-modified variant, fragment or their functional equivalents or biosimilars.

[0163] In some other embodiments of the combination therapy comprising the induced cells and at least one antibody that inhibits checkpoint molecules provided herein, the antibody is not produced by or within the induced cells and is further administered before, simultaneously with, or after administration of the induced cells having the genotypes listed in Table 1. In some embodiments, the administration of one, two, three, or more checkpoint inhibitors in combination therapy with the provided induced NK cells or induced T cells is simultaneous or sequential. In one embodiment of combination therapy comprising induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and one or more of their humanized, or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of combination therapy comprising induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is atezolizumab, or its humanized or Fc-modified variant, fragment and its functional equivalent or biosimilar. In some embodiments of combination therapy comprising induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is nivolumab, or its humanized or Fc-modified variant, fragment and its functional equivalent or biosimilar. In some embodiments of combination therapy comprising induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is pembrolizumab, or its humanized or Fc-modified variant, fragment and its functional equivalent or biosimilar.

[0164] II. Methods of Targeted Genome Editing at Selected Loci of iPSCs Genome editing, also referred to interchangeably herein as genome editing or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, and / or substituted in the genome of a target cell. Targeted genome editing (which is interchangeable with "targeted genome editing" or "targeted gene editing") enables insertion, deletion, or substitution at a pre-selected site within the genome. When an endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down due to the sequence deletion. Thus, targeted editing can also be used to precisely disrupt endogenous gene expression. The term "targeted integration" as used herein similarly refers to a process that involves the insertion of one or more exogenous sequences, with or without deletion of the endogenous sequence at the insertion site. In contrast, randomly integrated genes are susceptible to the effects of position effects and silencing, and their expression is unreliable and unpredictable. For example, centromeric and subtelomeric regions are particularly prone to transgene silencing. Newly integrated genes can affect the surrounding endogenous genes and chromatin, potentially altering cell behavior or promoting cell transformation. Therefore, inserting exogenous DNA into pre-selected loci such as safe harbor loci or genomic safe harbors (GSH) is important for safe, efficient, copy number control, and reliable gene response control.

[0165] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking the exogenous polynucleotide to be inserted, via the enzymatic machinery of the host cell.

[0166] Alternatively, higher-frequency targeted editing can be achieved by the specific introduction of double-strand breaks (DSBs) by a specific rare-cut endonuclease. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non-homologous end joining (NHEJ) that occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often causes random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast, when a donor vector containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration".

[0167] Available endonucleases capable of introducing specific targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) systems. Additionally, the DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxb1 integrases is also a promising tool for targeted integration.

[0168] ZFNs are targeted nucleases containing a nuclease fused to a zinc finger DNA-binding domain. The "zinc finger DNA-binding domain" or "ZFBD" refers to a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within a zinc finger binding domain whose structure is stabilized by coordination of a zinc ion. Examples of zinc fingers include C 2 H 2 zinc fingers, C 3 H zinc fingers, and C 4Zinc fingers are included, but not limited to these. "Designed" zinc finger domains are non-naturally occurring domains, and their design / construction is mainly due to the application of rational criteria, such as substitution rules and computerized algorithms for processing information in databases storing information on existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261. See also WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496. "Selected" zinc finger domains are non-naturally occurring domains whose production results mainly from empirical processes such as phage display, interaction trap, or hybrid selection. ZFNs are described in detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFNs in the art is the fusion of the FokI nuclease and the zinc finger DNA binding domain.

[0169] TALEN is a targeted nuclease that includes a nuclease fused to a TAL effector DNA binding domain. The "transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" means the polypeptide domain of a TAL effector protein that is involved in the binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA binding domain depends on the effector variable number of imperfect 34-amino acid repeats that contain polymorphisms at selected repeat positions called repeat variable diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.

[0170] Another example of a targeted nuclease used in the methods of the present invention is a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA binding domain having specificity for a target DNA sequence, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. See, for example, U.S. Patent Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0171] Further examples of targeted nucleases suitable for use in the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used individually or in combination.

[0172] Other non-limiting examples of target nucleases include naturally occurring and recombinant nucleases, CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like.

[0173] As an illustrative example, CRISPR / Cas9 requires two main components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to a target DNA sequence containing a PAM and a PAM-proximal seeding region. Combining the crRNA and tracrRNA forms a chimeric guide RNA (gRNA), which can direct Cas9 to the selected sequence. These two components can be delivered into mammalian cells via transfection or transduction.

[0174] Insertion via DICE provides for unidirectional integration of exogenous DNA, which is tightly restricted to small attB and attP recognition sites of each enzyme itself, using pairs of recombinases such as phiC31 and Bxb1. Since these targeted att sites do not naturally occur in the mammalian genome, they need to be first introduced into the genome at the desired integration site. See, for example, U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0175] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct into a cell to enable site-specific homologous recombination by the cell host enzymatic machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to enable insertion via ZFN. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to enable insertion via TALEN. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a gRNA comprising a Cas9 expression cassette and a guide sequence specific to a desired integration site into the cell to enable insertion via Cas9. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases into a desired integration site within the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable targeted integration via DICE.

[0176] Promising sites for targeted integration are within or outside genes in the human genome and include, but are not limited to, safe harbor loci, or genomic safe harbors (GSH), that can accommodate predictable expression of newly integrated DNA without theoretically adversely affecting the host cell or organism. Useful safe harbors need to allow sufficient transgene expression to produce desirable levels of proteins or non-coding RNAs encoded by the vector. Safe harbors also should not predispose cells to malignant transformation or alter cell function. For a recombination site to be a promising safe harbor locus, it ideally needs to meet conditions including, but not limited to: no disruption of regulatory elements or genes as determined by sequence annotation; an intergenic region within a gene-dense region or a convergent position between two genes transcribed in opposite directions; maintain distance to minimize the potential for long-range interactions between the promoter of a gene adjacent to a transcription activator encoded by the vector, particularly cancer-related genes and microRNA genes; have clearly ubiquitous transcriptional activity as reflected by a broad spatial and temporal expression sequence tag (EST) expression pattern indicative of ubiquitous transcriptional activity. This latter feature is particularly important in stem cells where chromatin remodeling during differentiation typically results in silencing of some loci and potential activation of other loci. Within regions suitable for exogenous insertion, the exact locus selected for insertion should lack repetitive elements and conserved sequences and primers for amplification of homology arms should be easily designed.

[0177] Sites suitable for human genome editing, or specifically targeted integration, include, but are not limited to, the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the human ortholog of the mouse ROSA26 locus. Additionally, the human ortholog of the mouse H11 locus can also be a suitable site for insertion using the compositions and methods of targeted integration disclosed herein. Further, the collagen and HTRP loci can also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be particularly necessary in stem cells for a particular integration event, and optimization of insertion strategies such as promoter selection, exogenous gene sequence and placement, and construct design is often required.

[0178] For targeted indels, the editing site is often included in an endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted indel is related to the regulation and modification of the immune response. In some other embodiments, the endogenous gene containing the targeted indel is related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins that suppress the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells, and cells derived therefrom.

[0179] Accordingly, one aspect of the present invention provides a method of targeted integration at a selected locus comprising a genomic safe harbor, or at a pre-selected locus known or proven to be safely and adequately regulated for constitutive or transient gene expression, such as the B2M, TAP1, TAP2, or tapasin loci as provided herein. In one embodiment, the genomic safe harbor for the method of targeted integration comprises one or more desirable integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of a genomic safe harbor. In one embodiment, the method of targeted integration into a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell a construct comprising a pair of homologous arms specific for the desired integration site and one or more exogenous sequences to enable site-specific homologous recombination by the cell host enzymatic machinery, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of a genomic safe harbor.

[0180] In other embodiments, the method of targeted integration into a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors. In yet another embodiment, the method of targeted integration into a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site to enable insertion via TALEN, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors. In another embodiment, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a Cas9 expression cassette and a gRNA comprising a guide sequence specific for a desired integration site to enable insertion via Cas9, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors.In yet another embodiment, a method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases into a desired integration site within the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for the DICE recombinase to enable DICE-mediated targeted integration, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors.

[0181] Furthermore, as provided herein, the above-described method for targeted integration in a safe harbor is used to insert a polynucleotide of interest, such as a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, and a polynucleotide encoding a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some other embodiments, a construct comprising one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the present invention is a suicide gene encoding a safety switch protein. Suicide gene systems suitable for inducing cell death include, but are not limited to, caspase 9 (or caspase 3 or 7) and AP1903, thymidine kinase (TK) and ganciclovir (GCV); cytosine deaminase (CD) and 5-fluorocytosine (5-FC). Furthermore, some suicide gene systems are cell-type specific. For example, genetic modification of T lymphocytes with the B cell molecule CD20 can eliminate them upon administration of the mAb rituximab. Furthermore, a modified EGFR comprising an epitope recognized by cetuximab can be used to deplete genetically engineered cells when the cells are exposed to cetuximab. Accordingly, one aspect of the present invention provides a method for targeted integration of one or more suicide genes encoding a safety switch protein selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0182] In some embodiments, one or more exogenous polynucleotides incorporated by the methods herein are driven by an exogenous promoter operably linked in a construct for targeted integration. The promoter can be inducible or constitutive and can be temporally specific, tissue specific or cell type specific. Constitutive promoters suitable for the methods of the invention include, but are not limited to, the cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0183] An exogenous polynucleotide incorporated by the method of this specification can be driven by an endogenous promoter in the host genome at the integration site. In one embodiment, the method of the invention is used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the method of the invention is used for targeted integration at the ROSA26 locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the method of the invention is used for targeted integration at the H11 locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the method of the invention is used for targeted integration at the collagen locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method of the invention is used for targeted integration at the HTRP locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous HTRP promoter. In theory, only correct insertion at the desired location will allow gene expression of an exogenous gene driven by an endogenous promoter.

[0184] In some embodiments, one or more exogenous polynucleotides included in a construct for a method of targeted integration are driven by one promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to enhance physical separation between the moieties and maximize access to enzymatic machinery. The linker peptide of the linker sequence can consist of amino acids selected to make the physical separation between the moieties (exogenous polynucleotides, and / or the proteins or peptides encoded therefrom) more flexible or more rigid, depending on the relevant function. The linker sequence can be cleavable by a protease or chemically cleavable, resulting in separate moieties. Examples of enzymatic cleavage sites in linkers include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is naturally produced by the host or is introduced exogenously. Alternatively, the cleavage site in the linker can be a site cleavable by a selected chemical, such as cyanogen bromide, hydroxylamine, or upon exposure to low pH. Any linker sequence can serve purposes other than providing a cleavage site. The linker sequence should enable effective placement of the moiety with respect to another adjacent moiety for the moieties to function properly. The linker can also be a simple amino acid sequence of sufficient length to prevent any steric hindrance between the moieties. Further, the linker sequence can provide post-translational modifications including, but not limited to, phosphorylation sites, biotinylation sites, sulfation sites, γ-carboxylation sites, etc. In some embodiments, the linker sequence is flexible such that it does not hold a biologically active peptide in a single unfavorable conformation. The linker can mainly consist of amino acids with small side chains such as glycine, alanine, and serine to provide flexibility. In some embodiments, about 80 or 90 percent or more of the linker sequence comprises glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, the G4S linker peptide separates the fusion protein's terminal processing and endonuclease domain. In other embodiments, the 2A linker sequence enables two separate proteins to be produced from a single translation. Suitable linker sequences can be readily identified empirically. Additionally, the appropriate size and sequence of the linker sequence can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry site (IRES) within the sequence. In some embodiments, any two consecutive linker sequences are different.

[0185] A method of introducing a construct containing an exogenous polynucleotide for targeted integration into a cell can be achieved using methods of gene transfer into cells known per se. In one embodiment, the construct comprises a viral vector backbone such as an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a Sendai viral vector, etc. In some embodiments, plasmid vectors are used to deliver and / or express exogenous polynucleotides into target cells (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo). In some other embodiments, episomal vectors are used to deliver exogenous polynucleotides into target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used in genetic manipulation to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV does not integrate into the host genome. Furthermore, episomal rAAV vectors mediate homologous-directed gene targeting at a much higher rate compared to transfection of conventional targeting plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the genome of iPSCs. In some embodiments, the genome-edited iPSCs and their derived cells obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0186] III. Methods for obtaining and maintaining genome-engineered iPSCs The present invention provides a method for obtaining and maintaining genomically engineered iPSCs that include one or more targeted edits at one or more desired sites, where the targeted edits continue to be intact and functional at each selected edit site in the expanded genomically engineered iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions into the genomes of the iPSCs and the cells derived therefrom, i.e., introduce targeted integration and / or indels at the selected sites. Among the many advantages of obtaining genomically engineered induced cells by editing and differentiating iPSCs as provided herein, as compared to directly manipulating primary effector cells derived from a patient's peripheral blood, include, but are not limited to: an unlimited source of engineered effector cells; no need to repeatedly manipulate the effector cells, especially when multiple engineered modalities are included; the resulting effector cells have elongated telomeres and are rejuvenated due to low exhaustion; the effector cell population is homogeneous in terms of no editing site, copy number, and allelic mutations, random mutations, and expression diversity, mainly due to the possibility of clone selection in the engineered iPSCs provided herein.

[0187] In certain embodiments, genomically engineered iPSCs that include one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells for extended periods in the cell culture medium shown in Table 2 as the Fate Maintenance Medium (FMM), where the iPSCs retain the targeted edits and functional modifications at the selected sites. The components of the medium may be present in the medium in amounts within the optimal ranges shown in Table 2. iPSCs cultured in FMM have been shown to remain undifferentiated, maintain genomic stability without the need for culturing washes or selections, with a basal or naive profile, and readily give rise to in vitro differentiation through all three somatic lineages, embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation through teratoma formation. See, for example, U.S. Patent Application No. 61 / 947,979, the disclosure of which is incorporated herein by reference.

Table 2

[0188] In some embodiments, genome-engineered iPSCs comprising one or more targeted integrations and / or indels are maintained, passaged, and expanded in a medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor and do not, or essentially do not, contain a TGFβ receptor / ALK5 inhibitor, wherein the iPSCs retain an intact and functional targeted edit at a selected site.

[0189] Another aspect of the invention provides a method of generating genome-engineered iPSCs through targeted editing of iPSCs or by first generating genome-engineered non-pluripotent cells by targeted editing and then reprogramming the selected / isolated genome-engineered non-pluripotent cells to obtain iPSCs comprising the same targeted edit as the non-pluripotent cells. A further aspect of the invention provides genome-engineered non-pluripotent cells that are undergoing reprogramming simultaneously by introduction of a targeted integration and / or a targeted indel into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming and the conditions for reprogramming include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genome engineering and reprogramming, the targeted integration and / or the targeted indel can be introduced into the non-targeted pluripotent cells before or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally small molecules.

[0190] In some embodiments, for the simultaneous genome engineering and reprogramming of non-pluripotent cells, targeted integration and / or indels can also be introduced into non-pluripotent cells after the multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, where the vector carrying the construct is introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes including, but not limited to, SSEA4, Tra1-81, and CD30.

[0191] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor and, optionally, a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 2). In some embodiments, genome-engineered iPSCs by any of the methods described above are further maintained and propagated using a mixture (FMM; Table 2) comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor.

[0192] In some embodiments of methods for generating genomically engineered iPSCs, the method comprises introducing one or more targeted integrations and / or indels into the iPSCs to genomically engineer the iPSCs and obtaining genomically engineered iPSCs having at least one genotype listed in Table 1. Alternatively, a method for generating genomically engineered iPSCs comprises (a) introducing one or more targeted edits into non-pluripotent cells to obtain genomically engineered non-pluripotent cells comprising a targeted integration and / or indel at a selected site, and (b) contacting the genomically engineered non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and, optionally, a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to obtain genomically engineered iPSCs comprising a targeted integration and / or indel at a selected site. Alternatively, a method for generating genomically engineered iPSCs comprises (a) contacting non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and, optionally, a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells, (b) introducing one or more targeted integrations and / or indels into the reprogramming non-pluripotent cells for genomic engineering, and (c) obtaining clonal genomically engineered iPSCs comprising a targeted integration and / or indel at a selected site.

[0193] The reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in PCT / US2015 / 018801 and PCT / US16 / 57136, the disclosures of which are incorporated herein by reference. One or more reprogramming factors can be in the form of a polypeptide. The reprogramming factors can also be in the form of a polynucleotide and thus are introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai viral vector. In some embodiments, one or more polynucleotides are introduced by a combination of plasmids. See, for example, U.S. Patent Application No. 62 / 571,105, the disclosure of which is incorporated herein by reference.

[0194] In some embodiments, non-pluripotent cells are introduced with multiple constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides can include suicide genes, or genes encoding targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival rate of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNAs including, but not limited to, siRNA, shRNA, miRNA, and antisense nucleic acids. These exogenous polynucleotides can be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, temporally specific promoters, and tissue-specific or cell-type specific promoters. Thus, the polynucleotide can be expressed under conditions that activate the promoter, for example, in the presence of an inducer, or in a specific differentiated cell type. In some embodiments, the polynucleotide is expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, one or more suicide genes are driven by a constitutive promoter, for example, caspase-9 driven by CAG. These constructs containing different exogenous polynucleotides and / or different promoters can be introduced into non-pluripotent cells simultaneously or sequentially. The non-pluripotent cells subjected to targeted integration of multiple constructs can be simultaneously contacted with one or more reprogramming factors to initiate reprogramming simultaneously with genome manipulation, thereby obtaining genome-manipulated iPSCs containing multiple targeted integrations in the same pool of cells. Thus, by this robust method, simultaneous reprogramming and engineering strategies can be used to derive clonal genome-engineered hiPSCs with multiple modalities integrated at one or more selected target sites.In some embodiments, the genome-edited iPSCs and their derivative cells obtained using the methods and compositions herein include at least one genotype listed in Table 1.

[0195] IV. Method for obtaining genetically engineered effector cells by differentiating genome-engineered iPSCs A further aspect of the invention provides a method for in vivo differentiation of genomically engineered iPSCs by teratoma formation, wherein the differentiated cells induced in vivo from the genomically engineered iPSCs retain intact and functional targeted editing including targeted integration and / or indels at desired sites. In some embodiments, the differentiated cells induced in vivo from genomically engineered iPSCs via teratomas contain one or more inducible suicide genes integrated at one or more desired sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors. In some other embodiments, the differentiated cells induced in vivo from genomically engineered iPSCs via teratomas contain a polynucleotide encoding a targeting modality, or a polynucleotide encoding a protein that promotes the transport, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells. In some embodiments, the differentiated cells induced in vivo from genomically engineered iPSCs via teratomas containing one or more inducible suicide genes further contain one or more indels of endogenous genes related to the regulation and mediation of the immune response. In some embodiments, the indels are contained in one or more endogenous checkpoint genes. In some embodiments, the indels are contained in one or more endogenous T cell receptor genes. In some embodiments, the indels are contained in one or more endogenous MHC class I suppressor genes. In some embodiments, the indels are contained in one or more endogenous genes related to the major histocompatibility complex. In some embodiments, the indels are contained in one or more endogenous genes including, but not limited to, B2M, PD1, TAP1, TAP2, tapasin, TCR genes. In one embodiment, the genomically engineered iPSCs containing one or more exogenous polynucleotides at a selected site further contain targeted editing in the gene encoding B2M (beta-2 microglobulin).

[0196] In certain embodiments, the genome-engineered iPSCs comprising one or more of the genetic modifications provided herein are used to induce hematopoietic cell lineages or other specific cell types in vitro, and the induced non-pluripotent cells retain functional genetic modifications including targeted editing at selected sites. In one embodiment, the genome-engineered iPSC-derived cells include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor (MPP) cells, T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, wherein these cells derived from the genome-engineered iPSCs retain functional genetic modifications including targeted editing at desired sites.

[0197] Applicable differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lineages include, for example, those described in International Application No. PCT / US2016 / 044122, the disclosure of which is incorporated herein by reference. As provided, the methods and compositions for generating hematopoietic cell lineages involve definitive hematopoietic endothelial (HE) derived from pluripotent stem cells, including hiPSCs, in a culture platform that is serum-free, feeder-free, and / or stroma-free, scalable, and does not require monolayer EB formation. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated and transdifferentiated cells, and various lineages of cells that have directly transitioned to a hematopoietic fate without passing through pluripotent intermediates. Similarly, cells generated by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0198] Methods for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture involve contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from the pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then subjected to contact with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain proliferating mesodermal cells with definitive hematopoietic endothelial (HE) potential without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF and, optionally, a ROCK inhibitor and / or a WNT pathway activator causes the mesodermal cells with definitive HE potential to differentiate into definitive HE cells that are similarly expanded during differentiation.

[0199] The methods provided herein for obtaining hematopoietic lineage cells are superior to EB-mediated pluripotent stem cell differentiation, as EB formation results in minimal cell proliferation from moderate proliferation and does not allow for monolayer culture, which is important for many applications requiring uniform proliferation, and uniform differentiation of cells within the population, and is cumbersome and inefficient.

[0200] The provided monolayer differentiation platform promotes differentiation into definitive hematopoietic endothelium, which leads to the derivation of hematopoietic stem cells and differentiated progeny such as T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion to enable the delivery of a therapeutically relevant number of pluripotent stem cell-derived hematopoietic cells for various therapeutic applications. Furthermore, monolayer culture using the methods provided herein yields functional hematopoietic lineage cells that enable the full spectrum of in vitro differentiation, ex vivo modification, and in vivo long-term hematopoietic self-renewal, reconstitution, and engraftment. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, definitive hematopoietic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0201] A method for directing the definitive hematopoietic lineage differentiation of pluripotent stem cells, comprising: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate the differentiation and proliferation of mesodermal cells from the pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, to initiate the differentiation and proliferation of mesodermal cells with definitive HE potential from the mesodermal cells; (iii) contacting the mesodermal cells with a definitive HE potential with a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator, and optionally not comprising a TGFβ receptor / ALK inhibitor, to initiate the differentiation and proliferation of definitive hematopoietic endothelium from the pluripotent stem cell-derived mesodermal cells with definitive hematopoietic endothelial potential.

[0202] In some embodiments, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not comprising a TGFβ receptor / ALK inhibitor, to seed and proliferate the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs comprising one or more genetic imprints, and the one or more genetic imprints comprised in the iPSCs are retained in the hematopoietic cells differentiated therefrom. In some embodiments of the method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of the hematopoietic lineage is in a monolayer culture format without embryoid body formation.

[0203] In some embodiments of the above-described method, the resulting definitive hematopoietic endothelial cells derived from pluripotent stem cells are CD34+. In some embodiments, the resulting definitive hematopoietic endothelial cells are CD34+CD43-. In some embodiments, the definitive hematopoietic endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the definitive hematopoietic endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the definitive hematopoietic endothelial cells are CD34+CD43-CD93-. In some embodiments, the definitive hematopoietic endothelial cells are CD34+CD93-.

[0204] In some embodiments of the above-described method, the method further comprises: (i) contacting the definitive hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, to initiate differentiation of the definitive hematopoietic endothelium into pre-T cell progenitor cells; and optionally (ii) contacting the pre-T cell progenitor cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells. In some embodiments of this method, the T cell progenitor cells derived from pluripotent stem cells are CD34+CD45+CD7+. In some embodiments of this method, the T cell progenitor cells derived from pluripotent stem cells are CD45+CD7+.

[0205] In some further embodiments of the above-described method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the method comprises: (i) contacting definitive hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator, to initiate the differentiation of the definitive hematopoietic endothelium into pre-NK cell progenitor cells; and optionally (ii) contacting pre-NK cell progenitor cells derived from pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 and not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate the differentiation of the pre-NK cell progenitor cells into NK cell progenitor cells or NK cells. In some embodiments, the NK progenitor cells derived from pluripotent stem cells are CD3−CD45+CD56+CD7+. In some embodiments, the NK cells derived from pluripotent stem cells are CD3−CD45+CD56+, and are further defined, optionally, by NKp46+, CD57+, and CD16+.

[0206] Thus, using the above-described differentiation methods, one or more populations of the following iPSC-derived hematopoietic cells can be obtained: (i) CD34+HE cells (iCD34) using one or more media selected from iMPC-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) definitive hematopoietic endothelium (iHE) using one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iii) definitive HSCs using one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iv) multipotent progenitor cells (iMPP) using iMPP-A; (v) T cell progenitor cells (ipro-T) using one or more media selected from iTC-A2 and iTC-B2; (vi) T cells (iTC) using iTC-B2; (vii) NK cell progenitor cells (ipro-NK) using one or more media selected from iNK-A2 and iNK-B2; and / or (viii) NK cells (iNK) and iNK-B2. In some embodiments, the media are as follows: a. iCD34-C contains one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator, and does not contain a TGFβ receptor / ALK inhibitor. b. iMPP-A contains a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. c. iTC-A2 contains one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, SCF, Flt3L, TPO, and IL7, and optionally a BMP activator. d. iTC-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. e.iNK-A2 contains a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator. f.iNK-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0207] In some embodiments, the genome-engineered iPSC-derived cells obtained from the methods described above contain one or more inducible suicide genes integrated at one or more desired integration sites including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors. In some other embodiments, the genome-engineered iPSC-derived cells contain polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the trafficking, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells. In some embodiments, the genome-engineered iPSC-derived cells containing one or more suicide genes further contain one or more indels in one or more endogenous genes related to immune response regulation and mediation, including but not limited to checkpoint genes, endogenous T cell receptor genes, and MHC class I inhibitory genes. In one embodiment, the genome-engineered iPSC-derived cells containing one or more suicide genes contain an indel in the B2M gene and B2M is knocked out.

[0208] Furthermore, applicable dedifferentiation methods and compositions for obtaining genomically engineered hematopoietic cells of a second fate from genomically engineered hematopoietic cells of a first fate include, for example, those shown in International Publication No. WO2011 / 159726, the disclosure of which is hereby incorporated by reference. The methods and compositions provided therein enable the partial reprogramming of starting non-pluripotent cells into non-pluripotent intermediate cells by restricting the expression of the endogenous Nanog gene during reprogramming, and subjecting the non-pluripotent intermediate cells to conditions for differentiating the intermediate cells into the desired cell type. In some embodiments, the genomically modified iPSCs and their derivative cells obtained using the methods and compositions herein include at least one genotype listed in Table 1.

[0209] V. Therapeutic use of induced immune cells with functional modalities differentiated from genetically engineered iPSCs In some embodiments, the present invention provides a composition comprising an isolated population or subpopulation of functionally enhanced derivative immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSCs comprise one or more targeted gene edits that can be retained in iPSC-derived immune cells, and the genetically engineered iPSCs and their derivative cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived CD34 cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro-T cells or T cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro-NK cells or NK cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived immunomodulatory cells or myeloid-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived genetically engineered immune cells are further modified ex vivo for improved therapeutic potential. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells derived from iPSCs comprises an increase in the number or ratio of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells derived from iPSCs comprises an increase in the number or ratio of type I NKT cells. In another embodiment, the isolated population or subpopulation of genetically engineered immune cells derived from iPSCs comprises an increase in the number or ratio of adaptive NK cells. In some embodiments, the isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, or myeloid-derived suppressor cells derived from iPSCs are allogeneic. In some other embodiments, the isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, or MDSCs derived from iPSCs are autologous.

[0210] In some embodiments, the iPSCs for differentiation contain genetic imprints selected to confer desirable therapeutic attributes in effector cells, on the condition that the developmental biology of the cells during differentiation is not disrupted, the genetic imprints are retained and functional in the differentiated hematopoietic cells derived from the iPSCs.

[0211] In some embodiments, the genetic imprint of the pluripotent stem cells comprises one or more gene modification modalities obtained by genomic insertion, deletion, or substitution in the genome of the pluripotent cells during or after reprogramming non-pluripotent cells to iPSCs, or (ii) one or more retainable therapeutic attributes of source-specific immune cells that are donor-specific, disease-specific, or treatment response-specific, the pluripotent cells are reprogrammed from source-specific immune cells, and the iPSCs retain the therapeutic attributes of the therapeutic attribute source that are also included in the hematopoietic cells derived from the iPSCs.

[0212] In some embodiments, the genetic modification modality includes one or more of a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, regulation of immune response and modulation, and / or survival rate of iPSCs or their derived cells. In some embodiments, the genetically modified iPSCs and their derived cells include the genotypes listed in Table 1. In some other embodiments, the genetically modified iPSCs and their derived cells that include the genotypes listed in Table 1 further include additional genetic modification modalities that include (1) deletion or reduced expression of one or more of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any gene in the chromosomal 6p21 region, and (2) introduction or increased expression of a surface trigger receptor for coupling with HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptor, or a bispecific or multispecific or universal engager.

[0213] In some further other embodiments, the hematopoietic lineage cells include the therapeutic attributes of source-specific immune cells for at least two of the following combinations: (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) resistance to the tumor microenvironment, (iv) recruitment and immune regulation of bystander immune cells, (iv) improved target specificity with reduced off-tumor effects, (v) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0214] In some embodiments, iPSC-derived hematopoietic cells comprising the genotypes listed in Table 1, and said cells express at least one cytokine and / or its receptor, or any modified protein thereof, comprising IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, and express at least a CAR. In some embodiments, the engineered expression of the cytokine and the CAR is NK cell-specific. In some other embodiments, the engineered expression of the cytokine and the CAR is T cell-specific. In one embodiment, the CAR comprises a CD38 binding domain. In some embodiments, the iPSC-derived hematopoietic effector cells are antigen-specific. In some embodiments, the antigen-specific induced effector cells target liquid tumors. In some embodiments, the antigen-specific induced effector cells target solid tumors. In some embodiments, the antigen-specific iPSC-derived hematopoietic effector cells can rescue tumor antigen escape.

[0215] By introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy, various diseases can be ameliorated. In some embodiments, the provided iPSC-derived hematopoietic cells are for allogeneic adoptive cell therapy. Further, the present invention provides, in some embodiments, the therapeutic use of the above-described therapeutic composition by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, or an infectious disease associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin disease, multiple myeloma, and myelodysplastic syndromes). Examples of solid cancers include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune diseases include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infectious diseases include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpesvirus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella-zoster virus), adenovirus-, lentivirus-, BK polyomavirus-related diseases.

[0216] Treatment using the induced hematopoietic system cells of the embodiments disclosed herein can be performed based on symptoms or for relapse prevention. Terms such as "treating", "treatment", etc. are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic with respect to completely or partially preventing a disease, and / or can be therapeutic with respect to partial or complete cure of the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any intervention in a subject's disease and includes: preventing a disease from occurring in a subject who is susceptible to the disease but has not yet been diagnosed as having it, inhibiting the disease, i.e., preventing its onset, or alleviating the disease, i.e., causing the disease to regress. The therapeutic agent or composition can be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease where the treatment stabilizes or reduces the patient's undesirable clinical symptoms is also particularly important. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury that can have at least one related symptom suppressed, improved, and / or ameliorated by cell therapy. Certain embodiments contemplate, but are not limited to, subjects who are candidates for bone marrow or stem cell transplantation, subjects who have received chemotherapy or radiation therapy, subjects who have or are at risk of having a hyperproliferative disorder or cancer, such as a hematopoietic hyperproliferative disorder or cancer, subjects who have or are at risk of developing a tumor, such as a solid tumor, subjects who have or are at risk of having a viral infection or a disease associated with a viral infection.

[0217] When evaluating the responsiveness to a treatment comprising the induced hematopoietic system cells of the embodiments disclosed herein, the response can be measured by clinical benefit rate, survival rate until death, pathological complete response, quantitative measurement of the version of the pathological response, clinical complete remission, clinical partial remission, clinically stable disease, relapse-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Response Evaluation Criteria In Solid Tumors) criteria.

[0218] A therapeutic composition comprising induced hematopoietic lineage cells as disclosed can be administered to a subject before, during, and / or after other treatments. Thus, methods of combination therapy can include administration or preparation of iPSC-derived immune cells before, during, and / or after use of an additional therapeutic agent. As described above, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small molecule RNAs, dsRNA (double-stranded RNA), monocytes, feeder cells, feeder cell components or their replenishing factors, vectors containing one or more polynucleotides of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). Administration of iPSC-derived immune cells can be temporally separated from administration of an additional therapeutic agent by hours, days, or weeks. Additionally or alternatively, administration can be combined with other bioactive agents or modalities such as, but not limited to, anti-tumor agents, non-pharmacological therapies such as surgery, and the like.

[0219] In some embodiments of the combinatorial cell therapy, the therapeutic combination includes the iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent that is an antibody or a fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody may be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability. In some embodiments, as an additional therapeutic agent for the administered iPSC-derived hematopoietic lineage cells, antibodies suitable for combinatorial therapy include anti-CD20 (e.g., rituximab, belimumab, ofatumumab, ublituximab, ocrelizumab, obinutuzumab), anti-HER2 (e.g., trastuzumab, pertuzumab), anti-CD52 (e.g., alemtuzumab), anti-EGFR (e.g., cetuximab), anti-GD2 (e.g., dinutuximab), anti-PDL1 (e.g., avelumab), anti-CD38 (e.g., daratumumab, isatuximab, MOR202), anti-CD123 (e.g., 7G3, CSL362), anti-SLAMF7 (elotuzumab), and their humanized or Fc-modified variants or fragments, or their functional equivalents or biosimilars, but are not limited thereto.

[0220] In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. Checkpoints refer to cellular molecules, often cell surface molecules, that can suppress or downregulate the immune response when not inhibited. A checkpoint inhibitor is an antagonist that can reduce the gene expression or gene product of a checkpoint or decrease the activity of a checkpoint molecule. Checkpoint inhibitors suitable for combination therapy with the induced effector cells, including NK cells or T cells, provided herein include, but are not limited to, PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and antagonists of inhibitory KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, 3DL2).

[0221] Some embodiments of the provided combination therapies that include induced effector cells further include at least one inhibitor that targets a checkpoint molecule. Some other embodiments of the combination therapies with the provided induced effector cells include two, three, or more inhibitors such that two, three, or more checkpoint molecules are targeted. In some embodiments, the effector cells for the combination therapies described herein are induced NK cells as provided. In some embodiments, the effector cells for the combination therapies described herein are induced T cells. In some embodiments, the induced NK cells or T cells for the combination therapies are functionally enhanced as provided herein. In some embodiments, the two, three, or more checkpoint inhibitors can be administered in the combination therapy simultaneously with, prior to, or following the administration of the induced effector cells. In some embodiments, the two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

[0222] In some embodiments, the antagonist that inhibits any of the above-described checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which can be more cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one or two or three or more checkpoint inhibitors include at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0223] Combination therapy containing inducible effector cells and one or more inhibitors of induced effector cells has been shown to be effective in treating cutaneous T-cell lymphoma, non-Hodgkin's lymphoma (NHL), mycosis fungoides, Paget's reticulosis, Sézary syndrome, granulomatous lax skin, lymphomatoid papulosis, chronic pityriasis lichenoides, acute pityriasis lichenoides, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennart's lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK cell lymphoma, and leukemia. The present invention can be applied to the treatment of liquid and solid cancers, including but not limited to alveolar lymphoma, B-cell lymphoma, Hodgkin's lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyosarcoma, Lewis lung carcinoma (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, hepatoma, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal carcinoma.

[0224] In some embodiments, other than the induced effector cells as provided herein, the combination for therapeutic use includes one or more additional therapeutic agents, including chemotherapeutic agents or radioactive moieties.Chemotherapeutic agents refer to cytotoxic antitumor agents, i.e., chemical agents that are found to preferentially kill tumor cells, or disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the proliferation of neoplastic cells.Chemotherapeutic agents may also be called antitumor or cytotoxic drugs or agents, and are well known in the art.

[0225] In some embodiments, the chemotherapeutic agent includes anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, mechlorethamine, melphalan, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisantrene, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine.Additional agents include aminoglutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzumab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, celecoxib, cetuximab, cladribine, clofarabine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, drostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegademase, pegaspargase, pentostatin, pipobroman, plicamycin, polyprrosan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including chemotherapeutic or radiation therapy agents known in the art.Such agents can be found in many standard medical and oncologist references (e.g., Goodman & Gilman’s The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, N.Y., 1995) or on the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated as needed.

[0226] Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide stimulate both NK cells and T cells. As provided herein, IMiDs can be used in combination with iPSC-derived therapeutic immune cells for cancer treatment.

[0227] In addition to the isolated population of iPSC-derived hematopoietic lineage cells included in the therapeutic composition, a composition suitable for administration to a patient can further comprise one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., a pharmaceutically acceptable medium such as a cell culture medium), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the particular composition being administered and by the particular method used to administer the therapeutic composition. Accordingly, there are a variety of suitable formulations of the therapeutic compositions of the present invention (see, e.g., Remington’s Pharmaceutical Sciences, 17 th th ed. 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0228] In one embodiment, the therapeutic composition comprises T cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises NK cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises CD34+HE cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises HSCs derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises MDSCs derived from pluripotent cells produced by the methods and compositions disclosed herein. Therapeutic compositions containing populations of iPSC-derived hematopoietic lineage cells disclosed herein can be administered individually or in combination with other suitable compounds by intravenous, intraperitoneal, enteral, or tracheal administration methods to affect the desired therapeutic goal.

[0229] These pharmaceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition at about 3 to about 10. Thus, the buffer can be as much as about 5% by weight of the total composition. Without limitation, electrolytes such as sodium chloride and potassium chloride can also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition ranges from about 4 to about 10. Alternatively, the pH of the therapeutic composition ranges from about 5 to about 9, from about 6 to about 9, or from about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH within one of the foregoing pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0230] The present invention also provides, in part, the use of a pharmaceutically acceptable cell culture medium in certain compositions and / or cultures of the present invention. Such compositions are suitable for administration to a human subject. Generally speaking, any medium that supports the maintenance, proliferation, and / or health of iPSC-derived immune cells according to embodiments of the present invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, the serum-free medium contains no animal components and may be protein-free if desired. Optionally, the medium may contain a pharmaceutically acceptable recombinant protein. A medium that contains no animal components refers to a medium whose components are derived from non-animal sources. The recombinant protein replaces the natural animal protein in a medium that contains no animals, and the nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a protein-free medium is defined as substantially free of protein. Those skilled in the art will understand that the examples of media described above are illustrative and in no way limit the formulation of media suitable for use in the present invention, and that there are many suitable media known and available to those skilled in the art.

[0231] Hematopoietic lineage cells derived from isolated pluripotent stem cells can have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34+HE cells, HSCs, B cells, myeloid-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, hematopoietic lineage cells derived from isolated pluripotent stem cells have from about 95% to about 100% T cells, NK cells, pro-T cells, pro-NK cells, CD34+HE cells, or myeloid-derived suppressor cells (MDSCs). In some embodiments, the present invention provides a therapeutic composition having purified T cells or NK cells, such as a composition having an isolated population of about 95% T cells, NK cells, pro-T cells, pro-NK cells, CD34+HE cells, or myeloid-derived suppressor cells (MDSCs) for treating a subject in need of cell therapy.

[0232] In one embodiment, the combination cell therapy comprises a population of NK cells derived from genomically engineered iPSCs comprising an anti-CD38 therapeutic protein or peptide and the genotypes listed in Table 1, wherein the induced NK cells comprise CD38 null. In another embodiment, the combination cell therapy comprises a population of T cells derived from genomically engineered iPSCs comprising an anti-CD38 therapeutic protein or peptide and the genotypes listed in Table 1, wherein the induced T cells comprise CD38 null. In some embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202, and a population of NK or T cells derived from genomically engineered iPSCs comprising the genotypes listed in Table 1, wherein the induced NK or T cells comprise CD38 null and hnCD16. In yet some other embodiments, the combination cell therapy comprises daratumumab, and a population of NK or T cells derived from genomically engineered iPSCs comprising the genotypes listed in Table 1, wherein the induced NK or T cells comprise CD38 null, hnCD16, and a CAR targeting CD19, BCMA, CD38, CD20, CD22, or CD123. In yet some additional embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202, and a population of NK or T cells derived from genomically engineered iPSCs comprising the genotypes listed in Table 1, wherein the induced NK or T cells comprise CD38 null, hnCD16, and a CAR as well as one or more exogenous cytokines.

[0233] As will be appreciated by those skilled in the art, both autologous and allogeneic hematopoietic cells derived from iPSCs based on the methods and compositions herein can be used in cell therapies as described above. In the case of autologous transplantation, the isolated population of induced hematopoietic cells is fully or partially HLA-matched to the patient. In another embodiment, the induced hematopoietic cells are not HLA-matched to the subject, wherein the induced hematopoietic cells are NK or T cells having HLA I null and HLA II null.

[0234] In some embodiments, the number of induced hematopoietic lineage cells in the therapeutic composition is at least 0.1×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 5×10 6 cells, at least 1×10 7 cells, at least 5×10 7 cells, at least 1×10 8 cells, at least 5×10 8 cells, at least 1×10 9 cells, or at least 5×10 9 cells. In some embodiments, the number of induced hematopoietic lineage cells in the therapeutic composition is about 0.1×10 5 cells to about 1×10 6 cells, about 0.5×10 6 cells to about 1×10 7 cells, about 0.5×10 7 cells to about 1×10 8 cells, about 0.5×10 8 cells to about 1×10 9 cells, about 1×10 9 cells to about 5×10 9 cells, about 0.5×10 9 cells to about 8×10 9 cells, about 3×10 9 cells to about 3×10 10 cells, or any range therebetween. Generally, for a 60 kg patient, 1×10 8 cells / dose is converted to 1.67×10 6 cells / kg.

[0235] In one embodiment, the number of induced hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a partial or single umbilical cord of blood, or at least 0.1×10 5 cells / kg body weight, at least 0.5×10 5 cells / kg body weight, at least 1×10 5 cells / kg body weight, at least 5×10 5cells / kg body weight, at least 10×10 5 cells / kg body weight, at least 0.75×10 6 cells / kg body weight, at least 1.25×10 6 cells / kg body weight, at least 1.5×10 6 cells / kg body weight, at least 1.75×10 6 cells / kg body weight, at least 2×10 6 cells / kg body weight, at least 2.5×10 6 cells / kg body weight, at least 3×10 6 cells / kg body weight, at least 4×10 6 cells / kg body weight, at least 5×10 6 cells / kg body weight, at least 10×10 6 cells / kg body weight, at least 15×10 6 cells / kg body weight, at least 20×10 6 cells / kg body weight, at least 25×10 6 cells / kg body weight, at least 30×10 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 cells / kg body weight, or 1×10 9 cells / kg body weight.

[0236] In one embodiment, a certain dosage of induced hematopoietic system cells is delivered to a subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2×10 6 cells / kg, at least 3×10 6 cells / kg, at least 4×10 6 cells / kg, at least 5×10 6 cells / kg, at least 6×10 6 cells / kg, at least 7×10 6 cells / kg, at least 8×10 6 cells / kg, at least 9×10 6 cells / kg, or at least 10×10 6 cells / kg, or more cells / kg, including all intervening cell dosages.

[0237] In another exemplary embodiment, the effective amount of cells provided to the subject is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, or about 10×10 6 cells / kg, or more cells / kg, including all intervening cell dosages.

[0238] In another exemplary embodiment, the effective amount of cells provided to the subject is about 2×10 6 cells / kg to about 10×10 6 cells / kg, about 3×10 6 cells / kg to about 10×10 6 cells / kg, about 4×10 6 cells / kg to about 10×10 6 cells / kg, about 5×10 6 cells / kg to about 10×10 6 cells / kg, 2×10 6 cells / kg to about 6×10 6 cells / kg, 2×10 6 cells / kg to about 7×10 6 cells / kg, 2×10 6 cells / kg to about 8×10 6 cells / kg, 3×10 6 cells / kg to about 6×10 6 cells / kg, 3×10 6 cells / kg to about 7×10 6 cells / kg, 3×10 6 cells / kg to about 8×10 6 cells / kg, 4×10 6 cells / kg to about 6×10 6 cells / kg, 4×10 6 cells / kg to about 7×10 6 cells / kg, 4×10 6 cells / kg to about 8×10 6 cells / kg, 5×10 6 cells / kg to about 6×106 cells / kg, 5×10 6 cells / kg to approximately 7×10 6 cells / kg, 5×10 6 cells / kg to approximately 8×10 6 cells / kg, or 6×10 6 cells / kg to approximately 8×10 6 cells / kg, including all intervening cell dosages.

[0239] In some embodiments, the therapeutic use of induced hematopoietic lineage cells is a single-dose treatment. In some embodiments, the therapeutic use of induced hematopoietic lineage cells is a multi-dose treatment. In some embodiments, the multi-dose treatment is one administration per day, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days, every 45 days, every 50 days, or any number of administrations on any number of days in between.

[0240] Compositions comprising a population of induced hematopoietic lineage cells of the present invention can be sterile, suitable for administration to a human patient, and can be administered immediately (i.e., administered without further processing). A cell-based composition that is administered immediately means that the composition does not require any further processing or manipulation prior to transplantation or administration to a subject. In other embodiments, the present invention provides an isolated population of induced hematopoietic lineage cells that are expanded and / or modified prior to administration of one or more agents. For induced hematopoietic lineage cells genetically engineered to express a recombinant TCR or CAR, the cells can be activated and expanded, for example, using the methods described in U.S. Patent No. 6,352,694.

[0241] In certain embodiments, the primary and co-stimulatory signals for inducing hematopoietic lineage cells can be provided by different protocols. For example, the agents providing each signal can be in solution or can be bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent can be bound to a surface and the other agent can be present in solution. In one embodiment, the agent providing the co-stimulatory signal can be bound to the cell surface and the agent providing the primary activation signal can be in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in a soluble form and then cross-linked to a surface such as a cell expressing an Fc receptor or an antibody, or another binding agent that binds to the agents disclosed in US Patent Application Publication Nos. 2004 / 0101519 and 20060034810 for use in the activation and proliferation of T lymphocytes in embodiments of the present invention, such as artificial antigen presenting cells (aAPCs).

[0242] Some variations in dosage, frequency, and protocol will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage, frequency, and protocol for an individual subject.

Examples

[0243] The following examples are provided for illustration and not for limitation.

[0244] Example 1 - Materials and Methods The applicant's own hiPSC platform, which enables single-cell passage and high-throughput 96-well plate-based flow cytometry sorting, was used to derive clonal hiPSCs by single or multiple gene modifications in order to effectively select suicide systems under the control of various promoters and test them in combination with various safe harbor locus integration strategies.

[0245] Maintenance of hiPSCs in small molecule culture: hiPSCs were routinely passaged as single cells when the confluence of the culture reached 75% - 90%. In the case of single cell dissociation, hiPSCs were washed once with PBS (Mediatech), treated with Accutase (Millipore) at 37°C for 3 - 5 minutes, and then pipetted to ensure single cell dissociation. Subsequently, the single cell suspension was mixed with an equal volume of the conventional medium, centrifuged at 225×g for 4 minutes, resuspended in FMM, and seeded onto the surface coated with Matrigel. The passage was usually at 1:6 - 1:8, transferred to a tissue culture plate pre-coated with Matrigel at 37°C for 2 - 4 hours, and supplied with FMM every 2 - 3 days. Cell culture was maintained in a humidified incubator set at 37°C and 5% CO2.

[0246] For genome editing via ZFNs, using human iPSC manipulation by ZFNs and CRISPR for targeted editing of the modality of interest as an example of ROSA26 targeted insertion, 2 million iPSCs were transfected with a mixture of 2.5 μg of ZFN-L (FTV893), 2.5 μg of ZFN-R (FTV894), and 5 μg of donor construct for AAVS1 targeted insertion. For genome editing via CRISPR, 2 million iPSCs were transfected with a mixture of 5 μg of ROSA26-gRNA / Cas9 (FTV922) and 5 μg of donor construct for ROSA26 targeted insertion. Transfection was performed using the Neon transfection system (Life Technologies) with parameters 1500 V, 10 ms, 3 pulses. On the second or third day after transfection, if the plasmid contained an artificial promoter-driven GFP and / or RFP expression cassette, flow cytometry was used to measure the transfection efficiency. On the fourth day after transfection, puromycin was added to the medium at a concentration of 0.1 μg / ml for the first 7 days and at a concentration of 0.2 μg / ml 7 days later to select for target cells. During puromycin selection, the cells were passaged into freshly Matrigel-coated wells on the tenth day. After the 16th day of puromycin selection, the surviving cells were analyzed by flow cytometry for the percentage of GFP+ iPS cells.

[0247] Bulk sorting and clone sorting of genome-edited iPSCs: iPSCs containing genome-targeted editing using ZFN or CRISPR-Cas9 were bulk sorted and clone sorted for GFP+SSEA4+TRA181+iPSCs 20 days after puromycin selection. A single-cell dissociation targeted iPSC pool was resuspended in a chilled staining buffer containing Hank's balanced salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1× penicillin / streptomycin (Mediatech), and 10 mM Hepes (Mediatech) freshly prepared for optimal performance. Conjugated primary antibodies such as SSEA4-PE, TRA181-Alexa Fluor-647 (BD Biosciences) were added to the cell solution and incubated on ice for 15 minutes. All antibodies were used at 7 μL in 100 μL of staining buffer per 1 million cells. The solution was washed once with staining buffer, spun down at 225 g for 4 minutes, resuspended in staining buffer containing 10 μM thiazobib, and maintained on ice for sorting by flow cytometry. Sorting by flow cytometry was performed using a FACS Aria II (BD Biosciences). For bulk sorting, gates were set on GFP+SSEA4+TRA181+ cells and sorted into 15 mL standard tubes filled with 7 mL of FMM. For clone sorting, using a 100 μM nozzle, sorted cells were directly dispensed into 96-well plates at a concentration of 3 events per well. Each well was pre-filled with 200 μL of FMM supplemented with 5 μg / mL fibronectin and 1× penicillin / streptomycin (Mediatech) and pre-coated with 5× Matrigel overnight. For 5× Matrigel pre-coating, one aliquot of Matrigel was added to 5 mL of DMEM / F12, incubated overnight at 4°C to resuspend properly, and finally added to 96-well plates at 50 μL per well and incubated overnight at 37°C. The 5× Matrigel was aspirated immediately before adding medium to each well. After sorting was completed, the 96-well plates were centrifuged at 225 g for 1-2 minutes and then incubated.The plate was left static for 7 days. On the 7th day, 150 μL of the medium was removed from each well and replaced with 100 μL of FMM. The wells were re-supplied with an additional 100 μL of FMM on the 10th day after sorting. Colony formation was detected as early as the 2nd day, and most colonies grew between 7 and 10 days after sorting. In the first passage, the wells were washed with PBS and dissociated with 30 μL of Accutase at 37 °C for approximately 10 minutes. The need for a long Accutase treatment reflects the compactness of the colonies that did not float during long-term culture. After the cells were observed to be dissociated, 200 μL of FMM was added to each well and pipetted several times to break up the colonies. The dissociated colonies were transferred to another well of a 96-well plate pre-coated with 5× Matrigel and centrifuged at 225 g for 2 minutes before incubation. This 1:1 passage was performed to expand the initial colonies before growth. Subsequent passages were routinely performed with a 3 - 5 minute Accutase treatment and a 1:4 - 1:8 expansion at 75 - 90% confluence into larger wells pre-coated with 1× Matrigel in FMM. Each clonal cell line was analyzed for GFP fluorescence levels and TRA1-81 expression levels. Clonal lines with GFP+ and TRA1-81+ levels close to 100% were selected for further PCR screening and analysis. Flow cytometry analysis was performed on a Guava EasyCyte 8 HT (Millipore) and analyzed using Flowjo (FlowJo, LLC).

[0248] Example 2 - CD38 Knockout in iPSCs Using Genome Editing via CRISPR / Cas9 Alt-R® SpCas9 D10A Nickase 3NLS, 100 μg, and Alt-R® CRISPR-Cas9 tracrRNA were purchased from IDT (Coralville, Iowa) and used for iPSC targeted editing. To perform allelic knockout of CD38 in iPSCs using Cas9 Nickase, screening for gNA (i.e., gD / RNA or guide polynucleotide) design and identified targeting sequence pairs (1A and 1B, 2A and 2B, 3A and 3B) are listed in Table 3.

Table 3

[0249] Subsequently, the genome-engineered iPSCs were characterized, and allelic CD38 knockout was confirmed.

[0250] Example 3 - CD38 - / - Verification of iPSCs and Derived Cells CD38 is known to be expressed at specific cell stages and plays an important role in effector cells. During hematopoiesis, CD38 is expressed in CD34 + stem cells, progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, and at the final stages of maturation of effector cells such as T cells and NK cells. Therefore, prior to this application, considering the CD38 expression profile and functionality, it was unclear and a concern whether iPSCs containing CD38 knockout would develop properly when exposed to the indicated differentiation conditions and whether the generated effector cells would function. CD38 null iPSCs containing allelic knockout of CD38 surprisingly maintained the ability to differentiate into induced cells. In one of the figures, three engineered iPSC clones gene-edited to result in allelic disruption of the CD38 gene and hnCD16 were differentiated into induced NK cells, and their phenotypes were analyzed. The flow profile in Figure 2 shows that each clone is CD56+ and CD38 negative.

[0251] To determine whether the cytotoxicity of iPSC-derived NK cells is retained when CD38 expression is knocked out, wild-type iPSC clones and three engineered iPSC clones with allelic disruption of the CD38 gene were differentiated into NK cells, and their ability to target and kill the tumor cell line RPMI-8266 was compared. As shown by the long-term killing assay in Figure 3, over 48 hours, the engineered CD38 - / - NK cells eliminated target cells in a manner similar to wild-type controls, indicating that CD38 is not essential for target cell recognition and killing. Thus, it is shown herein that complete loss of CD38 in iPSCs does not affect hematopoietic cell derivation or the cytotoxicity of effector cells.

[0252] Daratumumab (Darzalex) is an anti-cancer agent. It binds to CD38, which is overexpressed in multiple myeloma cells. Cell killing of multiple myeloma via daratumumab is partially dependent on ADCC and highly dependent on NK effector cells. However, CD38 is also expressed in NK cells, and as a result, daratumumab may induce ADCC (fratricide) against NK cells, significantly reducing the efficacy of daratumumab. Here, in Figure 6, CD38 - / - iPSC-derived NK cells have been shown to maintain their ADCC function when stimulated by the tumor cell line RPMI-8266 in the presence of daratumumab. CD38 - / - Figure 7 further shows that the viability of iPSC-derived NK cells (Figure 7B) is maintained for at least about 48 hours in culture in the presence of daratumumab compared to iPSC-derived NK cells expressing CD38 (Figure 7A). Furthermore, as shown in Figure 7, CD38 - / - iPSC-derived NK cells do not show degranulation and produce fewer cytokines than iNK cells expressing CD38 when stimulated in the presence of daratumumab.

[0253] In addition to CD38 null, induced pluripotent stem cells have also been successively engineered to obtain expression of high-affinity non-cleavable CD16, loss of HLA-I by knockout of the B2M gene, loss of HLA-II by knockout of CIITA, overexpression of the non-classical HLA molecule HLA-G, and expression of a linked IL15 / IL15 receptor alpha construct. After each manipulation step and before the next manipulation step, the iPSCs were sorted for the desired phenotype. The engineered iPSCs can be maintained for in vitro or induced cell generation. Figure 4 shows hnCD16 expression, B2M knockout, HLA-G expression, and IL15 / IL15Rα expression in iPSC-derived NK cells. Figure 7 shows the introduction of hnCD16 in combination with CD38 knockout in iPSC-derived NK cells. These data indicate that these genetically engineered modalities are maintained without disturbing the directed development of cells to the desired cell fate in vitro during hematopoietic differentiation.

[0254] Telomere shortening occurs with cellular aging and is associated with stem cell dysfunction and cellular senescence. Here, it has been shown that mature iNK cells maintain longer telomeres compared to adult peripheral blood NK cells. Telomere length was determined by flow cytometry of iPSCs, adult peripheral blood NK cells, and iPSC-derived NK cells, using the 1301 T cell leukemia cell line as a control (100%) to correct for the DNA index of the cells. As shown in Figure 5, iPSC-derived NK cells maintain significantly longer telomere lengths compared to adult peripheral blood NK cells (p =.105, ANOVA), indicating a higher potential for proliferation, survival, and persistence of iPSC-derived NK cells. 0 / 1 Determined using the 1301 T cell leukemia cell line as a control (100%) to correct for the DNA index of the cells. As shown in Figure 5, iPSC-derived NK cells maintain significantly longer telomere lengths compared to adult peripheral blood NK cells (p =.105, ANOVA), indicating a higher potential for proliferation, survival, and persistence of iPSC-derived NK cells.

[0255] Example 4 - Functional Profiling of CD38 Null-Induced NK Cells The phenotype of hnCD16 CD38- / - iNK cells, including the expression of NKG2A, NKp46, and KIR2DL2 / 3, and calcium flux were evaluated, and the phenotype of hnCD16 iNK was maintained after CD38 knockout (Figures 10A - 10D). The ADCC function of hnCD16 iNK with CD38 knockout was examined against the HER2-expressing ovarian cell line SKOV3 by Incucyte live cell imaging, and CD38 knockout did not affect the ADCC function of hnCD16 iNK (Figure 10E). Next, the specific cytotoxicity of daratumumab against various NK cell populations: peripheral blood NK cells (with CD16 shedding), hnCD16 iNK cells (with high-affinity non-cleavable CD16), non-modified iNK cells (with low CD16 expression), and hnCD16 CD38- / - iNK cells were measured after incubating each cell population with different concentrations of daratumumab for 4 hours. As shown in Figure 11, CD38 deficiency protected hnCD16 CD38- / - iNK cells from fratricide mediated by increasing concentrations of daratumumab compared to other cell populations without CD38 knockout. Thus, loss of CD38 prevents daratumumab-mediated NK cell fratricide in the presence of CD38-specific antibodies.

[0256] To evaluate the cytotoxicity of hnCD16 CD38− / − iNK cells compared to hnCD16 iNK cells, each cell population was incubated with MM.1S myeloma target cells for 18 hours, and then the viability of the tumor cells was evaluated by flow cytometry using annexin V and a live / dead viability marker. As shown in Figure 12, hnCD16 iNK cells mediate potent anti-myeloma activity with daratumumab, which is further enhanced by CD38 loss. Furthermore, in a 7-day cytotoxicity assay against RMPI-8226 tumor spheroids, hnCD16 CD38− / − iNK cells showed superior tumor cell clearance as measured by the number of target cells remaining at the end of the assay in the presence of a CD38-specific antibody and compared to hnCD16 iNK and unmodified iNK cells under the same conditions (Figure 13A). Additionally, the lack of NK cell fratricide improved the survival of hnCD16 CD38− / − iNK cells as shown by the improved persistence of these cells in the 7-day cytotoxicity assay shown in Figure 13B. Thus, hnCD16 CD38− / − iNK cells exhibit enhanced long-term anti-myeloma activity and persistence with CD38-specific antibodies such as daratumumab. As shown in Figure 14, hnD16 iNK cells lacking CD38 also demonstrate more durable ADCC with increased serial killing ability in the presence of a CD38-specific antibody. In this assay, hnCD16 or hnCD16 CD38− / − iNK cells were incubated with MM.1S myeloma target cells and daratumumab for 48 hours (stimulation round 1), and the number of MM.1S cells was quantified by Incucyte™ imaging. After 48 hours, the effector cells were removed and transferred to fresh target cells for a second round of stimulation and target cell killing with daratumumab (stimulation round 2). (Figure 14)

[0257] In light of the above, CD38-targeted knockout does not affect the phenotype of derivative NK cells or general cellular functions, and the resulting CD38-deficient induced NK cells were found to be protected from CD38-specific antibodies, daratumumab, such as mediated fratricide. Due to the synergistic effect of hnCD16 and CD38− / −, in combination with CD38-specific antagonists including monoclonal antibodies such as daratumumab, enhanced anti-myeloma activity and durable ADCC of induced NK cells are provided. Based on these findings, a clinical strategy has been proposed to overcome the NK cell depletion effect of CD38-targeted agents and improve the outcome of myeloma patients by combining off-the-shelf hnCD16CD38− / − iNK cells with daratumumab.

[0258] Example 5 - Use of CD38-specific antagonists to protect allogeneic effector cells from allogeneic rejection Engineered iNK cells with enhanced CD16 efficacy and CD38 depletion are resistant to CD38-targeted antibody-induced fratricide and mediate more potent anti-myeloma activity in combination with daratumumab. In recipients of allogeneic effector cells lacking CD38, by suppressing the activation of these lymphocytes using CD38-specific antagonists including monoclonal antibodies, due to the fact that CD38 is upregulated in activated lymphocytes such as T cells or B cells, the allogeneic rejection of these effector cells is reduced and / or prevented, thereby increasing the survival rate and persistence of the effector cells. To demonstrate the feasibility of this strategy, a mixed lymphocyte reaction (MLR, i.e., co-incubation of effector cell products with allogeneic PBMC) was performed to test the lifespan of the induced effector cells of the present invention in an allogeneic environment in relation to CD38 knockout and in the presence or absence of an anti-CD38 monoclonal antibody (e.g., daratumumab).

[0259] The hnCD16+ iNK cell populations (with and without CD38KO) are labeled with a cell-impermeant dye (Celltrace Violet™ or similar Incucyte™-compatible reagent) immediately prior to the assay. Different concentrations of hnCD16+ and hnCD16 CD38− / − iNK cells are incubated with a fixed number of PBMCs from random healthy donors (n = 3 - 4, not pooled) in the presence or absence of daratumumab (at an effective concentration, titrated prior to the assay). The viability of iNK cells in each population is monitored over time by Incucyte™ in long-term culture. Viability is also monitored by flow cytometry, and the staining panel further tracks CD38 upregulation of PBMC subpopulations and clearance of PBMC subpopulations by iNK cells based on ADCC mediated by daratumumab. Total clearance of iNK cells as a control is achieved using Venetoclax (an MCL-1 inhibitor for specific depletion of NK cells).

[0260] The extended lifespan of hnCD16 CD38KO iNK cells compared to wild-type hnCD16 iNK cells in the presence of anti-CD38, and the associated clearance of CD38+ subpopulations (peripheral NK cells, activated B cells, and T cells) from PBMC samples, demonstrate the ability of a CD38-specific antagonist to suppress activated peripheral T or B cells by targeting their upregulated CD38, thereby reducing allogeneic rejection of allogeneic effector cells by these activated peripheral T or B cells in recipients of effector cells comprising hnCD16 and CD38− / − as provided herein. The CD38-specific antagonist is a CD38-specific antibody, a CD38-specific engager, or a CD38 chimeric antigen receptor (CAR).

[0261] Furthermore, the expression of an exogenous truncated IL15 / IL15Rα fusion protein lacking the intracellular domain of IL15Rα has been shown to support the survival of iPSC-derived NK cells in vitro, regardless of the addition of soluble exogenous IL2. IL15Rα without an intracellular domain was fused to IL15 at the C-terminus via a linker to generate a truncated IL15 / IL15Ra fusion (or, in this application, called "IL15Δ") construct without a signaling domain. Exemplary IL15Δ provided herein includes those having a structure such as Design 3 or 4 in Figure 1. As shown in Figure 15, iNK cells were transduced with a lentiviral overexpression vector expressing either GFP (squares; negative control), full-length IL15 / IL15Ra fusion construct (black circles; positive control; Design 2 in Figure 1), or truncated IL15 / IL15Ra fusion construct (white circles; Design 3 in Figure 1). Neither the IL15 construct nor GFP showed enrichment in the presence of exogenous IL2 (Figure 15A), indicating that transduced cells survived at a rate comparable to non-transduced cells. In the absence of exogenous IL2, cells transduced with either IL15 / IL15Ra fusion construct were enriched over time, but not GFP-transduced cells. In the absence of IL2, cells transduced with either IL15 / IL15Ra construct had a survival advantage compared to non-transduced cells under the same culture conditions (Figure 15B). Furthermore, since the intracellular domain of IL15Rα is considered important for the receptor to be expressed and respond in IL15-responsive cells and for the cells to proliferate and function, it is surprising that the truncated IL15 / IL15Ra fusion construct with a shortened intracellular domain not only stably expressed in transduced iNK cells, but also supported iNK cells at a higher proliferation rate than the full-length IL15 / IL15Ra fusion construct, as shown in Figure 15B. Thus, the IL15Δ provided herein can express and maintain IL15 in a membrane-bound form and replace the full-length IL15 / IL15Ra fusion protein to provide trans-presentation of IL15 intracellularly.Although the fundamental mechanism is not fully understood, deleting the intracellular domain of IL15R appears to confer additional viability, fitness, or particular advantages in terms of survival, proliferation, and persistence to the responding cells, perhaps by completely eliminating cis-presentation mediated by normal IL15R via that intracellular domain and / or other potential signaling pathways.

[0262] One of ordinary skill in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended as limitations on the scope of the invention. It will be readily apparent to one of ordinary skill in the art that various substitutions and modifications can be made to the present disclosure without departing from the scope and spirit of the invention.

[0263] All patents and publications referred to herein represent the level of skill of those in the art to which the present disclosure pertains. All patents and publications are hereby incorporated by reference into this specification as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0264] The present disclosure, as exemplified herein, can be practiced without any one or more of the elements, limitations, or plurality of limitations not specifically disclosed herein. Thus, for example, in each instance herein, the terms "comprising," "consisting essentially of," and "consisting of" can each be replaced with either of the other two terms. The terms and expressions used are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, to exclude any equivalents of the features shown and described or any portion thereof, but it is recognized that various modifications are possible within the scope of the claimed present disclosure. Accordingly, although the present disclosure is specifically disclosed by preferred embodiments and optional features as necessary, modifications and variations of the concepts disclosed herein will often occur to those of ordinary skill in the art, and such modifications and variations are within the scope of the invention as defined by the appended claims.

Claims

1. A cell or population thereof, (i) the cell is (a) an induced pluripotent cell (iPSC), a clonal iPSC, or an iPS cell line cell, or (b) an induced cell obtained by differentiation of the cell of (a); (ii) A cell or a population thereof, wherein the cell comprises a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) that has a CD38 knockout or no intracellular domain.

2. The cell or population thereof of claim 1, wherein the derived cells of (i)(b) are hematopoietic cells and contain longer telomeres compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue.

3. The cells, (i) B2M null or low; (ii) CIITA null or low; (iii) induced expression of HLA-G or non-cleavable HLA-G; (iv) high affinity non-cleavable CD16 (hnCD16) or a variant thereof; (v) chimeric antigen receptor (CAR), (vi) a partial or complete peptide of a cell surface-expressed exogenous cytokine or its receptor; (vii) at least one of the genotypes listed in Table 1; (viii) deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene of the chromosome 6p21 region; (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ The cell or population thereof of claim 1, further comprising one or more of the following: an R, a CAR, a TCR, an Fc receptor, an engager, and an introduced or increased expression in at least one of a surface triggering receptor for binding to a bispecific or multispecific or universal engager.

4. The cells are induced NK or T cells, and compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue, (i) improved persistence and / or survival; (ii) increased resistance to natural immune cells; (iii) increased cytotoxicity; (iv) improved tumor penetration; (v) enhanced or gained ADCC; (vi) an enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site; (vii) enhanced ability to reduce tumor immunosuppression, and (viii) improved ability to rescue tumor antigen escape; (ix) reduced fratricide; The cell or population thereof according to claim 1 or 3, having at least one of the following characteristics:

5. The cell or population thereof described in claim 3, wherein the cell further comprises high affinity non-cleavable CD16 (hnCD16) or a variant thereof.

6. The high affinity non-cleavable CD16 (hnCD16) or a variant thereof, (a) F176V and S197P in the ectodomain of CD16; (b) a complete or partial ectodomain derived from CD64; (c) a non-native (or non-CD16) transmembrane domain; (d) a non-native (or non-CD16) intracellular domain; (e) a non-native (or non-CD16) signaling domain; (f) a non-naturally occurring stimulatory domain, and (g) the cell or population thereof described in claim 5, comprising at least one of the following transmembrane, signaling, and stimulatory domains not derived from CD16 and derived from the same or a different polypeptide.

7. (a) the non-native transmembrane domain is derived from a CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide; (b) the non-native stimulatory domain is derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide; (c) the non-native signaling domain is derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide; or (d) the non-native transmembrane domain is derived from NKG2D, the non-native stimulatory domain is derived from 2B4, and the non-native signaling domain is derived from CD3ζ, the cell or population thereof of claim 6.

8. The cell further comprises a chimeric antigen receptor (CAR), the CAR comprising: (i) T cell-specific or NK cell-specific; (ii) bispecific antigen binding CAR; (iii) a switchable CAR; (iv) a dimerized CAR, (v) split CAR, (vi) multi-chain CAR; (vii) an inducible CAR; (viii) co-expressed with another CAR; (ix) co-expressed with cell surface-expressed exogenous cytokines or partial or complete peptides of their receptors, optionally in separate or bicistronic constructs; (xi) optionally co-expressed with a checkpoint inhibitor, either in a separate construct or in a bicistronic construct; (xii) specific for CD19 or BCMA, and / or (xiii) ADGRE2, carbonic anhydrase IX (CA1X), CCRI, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, cytomegalovirus (CMV)-infected cell antigen, epithelial glycoprotein (EGF) epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase human erythrocyte serine / cytoplasmic reticulum (hTERT), interleukin-13 reverse transcriptase (ICAM-1), integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSL N), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigens; 4. The cell or population thereof of claim 3, wherein the CAR of any one of (i) to (xiii) is optionally inserted into a TRAC locus and / or is driven by the endogenous promoter of a TCR and / or the TCR is knocked out by the CAR insertion.

9. The cell further comprises a partial or complete peptide of a cell surface-expressed exogenous cytokine or its receptor, the exogenous cytokine or its receptor being (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors; or (b) (i) Co-expression of IL15 and IL15Rα by using a self-cleaving peptide; (ii) a fusion protein of IL15 and IL15Rα; (iii) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα has been truncated; (iv) a fusion protein of the membrane-bound Sushi domain of IL15 and IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and common receptor γC, said common receptor γC being natural or modified; (vii) a homodimer of IL15Rβ, wherein any one of (i)-(vii) may be co-expressed with the CAR in a separate construct or in a bicistronic construct; as needed, (c) A cell or population thereof according to claim 3, which expresses transiently.

10. 4. The cell or population thereof of claim 3, wherein the cell is an induced NK cell or an induced T cell, the induced NK cell being capable of recruiting and / or migrating T cells to a tumor site, and the induced NK cell or the induced T cell being capable of reducing tumor immune suppression in the presence of one or more checkpoint inhibitors.

11. 11. The cell or population thereof of claim 8 or 10, wherein the checkpoint inhibitor is an antagonist against one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR.

12. The checkpoint inhibitor, (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or The cell or population thereof of claim 11, comprising (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

13. The cell or population thereof of claim 2, wherein the induced cells comprise induced CD34 cells, induced hematopoietic stem and progenitor cells, induced hematopoietic multipotent progenitor cells, induced T cell progenitors, induced NK cell progenitors, induced T cells, induced NKT cells, induced NK cells, or induced B cells.

14. The cells, (i) one or more exogenous polynucleotides integrated into a safe harbor locus; or (ii) more than two exogenous polynucleotides integrated into different safe harbor loci; or (iii) a polynucleotide encoding IL15Δ comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21.

15. The cell or population thereof of claim 14, wherein the safe harbor loci include at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1.

16. The cell or population thereof of claim 15, wherein the safe harbor locus TCR is a TCR alpha constant region.

17. A composition comprising a cell or a population thereof according to any one of claims 1 to 16.

18. A composition for therapeutic use comprising the induced cells of any one of claims 1 to 16 and one or more therapeutic agents.

19. 20. The composition of claim 18, wherein the therapeutic agent comprises a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a supplement thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

20. (1) The checkpoint inhibitor, (a) one or more antagonist checkpoint molecules, including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; (c) at least one of atezolizumab, nivolumab, and pembrolizumab; (2) The composition of claim 19, wherein the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide.

21. The antibody, (a) anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies; (b) one or more of rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, certuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof; or 20. The composition of claim 19, wherein (c) the composition comprises daratumumab, and the induced hematopoietic cells comprise induced NK cells or induced T cells comprising a CD38 knockout and optionally expression of hnCD16 or a variant thereof.

22. 22. Therapeutic use of a therapeutic composition according to any one of claims 17 to 21 by introducing said composition into a subject suitable for adoptive cell therapy, said subject having an autoimmune disease, a hematological malignancy, a solid tumor, cancer, or a viral infection.

23. A method for producing the induced cell according to any one of claims 1 to 16, comprising differentiating iPSCs, the iPSCs harboring a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) in which CD38 has been knocked out or the intracellular domain of IL15Rα has been truncated, and optionally: (i) B2M null or low; (ii) CIITA null or low; (iii) induced expression of HLA-G or non-cleavable HLA-G; (iv) high affinity non-cleavable CD16 (hnCD16) or a variant thereof; (v) chimeric antigen receptor (CAR), (vi) a partial or complete peptide of a cell surface-expressed exogenous cytokine or its receptor; (vii) at least one of the genotypes listed in Table 1; (viii) deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene of the chromosome 6p21 region; (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ A method of producing an inducer cell comprising one or more of the following: an R, a CAR, a TCR, an Fc receptor, an engager, and the introduction or increased expression of at least one of a surface triggering receptor for binding to a bispecific or multispecific or universal engager.

24. To knock out CD38 or to knock in a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) that does not have an intracellular domain, and optionally (i) to knock out B2M and CIITA, or (ii) further comprising genomic engineering the clonal iPSCs to introduce expression of a partial or complete peptide of HLA-G or non-cleavable HLA-G, high affinity non-cleavable CD16 or a variant thereof, a CAR, and / or cell surface expression of an exogenous cytokine or its receptor; 24. The method of producing an induced cell of claim 23, wherein the CAR and the cell surface expressed partial or complete peptide of an exogenous cytokine or its receptor are co-expressed in separate constructs or in a bicistronic construct.

25. 24. The method of producing an induced cell of claim 23, wherein said genome engineering comprises targeted editing.

26. 26. The method of producing an induced cell of claim 25, wherein the targeted editing comprises a deletion, insertion, or indel, and the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

27. 1. CRISPR-mediated editing of clonal iPSCs, said editing comprising a CD38 knockout or a knock-in of a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) lacking an intracellular domain, said edited clonal iPSCs comprising at least one of the genotypes listed in Table 1.

28. (i) the CD38 knockout is biallelic, or (ii) the CD38 knockout is a nucleic acid cleavage between a first and a second target sequence, the targeting sequences comprising SEQ ID NO:3 and SEQ ID NO:4, respectively; or (iii) The CRISPR-mediated editing of claim 27, wherein the polynucleotide encoding IL15Δ comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 17, 19 or 21.

29. 28. The CRISPR-mediated editing of claim 27, wherein the editing further comprises the insertion of a CAR at the TRAC locus, and / or the CAR is driven by the endogenous promoter of a TCR, and / or the TCR is knocked out by the insertion of the CAR.

30. A method of improving anti-CD38 antibody therapy comprising administering effector cells without CD38 expression to a subject under treatment.

31. 31. The method of claim 30, wherein the anti-CD38 antibody is any of daratumumab, isatuximab, or MOR202, or a humanized or Fc-modified variant or fragment, functional equivalent, and biosimilar thereof.

32. The effector cells comprise induced hematopoietic cells, including induced NK cells or induced T cells, the induced NK cells or induced T cells comprising CD38 knockout, high affinity non-cleavable CD16 or a variant thereof, and optionally (i) B2M and CIITA knockout, (ii) the introduced expression of HLA-G or non-cleavable HLA-G, a CAR, and / or a partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface, wherein the CAR and the partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface are co-expressed in separate or bicistronic constructs, and / or 31. The method of claim 30, comprising: (iii) at least one of the genotypes listed in Table 1.

33. 31. The method of claim 30, wherein the method reduces anti-CD38 antibody-induced effector cell depletion in the subject during treatment.

34. A method of reducing or preventing allorejection responses to allogeneic effector cells by using a CD38-specific antagonist, wherein the allogeneic effector cells comprise a CD38 knockout, and the CD38-specific antagonist is capable of suppressing activated T cells and B cells in a recipient of the allogeneic effector cells.

35. 35. The method of claim 34, wherein the CD38-specific antagonist is an anti-CD38 antibody, a CD38-specific engager, or a CD38 chimeric antigen receptor (CAR).

36. 36. The method of claim 35, wherein said anti-CD38 antibody is any of daratumumab, isatuximab, or MOR202, or a humanized or Fc-modified variant or fragment, functional equivalent, and biosimilar thereof.

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