Engineered immune effector cells and use thereof

Abstract

To provide methods and compositions to obtain functionally enhanced derivative effector cells obtained from directed differentiation of a genomically engineered iPSC.SOLUTION: Disclosed is a construct for engineering a cell to obtain a genomically edited cell, the construct comprising: (i) a left homology arm and a right homology arm targeting CD38 at a selected position in CD38 locus in the cell genome, operatively linked to (ii) one or more nucleic acid sequences encoding one or more exogenous proteins; where the genomically edited cell is an induced pluripotent cell (iPSC), a clonal iPSC, or an iPS cell line cell; where the genomically edited cell comprises a targeted integration of the one or more nucleic acid sequences encoding one or more exogenous proteins at the selected position in CD38 locus, and CD38 knockout; and where the genomically edited cell is an iPSC capable of differentiating into an iPSC-derived hematopoietic cell.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 649,781, filed March 29, 2018, U.S. Provisional Patent Application No. 62 / 774,052, filed November 30, 2018, and International Application No. PCT / US18 / 67289, filed December 21, 2018, the disclosures of which are incorporated by reference in their entireties herein.

[0002] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING This application incorporates by reference the computer readable form (CRF) of the Sequence Listing in ASCII text format entitled 13601-204-228_SEQ_LISTING.txt, created on Mar. 28, 2018, which was submitted herewith and is 45,867 bytes in size.

[0003] The present disclosure relates generally to the field of off-the-shelf immune cell products. More specifically, 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 critical limitations of patient-derived cell therapy. [Background technology]

[0004] The field of adoptive cell therapy is currently focused on using patient- and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapies and to provide therapy 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 potent antitumor effectors that play a key role in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains challenging and there is an unmet demand for improvement. Thus, significant opportunities remain to fully harness the potential of T cells and NK cells, or other lymphocytes, in adoptive immunotherapy. Summary of the Invention

[0005] There is a need for functionally improved effector cells that address a variety of issues, including response rates, cell attrition, loss of infused cells (viability and / or persistence), tumor escape due to target loss or lineage switching, precision of tumor targeting, off-target toxicity, off-tumor effects, efficacy against solid tumors, i.e. the tumor microenvironment and associated immune suppression, recruitment, trafficking, and invasion.

[0006] 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) clonal line, which contains one or several genetic modifications in its genome, including DNA insertions, deletions, and substitutions, which remain retained and functional in the subsequent derived cells after differentiation, expansion, passage, and / or transplantation.

[0007] The non-pluripotent cells derived from iPSCs of the present application include, but are not limited to, CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells. The non-pluripotent cells derived from iPSCs of the present application contain one or several genetic modifications in their genomes through differentiation from iPSCs containing identical genetic modifications. The engineered clonal iPSC differentiation strategy to obtain genetically engineered derived cells also requires that the developmental potential of iPSCs in the directed differentiation is not adversely affected by the engineered modality of iPSCs, and that the engineered modality functions as intended in the derived cells. Furthermore, this strategy overcomes the current barriers in engineering primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely, that such cells are often difficult to engineer, resulting in cells that lack reproducibility and uniformity and exhibit poor cell persistence with high cell death and low cell proliferation. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations that are otherwise obtained using initially heterogeneous primary cell sources.

[0008] Some embodiments of the present invention provide genomically engineered iPSCs obtained following, simultaneously with, and prior to the reprogramming process using methods that reflect strategies of genomic engineering including (I), (II), or (III), respectively:

[0009] (I): genetically engineering iPSCs with one or both of (i) and (ii) in any order: (i) introducing one or more construct(s) into the iPSCs to allow targeted integration at the selected site(s); (ii)(a) introducing one or more double-stranded break(s) into the iPSCs at the selected site(s) using one or more endonucleases capable of recognition of the selected sites; (b) culturing the iPSCs of step (I)(ii)(a) to allow endogenous DNA repair to generate targeted indels at the selected site(s); thereby obtaining genomically engineered iPSCs capable of differentiation into partially or fully differentiated cells.

[0010] (II): Genetically engineering the reprogrammed non-pluripotent cells to obtain genomically engineered iPSCs: (i) contacting 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) introducing into the reprogrammed non-pluripotent cells of step (II)(i) one or both of (a) and (b) in any order: (a) targeted integration at the selected site(s); (b) creating one or more double stranded break(s) at the selected site(s) using at least one endonuclease capable of recognition of the selected site(s), and then culturing the cells of step (II)(ii)(b) for endogenous DNA repair to allow for the generation of targeted indels at the selected site(s); thus, the obtained genomically engineered iPSCs contain at least one functional targeted genome edit, and said genomically engineered iPSCs are capable of being differentiated into partially or fully differentiated cells.

[0011] (III): Genetically engineering non-pluripotent cells for reprogramming to obtain genomically engineered iPSCs comprising (i) and (ii): (i) introducing into the non-pluripotent cells one or both of (a) and (b) in any order: (a) one or more construct(s) enabling targeted integration at the selected site(s); (b) making one or more double-strand break(s) at the selected site(s) using at least one endonuclease capable of recognition of the selected site(s), wherein the cells of step (III)(i)(b) are cultured to allow endogenous DNA repair to occur at the selected site(s). (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 a targeted edit at the selected site, thereby obtaining genomically engineered iPSCs comprising at least one functional targeted genomic edit at the selected site, wherein said genomically engineered iPSCs can be differentiated into partially differentiated cells or fully differentiated cells.

[0012] In one embodiment of the above-mentioned method, at least one targeted genome editing at one or more selected sites includes the insertion of one or more exogenous polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacoactive proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of genomically engineered iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides for insertion are operably linked to one or more exogenous promoters, including (1) CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, transient, tissue-specific, or cell type-specific promoters; or (2) AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of genome safe harbor, contained in the selected sites. In some embodiments, the genomically engineered iPSCs generated using the methods described above contain one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, where if the genomically engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into different safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotides encode partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors. In some embodiments, the partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors encoded by the exogenous polynucleotide are in the form of a fusion protein.

[0013] In some other embodiments, the genomically engineered iPSCs generated using the methods provided herein contain indels in one or more endogenous genes associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response regulation and modulation, or proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or viability 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.

[0014] In yet some other embodiments, the genomically engineered iPSCs generated using the methods provided herein comprise an exogenous polynucleotide encoding a caspase at the AAVS1 locus and an exogenous polynucleotide encoding a thymidine kinase at the H11 locus.

[0015] In yet some other embodiments, approaches (I), (II) and / or (III) further comprise contacting the genomically engineered iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain pluripotency of the genomically engineered iPSCs. In one embodiment, the obtained genomically engineered iPSCs containing at least one targeted genome edit are functional, differentiation competent, and capable of differentiating into non-pluripotent cells containing the same functional genome edit.

[0016] The present invention also provides the following:

[0017] One aspect of the present application is a cell or population thereof, wherein the cell is an induced pluripotent cell (iPSC), a clonal iPSC, or an iPS cell line cell, or a derived cell obtained from differentiating any of the above iPSCs; any of the above cells comprising at least one CD38 knockout, or a polynucleotide encoding an IL15 / IL15Rα fusion protein without the intracellular domain (IL15Δ). In some embodiments of derived cells obtained from iPSC differentiation, the derived cells are hematopoietic cells, including, but not limited to, CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells; the hematopoietic cells (i.e., induced CD34 cells, induced hemogenic endothelial cells, induced hematopoietic stem and progenitor cells, induced hematopoietic pluripotent 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 native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue.

[0018] In some embodiments of the above-described iPSCs and derived cells thereof comprising a polynucleotide encoding a CD38 knockout, or an IL15 / IL15Rα fusion protein without an intracellular domain (IL15Δ), the cells further comprise one or more of the following genomic edits: (i) B2M null or low, (ii) CIITA (iii) introduced expression of HLA-G or non-cleavable HLA-G, (iv) high affinity non-cleavable CD16 (hnCD16) or a variant thereof, (v) a chimeric antigen receptor (CAR), (vi) cell surface expression of an exogenous cytokine or a partial or complete peptide of 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, and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Induced or increased expression of at least one of the following surface triggering receptors for binding to R, CAR, TCR, Fc receptor, engager, and bispecific or multispecific or universal engager.

[0019] In some embodiments of the iPSCs and derived cells thereof comprising at least a polynucleotide encoding an IL15 / IL15Rα fusion protein without CD38 knockout or intracellular domain (IL15Δ), and optionally additional genome editing as described above and throughout this application, the cells may comprise (i) one or more exogenous polynucleotides integrated into one safe harbor locus, or (ii) three or more exogenous polynucleotides integrated into different safe harbor loci, or (iii) an IL15Δ-encoding polynucleotide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical 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 one particular embodiment, the safe harbor locus TCR is the constant region of TCR alpha.

[0020] In some embodiments of the cells or populations thereof, the cells comprising at least a CD38 knockout or IL15Δ, and one or more of the additional genomic edits described above, are induced NK or induced T cells, which include, but are not limited to, at least one of the following characteristics: (i) improved persistence and / or survival, (ii) increased resistance to natural immune cells, (iii) increased cytotoxicity, (iv) improved tumor penetration, (v) enhanced or acquired ADCC, (vi) enhanced ability to migrate and / or activate or recruit bystander immune cells to tumor sites, (vii) enhanced ability to reduce tumor immune suppression, (viii) improved ability to rescue tumor antigen escape, and (ix) reduced fratricide, when compared to their native counterpart NK or T cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue.

[0021] In one embodiment of the cell or population thereof, the CD38 knockout or IL15Δ-containing cell further comprises 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 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-native stimulatory domain, and (g) transmembrane, signaling, and stimulatory domains not derived from CD16 and derived from the same or a different polypeptide. In some embodiments, the non-native transmembrane domain is derived from a CD3D, CD3E, CD3G, CD3zeta, 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. In some embodiments, 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. In some other embodiments, the non-native signaling domain is derived from a CD3zeta, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In some particular embodiments of the hnCD16 variant, 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 CD3zeta.

[0022] In one embodiment of the cell or population thereof, the CD38 knockout or IL15Δ comprising cell further comprises a chimeric antigen receptor (CAR), which 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) multi-chain CAR, (vii) inducible CAR, (viii) co-expressed with another CAR, (ix) co-expressed with a cell surface expressed exogenous cytokine or partial or complete peptide of its receptor, 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, cytomegalovirus (CMV)-infected cell antigen, 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), 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), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal 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.

[0023] In some embodiments in which a 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, Foxp1, 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-mentioned checkpoint molecules, or a humanized or Fc-modified variant or fragment, as well as functional equivalents and biosimilars thereof. In some embodiments, any one of the CARs (i)-(ix) can be inserted into the TRAC locus. In some embodiments, any one of the CARs (i)-(ix) inserted into the TRAC locus can be driven by the endogenous promoter of the TCR. In some embodiments, the insertion of any one of the CARs (i)-(ix) at the TRAC locus results in a TCR knockout.

[0024] In one embodiment of the cell or population thereof, the cell comprising a CD38 knockout further comprises a partial or complete peptide of a cell surface expressed exogenous cytokine or its receptor, which may include 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) IL15 The CAR may comprise at least one of: (i) an IL15 / IL15Rα fusion protein in which the intracellular domain of Rα is truncated; (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 common receptor γC, where common receptor γC is native or modified; (vii) a homodimer of IL15Rβ, where any one of (i)-(vii) may be co-expressed with the CAR in a separate construct or in a bicistronic construct. In some embodiments, partial or complete peptides of exogenous cytokines or receptors on the cell surface are transiently expressed in the cells provided herein.

[0025] In another embodiment of the cell or population thereof, the cell comprises a partial or complete peptide of a cell surface expressed exogenous cytokine or receptor, and the cytokine may include at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors. In embodiments of cells or populations thereof comprising an IL15 cytokine or receptor, the cells may comprise at least one of: (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 in which the intracellular domain of IL15Rα has been truncated; (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 common receptor γC, where common receptor γC is native or modified; or (vii) a homodimer of IL15Rβ, where any one of (i)-(vii) may be co-expressed with the CAR in a separate construct or in a bicistronic construct. In some embodiments, partial or complete peptides of exogenous cytokines or receptors on the cell surface are 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 at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:17, 19 or 21.In one embodiment of a cell or population thereof, the cell comprising IL15Δ is a cell that is a member of a cell line comprising 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), cell surface expression of an exogenous cytokine or a partial or complete peptide of its receptor (wherein the cytokine is not IL15), at least one of the genotypes listed in Table 1, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and a chromosome. The cells or populations thereof may further comprise one or more of a deletion or reduced expression of at least one of any gene in the 6p21 region, and an introduced or increased expression of at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptor, engager, and surface triggering receptor for binding with a bispecific or multispecific or universal engager. In embodiments of cells or populations thereof comprising both IL15Δ and a CAR, IL15Δ may be co-expressed with the CAR in a separate construct or in a bicistronic construct.

[0026] In one embodiment of the cell or population thereof, the CD38 knockout or IL15Δ comprising cells are induced NK cells or induced T cells, and the induced NK cells are capable of recruiting and / or migrating T cells to a tumor site, where the induced NK cells or induced T cells are capable of reducing tumor immune suppression 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 any of (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0027] Another aspect of the present application provides compositions comprising any of the cells or populations thereof described above and throughout the present application. In some embodiments, the iPSCs or iPSC-derived cells (derived cells) may comprise any one of the genotypes listed in Table 1 of the present application. In some embodiments, the iPSCs or derived cells therefrom comprise CD38 knockout (CD38- / -). In some embodiments, the iPSCs or derived cells therefrom comprise IL15Δ. In some embodiments, the iPSCs or derived cells therefrom comprise hnCD16 and CD38 knockout. In some embodiments, the iPSCs or derived cells therefrom comprise hnCD16 and IL15Δ. In some embodiments, the iPSCs or derived cells therefrom comprise hnCD16, CD38 knockout, and IL15Δ. In some embodiments, the iPSCs or derived cells therefrom comprise hnCD16, CD38- / -, and CAR. In some embodiments, the iPSCs or derived cells therefrom comprise hnCD16, IL15Δ, and CAR. In some embodiments, the iPSC or derived cells comprise hnCD16, IL15Δ, CD38− / −, and CAR. In some embodiments, the iPSC or derived cells comprise hnCD16, CD38− / −, CAR, and cell surface expression of a partial or complete peptide of an exogenous cytokine or its receptor, as described above and throughout this 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 a CAR, the CAR is specific for CD269 (BCMA). In yet some other embodiments, the CAR is specific for 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, an antigen 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 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), 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) ), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal 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 any one of a variety of pathogen antigens known in the art.

[0028] Thus, a further aspect of the present application provides compositions for therapeutic use that include, in addition to any of the induced cells provided herein, one or more therapeutic agents. In some embodiments of compositions for therapeutic use, the therapeutic agent includes 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). In some embodiments of compositions for therapeutic use, the checkpoint inhibitors used with the provided cells comprise 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, Foxp1, 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 compositions for therapeutic use, the checkpoint inhibitors used with the provided cells include one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof. In some other embodiments of the compositions for therapeutic use, the checkpoint inhibitors used with the provided cells include at least one of atezolizumab, nivolumab, and pembrolizumab. In some embodiments of the compositions for therapeutic use, the therapeutic agent includes one or more of venetoclax, azacitidine, and pomalidomide.

[0029] In some embodiments of the compositions for therapeutic use, the antibodies used with the provided cells include 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 compositions for therapeutic use, the antibodies used with the provided cells include 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. In yet some other embodiments of the compositions for therapeutic use, the antibody used with the provided cells comprises daratumumab.

[0030] The present application also provides therapeutic uses of the cells or therapeutic compositions 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 hematological malignancy, a solid tumor, cancer, or a viral infection.

[0031] A further aspect of the present application provides a method of producing the induced cells described herein, comprising differentiating iPSCs comprising CD38 knockout or IL15Δ, and optionally one or more of the following: (i) B2M null or low, (ii) CIITA (iii) introduced expression of HLA-G or non-cleavable HLA-G, (iv) high affinity non-cleavable CD16 (hnCD16) or a variant thereof, (v) a chimeric antigen receptor (CAR), (vi) cell surface expression of an exogenous cytokine or a partial or complete peptide of 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, and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Induced or increased expression of at least one of the following surface triggering receptors for binding to R, CAR, TCR, Fc receptor, engager, and bispecific or multispecific or universal engager.

[0032] In some embodiments of the method of production, the method further comprises genomic engineering of the clonal iPSCs to knock out CD38 or knock in IL15Δ, and optionally knock out B2M and CIITA, or introduce expression of HLA-G or non-cleavable HLA-G, high affinity non-cleavable CD16 or variants thereof, CAR, and / or partial or complete peptide of cell surface expression of exogenous cytokine or its receptor, wherein the CAR and partial or complete peptide of cell surface expression of exogenous cytokine or its receptor are co-expressed in separate constructs or bicistronic constructs. In some embodiments of the method of production, the genomic engineering of the iPSCs comprises targeted editing. In some embodiments, the targeted editing comprises deletion, insertion, or indel. In some embodiments, the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0033] The present application further provides CRISPR-mediated editing of clonal iPSCs, thereby generating edited clonal iPSCs comprising CD38 knockout or IL15Δ knockin, or at least one of the genotypes listed in Table 1. In some embodiments of CRISPR-mediated editing, the obtained CD38 knockout is biallelic. In some embodiments of CRISPR-mediated editing, the CD38 knockout is a nucleic acid cleavage between a first and a second target sequence, and the targeting sequences comprise SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In some embodiments of CRISPR-mediated editing, the obtained IL15Δ knockin comprises 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. In some embodiments of the above CRISPR-mediated editing, the editing further comprises the 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.

[0034] An additional aspect of the present application provides a method of improving anti-CD38 antibody therapy, comprising 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 thereof, functional equivalents, and biosimilars thereof. In some embodiments, effector cells provided for the method of improving anti-CD38 antibody therapy comprise induced hematopoietic cells including induced NK cells or induced T cells, where the induced NK cells or induced T cells comprise a CD38 knockout, a high affinity non-cleavable CD16 or variant thereof, and optionally (i) a B2M and CIITA knockout, (ii) introduced expression of HLA-G or non-cleavable HLA-G, a CAR, and / or a partial or complete peptide of a cell surface expressed exogenous cytokine or its receptor, where the CAR and the partial or complete peptide of a cell surface expressed exogenous cytokine or its receptor are co-expressed in separate or bicistronic constructs, 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 anti-CD38 antibody-induced effector cell loss in a subject under such treatment.

[0035] Yet another aspect of the present application provides a method of reducing or preventing allorejection against allogeneic effector cells by using a CD38-specific antagonist, wherein the allogeneic effector cells comprise a CD38 knockout, and the CD38-specific antagonist can suppress 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 any of daratumumab, isatuximab, or MOR202, or their humanized or Fc-modified variants or fragments, functional equivalents, and biosimilars. In yet another embodiment, the anti-CD38 antibody is daratumumab, and the methods provided herein provide a novel use of daratumumab.

[0036] A further aspect of the present application provides a construct for modifying a cell to obtain a genome-edited cell, comprising: (i) a left homology arm and a right homology arm targeting CD38 at a selected position of the CD38 locus (LHA / CD38, RHA / CD38) in the genome of the cell, which is (ii) operably linked to one or more nucleic acid sequences encoding one or more exogenous proteins, wherein the genome-edited cell is an induced pluripotent cell (iPSC), a clonal iPSC, or a cell of an iPS cell line, wherein the genome-edited cell comprises targeted integration of one or more nucleic acid sequences encoding one or more exogenous proteins at a selected position of the CD38 locus, and a CD38 knockout, and wherein the genome-edited cell is an iPSC capable of differentiating into an iPSC-derived hematopoietic cell. In one embodiment of the construct, one or more nucleic acid sequences encoding one or more exogenous proteins are operably linked to and driven by the endogenous promoter of CD38 upon integration, or at selected positions in the CD38 locus, in an exon of CD38.

[0037] In some embodiments of the construct, the one or more exogenous proteins comprise at least one or both of the following: (i) high affinity non-cleavable CD16 (hnCD16) or a variant thereof, (ii) exogenous IL15 / IL15 receptor fusion protein (IL15RF) or a variant thereof expressed on the cell surface. Thus, the genome edited cells and derived hematopoietic cells therefrom comprise one or more nucleic acid sequences encoding hnCD16 and IL15RF, or variants thereof. In some embodiments of the construct, hnCD16 or a variant thereof comprises at least one of the following: (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-native stimulatory domain, and (g) transmembrane, signaling, and stimulatory domains not derived from CD16 and derived from the same or a different polypeptide. In some other embodiments of the construct, IL15RF or a variant thereof comprises at least one of the following: (a) co-expression of IL15 and IL15Rα using a self-cleaving peptide; (b) a fusion protein of IL15 and IL15Rα; (c) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα has been truncated; (d) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα; (e) a fusion protein of IL15 and IL15Rβ; (f) a fusion protein of IL15 and common receptor γC, where common receptor γC is native or modified; (g) a homodimer of IL15Rβ; or (h) a polynucleotide encoding IL15RF comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 17, 19, or 21.In some embodiments of the high affinity non-cleavable CD16 (hnCD16) or variant thereof included in the construct, the hnCD16 comprises one or more of the following: (i) a non-native transmembrane domain derived from a CD3D, CD3E, CD3G, CD3zeta, 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; (ii) a non-native stimulatory domain derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide; (iii) a non-native signaling domain derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide; or (iv) 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.

[0038] In some embodiments, the genome edited cells and induced hematopoietic cells obtained from using the provided constructs further comprise a chimeric antigen receptor (CAR), the CAR having at least one of the following characteristics: (i) T cell specific or NK cell specific, (ii) bispecific antigen binding CAR, (iii) switchable CAR, (iv) dimerized CAR, (v) split CAR, (vi) multi-chain CAR, (vii) inducible CAR, (viii) co-expressed with another CAR, (ix) co-expressed with a cell surface expressed exogenous cytokine or partial or complete peptide of its receptor, optionally in a separate construct or in a bicistronic construct, (xi) co-expressed with a checkpoint inhibitor, (xii) encoded by a nucleic acid further comprised in the provided construct, wherein the CAR is inserted at a selected position in the CD38 locus, (xiii) inserted in the TRAC locus and / or driven by the endogenous promoter of the TCR and / or the TCR is knocked out by the CAR insertion, (xii) i) specific for CD19 or BCMA, and / or (xiv) ADGRE2, carbonic anhydrase IX (CAlX), 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, 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),Specific for any one of the following: 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), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal 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.

[0039] In another embodiment of the construct, the construct further comprises one or more of the following: (i) an exogenous promoter driving expression of one or more nucleic acid sequences encoding one or more exogenous proteins, where the exogenous promoter is constitutive, inducible, time-specific, tissue-specific, and / or cell-type specific; (ii) a linker sequence inserted between the two nucleic acid sequences encoding the exogenous proteins; (iii) an insulator at the 3' end of LHA / CD38 and an insulator at the 5' end of RHA / CD38; and (iv) a polyA signal. In some embodiments of the construct, the exogenous promoter may be any one of CMV, EF1α, PGK, CAG, or UBC, or the linker sequence is a 2A sequence encoding a self-cleaving 2A peptide, or the linker sequence is an internal ribosome entry sequence (IRES).

[0040] In some embodiments of the constructs, the resulting genome-edited cells that are iPSCs or iPSC-derived hematopoietic cells have one or more of the following phenotypes: (i) contain longer telomeres compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue; (ii) are CD38 - / - CD16, (iii)CD38- / - IL15, and / or (iv) CD38 - / - In some embodiments of the construct, the resulting genome-edited cells or iPSC-derived hematopoietic cells that are iPSCs further comprise one or more of the following: (i) B2M null or low, (ii) CIITA null or low, (iii) introduced expression of HLA-G or uncleaved HLA-G, (iv) deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region, and (v) HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Induced or increased expression of at least one of R, CAR, Fc receptor, engager, and surface triggering receptor for binding with bispecific or multispecific or universal engager.

[0041] In some embodiments of the construct, the hematopoietic cells derived from differentiating the resulting genome-edited iPSCs 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. In one embodiment, the resulting hematopoietic cells derived from genome-edited iPSCs, including induced NK or induced T cells, have at least one characteristic, when compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue, including: (i) improved persistence and / or survival, (ii) increased resistance to native immune cells, (iii) increased cytotoxicity, (iv) improved tumor penetration, (v) enhanced or acquired ADCC, (vi) enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site, (vii) enhanced ability to reduce tumor immune suppression, (viii) improved ability to rescue tumor antigen escape, and (ix) reduced fratricide in the presence of an anti-CD38 antibody or CD38 CAR. In some embodiments, the anti-CD38 antibody is any of daratumumab, isatuximab, or MOR202, or humanized or Fc-engineered variants or fragments, functional equivalents, and biosimilars thereof.

[0042] Also provided in the present application is an expression vector comprising the construct described herein.Thus, further provided is a host cell comprising the construct or the expression vector described herein.Further provided is a composition for producing genome-edited iPSCs or derived hematopoietic cells thereof, the composition comprising the construct or expression vector described herein.Further provided is a kit, the kit comprising the construct, expression vector, or host cell described herein, and one or more media for culturing, maintaining, or differentiating iPSCs.Further included are the construct, expression vector, and host cell for pharmaceutical use.

[0043] An additional aspect of the present application provides a method for producing genome-edited iPSCs or hematopoietic cells derived therefrom using the above described constructs, the method comprising (i) introducing the construct into iPSCs, and (ii) incubating the iPSCs for a sufficient time to allow targeted integration of one or more nucleic acid sequences encoding one or more exogenous proteins at the CD38 locus, thereby knocking out the expression of CD38 in the iPSCs, to obtain genome-edited iPSCs. In some embodiments, the method may further comprise inducing differentiation of the resulting genome-edited iPSCs into hematopoietic cells, including targeted integration of one or more nucleic acid sequences encoding one or more exogenous proteins at the same CD38 locus as the genome-edited iPSCs and CD38 knockout. In some embodiments of the method, the method further comprises introducing CRISPR-cas / gNA (guide nucleic acid), ZFN, TALEN, homing nuclease, or any other functional mutation thereof. In some embodiments, genome-edited iPSCs obtained using the above methods have improved genomic stability over iPSCs that undergo integration and knockout in separate stepwise editing events.

[0044] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description, taken in conjunction with the accompanying drawings, in which are set forth by way of illustration and example certain embodiments of the invention. [Brief description of the drawings]

[0045] [Figure 1] Illustrative of several construct designs for cell surface expression of cytokines in iPSC-derived cells. IL15 is used as an illustrative example that can be replaced with other desired cytokines. [Diagram 2] Phenotypic profiling of CD38- / -induced NK cells obtained from each of the three CD38- / - iPSC clones using flow cytometry is shown. [Diagram 3]We show that CD38- / - iPSC-induced NK cells exhibit the same cytotoxic capacity as wild-type iPSC-derived NK cells. [Figure 4] Graphical representation of flow cytometry of mature iPSC-derived NK cells showing stepwise manipulation of hnCD16 expression, B2M knockout (loss of HLA-A2 expression), HLA-G expression, and IL-15 / IL-15ra (LNGFR) construct expression. [Diagram 5] Graphical representation of telomere length determined by flow cytometry, showing that iPSC-derived mature induced NK cells maintain longer telomeres compared to adult peripheral blood NK cells. [Figure 6] We show that CD38- / - iPSC-derived NK cells maintain ADCC (antibody-dependent cell-mediated cytotoxicity) function when stimulated with the tumor cell line RPMI-8266 in the presence of the CD38 antibody daratumumab. [Figure 7] FIG. 7 shows that compared to CD38-expressing iPSC-derived NK cells (A), the viability of CD38- / - iPSC-derived NK cells (B) is maintained for at least about 48 hours in culture in the presence of the CD38 antibody, daratumumab. [Figure 8] Figure 7 shows that CD38- / - iPSC-derived NK cells (lower panel) did not exhibit degranulation and produced less cytokines than CD38-expressing iNK cells (upper panel) when stimulated in the presence of the CD38 antibody daratumumab. [Figure 9] FIG. 7 shows the introduction of hnCD16 into iPSC-derived NK cells (A) and iPSC-derived NK cells with both exogenous hnCD16 and CD38 knockout (B). [Figure 10] Phenotypic and functional analysis of hnCD16-CD38- / - induced NK cells compared to hnCD16 induced NK cells: (A) NKG2A expression by flow cytometry, (B) NKp46 expression by flow cytometry, (C) KIR2DL2 / 3 expression by flow cytometry, (D) calcium flux by flow, (E) ADCC against the HER2 expressing ovarian cell line SKOV3 by Incucyte™ live cell imaging. [Figure 11] Daratumumab-mediated NK cell fratricide in induced NK cells with or without CD38 knockout compared to peripheral blood NK cells. The specific cytotoxicity of daratumumab against different NK cell populations was measured after 4 hours incubation of the indicated iNK cells in the presence of increasing concentrations of daratumumab. [Figure 12] Enhanced daratumumab anti-myeloma activity mediated by CD38 null hnCD16-induced NK cells. The indicated iNK cells were incubated with MM.1S myeloma target cells for 18 hours, after which tumor cell viability was assessed by flow cytometry with Annexin V and live / dead viability markers. [Figure 13] Figure 1 shows that hnCD16 CD38- / - iNK cells exhibit enhanced and long-term daratumumab-induced anti-myeloma activity and persistence. (A) Tumor cell clearance as measured by the number of target cells remaining at the end of the 7-day cytotoxicity assay against RMPI-8226 tumor spheroids. (B) Lack of NK cell fratricide improved survival of hnCD16 CD38- / - iNK cells over the 7-day cytotoxicity assay. [Figure 14] We show that iNK cells lacking CD38 have a more durable ADCC with increased potential for sequential killing in the presence of daratumumab. [Figure 15] iNK cells transduced with full-length IL15 / IL15Rα fusion constructs (closed circles; positive control) or truncated IL15 / IL15Rα fusion constructs lacking the cytoplasmic signaling domain (open circles) had a survival advantage compared to untransduced or GFP-transduced cells in the same cultures, independent of exogenous soluble IL2. A: in the presence of exogenous IL2; B: in the absence of exogenous IL2. [Figure 16]Figures 16A-16D show various compositions of CD38-targeted transgene knock-in constructs (A and B vs. C and D) with one or more transgenes driven by an exogenous promoter or the CD38 endogenous promoter (B and D vs. A and C) to generate CD38- / - transgene+ pluripotent stem cells and effector cells derived therefrom. [Figure 17-1] FIG. 1 shows exemplary nucleic acid sequences included in an exogenous promoter-driven CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct for generating CD38- / -CD16 IL15 effector cells derived from engineered pluripotent stem cells using the construct and variants thereof. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above. [Figure 18-1] FIG. 1 shows exemplary nucleic acid sequences included in a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct driven by the CD38 endogenous promoter to generate CD38- / -CD16+IL15+ effector cells derived from engineered pluripotent stem cells using the construct and variants thereof. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 19] 1 shows flow cytometry phenotyping of unmanipulated iNK cells or CD38- / -CD16 IL15 iNK cells engineered with a CD38-targeting IL15 / IL15ra-2A-hnCD16 knock-in construct. [Figure 20] We show that CD38- / -CD16 IL15 iNK cells derived from iPSCs engineered with a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct have improved in vitro persistence compared to non-engineered iNK (FTi250). Cells were cultured for 4 days in the absence of IL-2 or IL-15 and the percentage of input cells was measured on days 2 and 4. [Figure 21] Figure 1 shows that CD38- / -CD16 IL15 iNK cells derived from iPSCs engineered with a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct avoid daratumumab-induced fratricide compared to non-engineered iNKs. Rituximab was used as a control antibody. [Figure 22] We show that CD38- / -CD16 IL15 iNK cells derived from iPSCs engineered with a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct have superior ADCC against Raji cells in combination with daratumumab antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Genomic modifications of iPSCs (induced pluripotent stem cells) include polynucleotide insertions, deletions, and substitutions. Exogenous gene expression in genomically engineered iPSCs often encounters problems such as gene silencing, or reduced gene expression after long-term clonal expansion of the original genomically engineered iPSCs, after cell differentiation, and in dedifferentiated cell types from cells derived from genomically engineered iPSCs. Meanwhile, it is difficult to directly engineer primary immune cells such as T cells or NK cells, which poses obstacles 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 incorporating one or more exogenous genes, including suicide genes and other functional modalities, which confer improved therapeutic properties with respect to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, longevity, self-renewal, persistence, and / or survival, into iPSC-derived cells, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T cells, NKT cells, and NK cells.

[0047] definition Unless otherwise 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 otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0048] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0049] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0050] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0051] The term "and / or" should be understood to mean either one or both of the alternatives.

[0052] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0053] As used herein, the term "substantially" or "essentially" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "substantially the same" or "essentially the same" refers to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about identical to the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0054] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that is free of a particular substance or source thereof, such as, for example, 95% free, 96% free, 97% free, 98% free, 99% free, etc., of the particular substance or source thereof, or is undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance is (1) not included in the composition at any concentration, or (2) included in the composition but at a low density and functionally inactive. A similar meaning may be applied to the term "absent," which refers to the absence of a particular substance or source thereof in a composition.

[0055] Throughout this specification, unless the context requires otherwise, "comprise", "comprises", and "comprising" will be understood to mean the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include", "having", "contain", and "comprise" are used interchangeably.

[0056] "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present.

[0057] "Consisting essentially of" means the inclusion of any elements listed following the phrase, limited to other elements that do not interfere with or contribute to the activity or operation specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or operation of the recited elements.

[0058] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such 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.

[0059] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of the body, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures include live cells or tissues taken from a living organism and cultured in a laboratory setup, usually under sterile conditions, typically for a few hours or up to about 24 hours, but up to 48 or 72 hours or more depending on the circumstances. In certain embodiments, such tissues or cells may be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using live cells or tissues are typically considered to be "in vitro", although in certain embodiments, the term can be used interchangeably with ex vivo.

[0060] The term "in vivo" generally refers to activities that take place inside a living organism.

[0061] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in a non-reprogrammed state.

[0062] As used herein, the term "differentiation" refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as, for example, blood or muscle cells. Differentiated or differentiation-induced cells are cells that assume a more specialized ("committed") position within the lineage of a cell. The term "committed" as applied to the process of differentiation refers to a cell that has progressed down a differentiation pathway to a position where it will continue to differentiate into a particular cell type or subset of cell types under normal circumstances and cannot, under normal circumstances, differentiate into a different cell type 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 an 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, ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs), which are unable to give rise to an entire organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells), which are able to give rise to an entire organism.

[0063] As used herein, the term "induced pluripotent stem cells" or iPSCs means that stem cells have been induced or altered, produced from differentiated adult, neonatal or fetal cells, i.e., reprogrammed into cells that can differentiate into all tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The produced iPSCs do not refer to naturally occurring cells.

[0064] As used herein, the term "embryonic stem cell" refers to the naturally occurring pluripotent stem cells of the inner cell mass of blastocyst. Embryonic stem cells are pluripotent and generate the derivatives of all three major germ layers, ectoderm, endoderm, and mesoderm. They do not contribute to extraembryonic membranes or placenta, i.e., they are not totipotent.

[0065] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate 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 hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell forms 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 a state of a cell that has a degree of developmental potential that is less than totipotency and pluripotency.

[0066] Pluripotency can be determined, in part, by assessing the pluripotency characteristics of a cell, including, but not limited to, (i) pluripotent stem cell morphology, (ii) unlimited self-renewal potential, (iii) 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) ability to differentiate into all three somatic lineages (ectoderm, mesoderm, endoderm), (v) formation of teratomas composed of the three somatic lineages, and (vi) formation of embryoid bodies composed of cells of the three somatic lineages.

[0067] Two types of pluripotency have been described so far: a "primed" or "metastable" state of pluripotency, similar to epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "ground" state, similar to the pluripotent early / inner cell mass of preimplantation blastocysts. While both pluripotent states exhibit characteristics as described above, the naive or ground state additionally exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival during single cell culture, (iii) an overall reduction in DNA methylation, (iv) reduced H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters, and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard methodologies of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, generally appear to have the characteristics of a primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in a primed state and exhibit characteristics of the ground state unless exogenous transgene expression is maintained.

[0068] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape, with a high nucleus-to-cytoplasm ratio, prominent presence of nucleoli, and typical intercellular spacing.

[0069] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0070] "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, but are not limited to, 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.

[0071] "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 each case singular "medium"), "supplements" and "medium supplements" refer to nutritional compositions in which cell cultures are cultivated.

[0072] "Culturing" or "maintaining" refers to sustaining, propagating (growing), and / or differentiating tissue or cells outside the body, for example, in sterile plastic (or coated plastic) cell culture dishes or flasks. "Culturing" or "maintaining" can utilize culture medium as a source of nutrients, hormones, and / or other factors that aid in the growth and / or maintenance of the cells.

[0073] As used herein, the term "mesoderm" refers to one of three germ layers that emerge during early embryonic development and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0074] As used herein, the term "secondary hemogenic endothelium" (HE) or "pluripotent stem cell-derived secondary hemogenic endothelium" (iHE) refers to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial hematopoietic conversion. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm through hemangioblasts to secondary hemogenic endothelial cells and hematopoietic progenitor cells.

[0075] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including multipotent hematopoietic stem cells (blood cells), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent 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) lines. As used herein, the term "secondary hematopoietic stem cells" refers to CD34+ hematopoietic cells that can give 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.

[0076] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cell that completes maturation in the thymus and has various roles in the immune system, including identifying specific foreign antigens in the body, and activating and inactivating other immune cells. The T cell can be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. The T cell can be a CD3+ cell. T cells can be any type of T cell and can be at any stage of development, including, but not limited to, 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 (γδ T cells), and the like. 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 or progenitor cells.

[0077] "CD4+ T cells" refers to a subset of T cells that express CD4 on their surface and are associated with cellular immune responses. They are characterized by their secretory profile after stimulation, which may include 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 for 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 a relevant recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, it defines a subset of helper / inducer.

[0078] "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 the associated recognition element of major histocompatibility complex class I restricted interactions.

[0079] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56+, express at least one of NKG2C and CD57, and optionally CD16, but lack expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, an isolated subpopulation of CD56+ NK cells comprises expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. CD56+ may be dim or bright expression.

[0080] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which 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 very limited TCR repertoire - a canonical α chain (Vα24-Jα18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, presents a more heterogeneous use of TCRαβ. Type I NKT cells are considered to be suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0081] 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 the isolated cell is substantially free of at least one component found in the environment in which the "non-isolated" reference cell resides. The term includes cells that have been removed from some or all components as found in their natural environment, e.g., isolated from a tissue or biopsy sample. The term also includes cells that have been removed from at least one, some, or all components as they are found in a non-native environment, e.g., 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, as found in nature or when grown, stored, or persisted in a non-native environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a medium that does not occur in nature. An isolated cell can be obtained from isolating a desired cell or population of cells from other substances or cells in the environment, or from removing one or more other cell populations or subpopulations from the environment.

[0082] As used herein, terms such as "purify" refer to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0083] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0084] A "construct" refers to a complex of polymers or molecules that includes a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct that can direct the delivery or transfer of foreign genetic material to a target cell and can replicate and / or express in the target cell. As used herein, the term "vector" includes the construct that is delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. 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.

[0085] "Integration" means that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence in the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted into the chromosome 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 the construct, with or without the deletion of endogenous sequences or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" can further include the replacement of the deleted nucleotide with the endogenous sequence or one or more inserted nucleotides.

[0086] 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. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the genetic material of the host, for example, by integrating into a chromosome of the host, or as non-chromosomal genetic material, for example, a plasmid. Thus, the term used in reference to the expression of an encoding nucleic acid refers to the introduction of the encoding 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 used in reference to the expression of an encoding nucleic acid, the term refers to the expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0087] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" 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. A gene of interest or a polynucleotide may include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may code for 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 that has less than 100% sequence identity with 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.

[0088] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide consists of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) in place of thymine when the polynucleotide is RNA. 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 polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to both double-stranded and single-stranded molecules.

[0089] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules 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, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, also commonly referred to in the art as polypeptides or proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0090] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment, where 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 a sense or antisense orientation.

[0091] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic attributes of source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can be further differentiated into specific cell types, including any hematopoietic lineage cells. Source cell-derived iPSCs, and cells differentiated therefrom, can be collectively referred to as "derived" 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, umbilical cord blood, or other donor tissues. As used herein, genetic imprints that confer preferential therapeutic attributes are incorporated into iPSCs by reprogramming selected source cells that are donor, disease, or therapeutic response specific, or by introducing genetic modification modalities into iPSCs using genome editing. In aspects of source cells obtained from specific selected donor, disease, or therapeutic contexts, genetic imprints that contribute preferential therapeutic attributes can include context-specific genetic or epigenetic that represent a retainable phenotype, i.e., preferential therapeutic attribute, that is passed on to derived cells of the selected source cells, regardless of whether the underlying molecular event has been identified. Donor, disease, or therapeutic response specific source cells can contain genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including, but not limited to, pre-arranged monospecific 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 the high affinity CD16 receptor of the selected donor, selected HLA-matched donor cells exhibiting predetermined HLA requirements, i.e., population-expanded haplotypes.As used herein, preferential therapeutic attributes include improved engraftment, trafficking, homing, viability, self-renewal, persistence, regulation and modulation of immune responses, survival, and cytotoxicity of the derived cells. Preferential therapeutic attributes may also be related to expression of antigen targeting receptors, HLA presentation or lack thereof, tolerance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity with reduced extratumoral effects, and resistance to treatments such as chemotherapy.

[0092] As used herein, the term "enhanced therapeutic properties" refers to therapeutic properties of a cell that are enhanced compared to typical immune cells of the same general cell type. For example, NK cells with "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, trafficking, homing, viability, self-renewal, persistence, regulation and modulation of immune responses, viability, and cytotoxicity. Therapeutic properties of immune cells are also manifested by expression of antigen targeting receptors, HLA presentation or lack thereof, resistance to tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity with reduced extratumoral effects, and resistance to treatments such as chemotherapy.

[0093] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between an immune cell, e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil, and a tumor cell and activate the immune cell. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers, or multispecific killer cell engagers, or universal engagers that can be compatible with multiple immune cell types.

[0094] As used herein, the term "surface triggering receptor" refers to a receptor that can induce or initiate an immune response, e.g., a cytotoxic response. Surface triggering receptors can be engineered and expressed on effector cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, neutrophils. In some embodiments, the surface triggering receptor promotes bispecific or multispecific antibody binding between an effector cell and a specific target cell, e.g., a tumor cell, regardless of the effector cell's native receptor and cell type. Using this approach, it is possible to generate iPSCs that contain a universal surface triggering receptor and differentiate such iPSCs into a population of various effector cell types that express the universal surface triggering receptor. By "universal," it is meant that the surface triggering receptor can be expressed and activated on any effector cell, regardless of cell type, and all effector cells that express the universal receptor can bind or ligate to an engager that has the same epitope that can be recognized by the surface triggering receptor, regardless of the tumor-binding specificity of the engager. In some embodiments, an engager with the same tumor-targeting specificity is used to bind to the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to the universal surface triggering receptor. Thus, one or more effector cell types may be used to kill one specific type of tumor cell or two or more types of tumors. Surface triggering receptors generally contain a costimulatory domain for effector cell activation and an anti-epitope specific to the epitope of the engager. Bispecific engagers are specific to the anti-epitope of the surface triggering receptor on one end and specific to the tumor antigen on the other end.

[0095] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of a cell therapy. In some examples, expression of the safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells that have permanently incorporated a gene encoding the safety switch protein into their genome. This conditional regulation can vary and can include control by post-translational activation via small molecules and tissue-specific and / or transient transcriptional regulation. The safety switch can mediate 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, e.g., a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cell. 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, a prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product and kills the transduced cells.

[0096] As used herein, the term "pharmaceutical active protein or peptide" refers to a protein or peptide that can achieve a biological and / or pharmaceutical effect on an organism. A pharmaceutical active protein has curative or palliative properties against a disease and can be administered to improve, alleviate, alleviate, reverse or reduce the severity of the disease. A pharmaceutical active protein also has prophylactic properties and is used to prevent the onset of a disease or to reduce the severity of such a disease or pathological condition if it appears. A pharmaceutical active protein includes the entire protein or peptide or a pharmaceutical active fragment thereof. It also includes pharmaceutical active analogs of the protein or peptide or analogs of fragments of the protein or peptide. The term pharmaceutical active protein also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharmaceutical active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0097] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces, or enhances cell signaling. Signaling refers to the transmission of a molecular signal in the form of a chemical modification by the recruitment of protein complexes along a pathway that ultimately leads to a biochemical event in the 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 gate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0098] As used herein, the term "targeting modality" refers to molecules, e.g., polypeptides, that are genetically incorporated into cells to promote antigen and / or epitope specificity, including but not limited to: i) antigen specificity when associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when associated with a monoclonal antibody or bispecific engager; iii) targeting of transformed cells; iv) targeting of cancer stem cells; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0099] 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 opposed to non-specific or non-selective binding.

[0100] As used herein, the term "adoptive cell therapy" refers to cell-based immunotherapy, as used herein, that involves the infusion of autologous or allogeneic lymphocytes, identified as T or B cells, genetically modified or unmodified, that are expanded ex vivo prior to infusion.

[0101] As used herein, a "therapeutically sufficient amount" includes within its meaning a non-toxic but sufficient and / or effective amount of the particular treatment and / or pharmaceutical composition to which it refers, to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the stage and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to improve, alleviate, and / or ameliorate at least one symptom associated with the disease or condition of the subject being treated.

[0102] Differentiation of pluripotent stem cells requires changes in the culture system, including stimuli in the medium and changes in the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a common and important intermediate to initiate lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development, giving rise to multiple lineages within a three-dimensional area. Throughout the differentiation process, which is usually hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and grow into cystic EBs, at which point they are further processed to continue differentiation, typically days to weeks. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multi-layered cluster of cells, which is typically achieved by one of several methods, including settling the pluripotent cells as droplets, allowing the cells to settle into "U" bottom well plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require further differentiation cues to promote EB growth. Therefore, pluripotent stem cell aggregates need to be transferred to differentiation medium that provides cue induction to the lineage of choice. EB-based culture of pluripotent stem cells typically generates differentiated cell populations (ectoderm, mesoderm, and endoderm germ layers) with moderate proliferation within the EB cell clusters. Although EBs have been proven to promote cell differentiation, they give rise to heterogeneous cells with various differentiation states due to inconsistent exposure of cells in three-dimensional structures to differentiation cues from the environment. Moreover, EBs are laborious to create and maintain. Moreover, cell differentiation by EBs is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0103] In contrast, "aggregate formation", unlike "EB formation", can be used to expand a population of pluripotent stem cell-derived cells. For example, during aggregate-based pluripotent stem cell expansion, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates forming larger aggregates, and these aggregates can be routinely mechanically or enzymatically dissociated into smaller aggregates to maintain cell proliferation and increase cell numbers in culture. Unlike EB culture, cells cultured within aggregates in maintenance culture maintain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0104] As used herein, "monolayer differentiation" refers to a method of differentiation that is different from differentiation by three-dimensional multi-layered clusters of cells, i.e., "EB formation". Among other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Since monolayer culture does not mimic embryonic development such as EB formation, differentiation into specific lineages is considered minimal compared to all three germ layer differentiation of EBs.

[0105] As used herein, "dissociated" cells refer to cells that are substantially separated or purified from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from animals or tissues 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 a culture plate or other surface. Thus, dissociation includes disrupting the extracellular matrix (ECM) and cellular interactions with the substrate (such as the culture surface) or disrupting the ECM between cells.

[0106] As used herein, "feeder cells" or "feeders" is a term that refers to one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, proliferate, or differentiate, where the feeder cells provide stimuli, growth factors, nutrients, and support 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 irradiation or treatment with antimitotic agents such as mitomycin to prevent them from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type can be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation to specific lineages, enhance proliferation capacity, and promote maturation into specialized cell types, such as effector cells.

[0107] As used herein, a "feeder-free" (FF) environment refers to an environment, such as culture conditions, cell culture or culture medium, that is essentially free of feeder or stromal cells and / or has not been preconditioned by culturing feeder cells. A "preconditioned" medium refers to a medium that has been harvested after feeder cells have been cultured in the medium for a period of time, such as at least one day. Preconditioned medium contains many mediator substances, including growth factors and cytokines secreted from feeder cells cultured in the medium. In some embodiments, the feeder-free environment does not contain either feeder cells or stromal cells, nor has it been preconditioned by culturing feeder cells.

[0108] "Functional" as used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, refers to (1) successful knock-in, knock-out, knock-down gene expression, transgenic or controlled gene expression at the genetic level, such as inducible or transient expression at a desired stage of cellular development, achieved by direct genome editing or modification, or by "passage" through differentiation or reprogramming from an initially genomically engineered starting cell, or (2) (i) the successful knock-in, knock-out, knock-down gene expression at the genetic level, such as inducible or transient expression at a desired stage of cellular development, achieved by direct genome editing or modification, or by "passage" through differentiation or reprogramming from an initially genomically engineered starting cell, or (2) (ii) the successful knock-in, knock-out, knock-down gene expression at the genetic level, such as inducible or transient expression at a desired stage of cellular development, achieved by direct genome editing or modification, or by "passage" through differentiation or reprogramming from an initially genomically engineered starting cell, or (3) the successful knock-in, knock-out, knock-down gene expression at the genetic level, such as inducible or transient expression at a desired stage of cellular development, achieved by direct genome editing or modification, or by "passage" through differentiation or reprogramming from an initially genomically engineered starting cell, or (4) the successful knock-in, knock-out, knock-down gene expression at the genetic level, such as inducible or transient expression at a desired stage of cellular development, achieved by direct genome editing or modification, or by " refers to the removal, addition, or alteration of cellular function / characteristics at the cellular level due to (i) gene expression modifications obtained from a cell, (ii) gene expression modifications that are maintained in the cell upon "passage" through differentiation or reprogramming from an original genomically engineered starting cell, (iii) downstream gene regulation of the cell as a result of gene expression modifications that are only present during the early developmental stages of the cell or only in the starting cell that gives rise to the cell via differentiation or reprogramming, or (iv) enhanced or newly achieved cellular function or attribute exhibited in a mature cell product originally derived from genomic editing or modifications performed in an iPSC, progenitor cell, or de-differentiated cellular source.

[0109] "HLA-deficient," including HLA class I-deficient, or HLA class II-deficient, or both, refers to cells that lack or no longer maintain levels of surface expression of complete MHC complexes comprising HLA class I protein heterodimers and / or HLA class II heterodimers, or that have reduced, diminished or decreased levels below those naturally detectable by other cells or synthetic methods.

[0110] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that are further modified by introducing genes expressing proteins associated with improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as, but not limited to, 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. "Modified HLA-deficient" cells also include cells other than iPSCs.

[0111] "Fc receptors", abbreviated as FcR, are classified based on the type of antibody they recognize. For example, those that bind the most common class of antibody IgG are called Fc-gamma receptors (FcγR), those that bind IgA are called Fc-alpha receptors (FcαR), and those that bind IgE are called Fc-epsilon receptors (FcεR). Classes of FcR are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and therefore different antibody affinities, including FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0112] "Chimeric Fc receptor", abbreviated as CFcR, is a term used to describe engineered Fc receptors in which the native transmembrane and / or intracellular signaling domains have been modified or replaced with non-native transmembrane and / or intracellular signaling domains. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane and signaling domains being non-native, one or more stimulatory domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance receptor-induced cell activation, proliferation, and function. Unlike chimeric antigen receptors (CARs), which contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to Fc fragments, or Fc regions of antibodies, or Fc regions contained in engagers or binding molecules, and bind to molecules to activate cell functions, with or without bringing the target cell into close proximity. For example, Fcγ receptors can be engineered to contain selected transmembrane domains, stimulatory domains, and / or signaling domains in the intracellular region that respond to the binding of IgG at the extracellular domain, thereby generating CFcR. In one example, CFcR is produced by engineering CD16, an Fcγ receptor, by replacing its transmembrane and / or intracellular domain. To further improve the binding affinity of 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 that include high affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or the extracellular domain of the receptor is replaced to be non-cleavable, i.e., not shed, thereby obtaining hnCD16-based CFcR.

[0113] Two isoforms of the FcγR receptor CD16 have been identified: Fc receptors 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 NK cells and promote antibody-dependent cellular 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 upon NK cell activation is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. 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) has been altered or eliminated do not shed. The cleavage site and membrane proximal region are described in detail in WO2015 / 148926, the full disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high affinity and non-cleavable. Another exemplary high affinity non-cleavable CD16 (hnCD16) variant is an engineered CD16 that contains an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0114] I. Cells and Compositions Useful for Adoptive Cell Therapy with Enhanced Properties Provided herein is a strategy to systematically manipulate the regulatory circuitry of clonal iPSCs without affecting the differentiation potential of iPSCs and the cellular developmental biology of iPSCs and their derived cells while enhancing the therapeutic properties of the derived cells. The derived cells are functionally improved and suitable for adoptive cell therapy after a combination of selective modalities is 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 gene edits provided still have the ability to enter cellular development while retaining coordinated activity, and / or to mature and generate functional differentiated cells. Unexpected failures during directed cell differentiation from iPSCs are due to aspects including, but not limited to, developmental stage specific gene expression or lack thereof, requirement for HLA complex presentation, protein shedding of the introduced surface expression modalities, and the need for reconstitution of the differentiation protocol to allow for alteration of the phenotype and / or function of the cells. The present application has demonstrated that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from individual or combined genomic modifications that are retained in the effector cells following iPSC differentiation.

[0115] 1.CD38 knockout The cell surface molecule CD38 is highly upregulated in multiple hematological malignancies of both lymphoid and myeloid origin, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated cancer cell depletion usually results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cellular cytotoxicity). In addition to ADCC, immune effector mechanisms in conjunction with therapeutic antibodies may also include phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0116] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells, as well as on NK cells, activated T cells, and B cells. During hematopoiesis, CD38 is expressed in association with CD34 + It is expressed in stem cells and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, as well as in the final stages of maturation that last up to the plasma cell stage. CD38, a type II transmembrane glycoprotein, serves cellular functions both as a receptor and as a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 mediates the synthesis and upregulation of NAD + CD38 catalyzes the hydrolysis of cadmium to ADP-ribose, thereby generating the second messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which is calcium-dependent and important for the process of cell adhesion. CD38 recognizes CD31 as a receptor and regulates cytokine release and cytotoxicity of activated NK cells. CD38 associates with cell surface proteins in lipid rafts and mediates the release of cytoplasmic Ca. 2+ It has also been reported to regulate flux and mediate signaling in lymphoid and myeloid cells.

[0117] In the treatment of malignant tumors, the systemic use of T cells transduced with CD38 antigen-binding receptors results in lysis of the CD38+ fraction of CD34+ hematopoietic progenitors, monocytes, NK cells, T cells, and B cells, impairing immune effector cell function in the recipient, resulting in incomplete therapeutic responses and reduced or eliminated efficacy. Furthermore, in multiple myeloma patients treated with the CD38-specific antibody daratumumab, a reduction in NK cells was observed in both bone marrow and peripheral blood, whereas other immune cell types such as T cells and B cells were unaffected 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 on activated lymphocytes such as T and B cells, inhibiting activation of these lymphocytes using CD38-specific antibodies such as daratumumab in recipients of allogeneic effector cells reduces and / or prevents allorejection of these effector cells, thereby increasing effector cell survival and persistence. Thus, the present application also provides strategies to enhance effector cell persistence and / or survival through reducing or preventing allorejection by using CD38-specific antibodies, secreted CD38-specific engagers or CD38 CARs (chimeric antigen receptors) for activation of recipient T and B cells. Specifically, the provided strategies include generating CD38 knockout iPSC lines and inducing differentiation of the engineered iPSC lines to CD38 null (CD38 - / - Prior to this application, given that CD38 plays many important roles in cell developmental biology and cell function as discussed above, it was unclear whether disrupting CD38 in iPSCs would disrupt aspects including iPSC differentiation, induced cell phenotype, and effector cell function.

[0118] 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 can undergo directed differentiation to produce functional derived hematopoietic cells, including but not limited to mesodermal cells with definitive hemogenic 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. In some embodiments, when anti-CD38 antibodies are used to induce ADCC or anti-CD38 CARs are used for targeted cell killing, CD38 - / - The iPSCs and / or their derived effector cells are not eliminated by anti-CD38 antibodies or anti-CD38 CARs, thereby increasing the persistence and / or viability of the 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 and / or after exposure to such therapeutic agents. In some embodiments, the CD38 null effector cells are NK cells derived from the iPSCs. In some embodiments, the CD38 null effector cells are T cells derived from the iPSCs. In some embodiments, the CD38 null iPSCs and derived cells include one or more additional genome edits as described herein, including but not limited to hnCD16 expression, CAR expression, cytokine / cytokine receptor expression, HLAI and / or HLAII knockout, and additional modalities provided.

[0119] 2.hnCD16 knock-in CD16 has been identified as two isoforms, the Fc receptors 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 cellular 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 NK cell activation is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V (also referred to as F158V in some publications) is an exemplary polymorphic variant of CD16 with high affinity, while the S197P variant is an exemplary engineered non-cleavable version of CD16. Engineered CD16 variants including both F176V and S197P have high affinity and are non-cleavable, as described in more detail in WO2015 / 148926, the full disclosure of which is incorporated herein by reference. Furthermore, a chimeric CD16 receptor in which the ectodomain of CD16 is essentially replaced with at least a portion of the ectodomain of CD64 can also achieve the desired high affinity and non-cleavable function of a CD16 receptor capable of ADCC. In some embodiments, the replaced 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).

[0120] Thus, in some embodiments, the high affinity non-cleavable CD16 receptor (hnCD16) comprises both F176V and S197P, and in some embodiments, F176V and the cleavage region is 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 therebetween, identity when compared to any of the exemplary sequences SEQ ID NOs: 7, 8, and 9, each of which comprises at least a portion of the CD64 ectodomain. SEQ ID NOs: 7, 8, and 9 are encoded by exemplary sequences SEQ ID NOs: 10-12, respectively. As used herein and throughout this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm recognized in the art.

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[0127] Thus, provided herein are clonal iPSCs engineered to contain a high affinity non-cleavable CD16 receptor (hnCD16), among other editing contemplated and described herein, which can differentiate into effector cells that contain hnCD16 introduced into the iPSC. In some embodiments, the induced effector cells that contain hnCD16 are NK cells. In some embodiments, the induced effector cells that contain hnCD16 are T cells. The exogenous hnCD16 expressed in the iPSCs or its derived cells exhibits high affinity for binding to ADCC antibodies or fragments thereof as well as bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domains of CD16 or CD64 of said hnCD16. Bispecific, trispecific, or multispecific engagers or binders are further described below in this application (see Section I.7). Thus, the present application provides induced effector cells, or populations of cells thereof, pre-loaded with one or more preselected ADCC antibodies via high affinity binding to the extracellular domain of hnCD16 expressed on the induced effector cells, said hnCD16 comprising the extracellular binding domain of CD64, or of CD16 having F176V and S197P, in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection as further detailed in Section V below.

[0128] In some other embodiments, the native CD16 transmembrane and / or intracellular domains of hnCD16 are further modified or replaced to produce chimeric Fc receptors (CFcRs) that contain non-native transmembrane domains, non-native stimulatory domains, and / or non-native signaling domains. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor than the receptor that provides the extracellular domain. Exemplary herein, CFcRs based on CD16 or variants thereof do not have transmembrane domains, stimulatory domains, or signaling domains derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR comprises a non-native transmembrane domain derived from a CD3D, CD3E, CD3G, CD3zeta, 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 comprises a non-natural stimulatory / inhibitory domain derived from a 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 comprises a non-natural signaling domain derived from a CD3zeta, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of hnCD16, the chimeric receptor provided comprises a transmembrane domain and a signaling domain that are both derived from one of the following polypeptides: IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D.One particular 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, the extracellular domain of hnCD16 being derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, the extracellular domain of CD16 comprising F176V and S197P. Another embodiment of the hnCD16-based chimeric Fc receptor comprises the transmembrane domain and the signaling domain of CD3ζ, the extracellular domain of hnCD16 being derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, the extracellular domain of CD16 comprising F176V and S197P.

[0129] Various embodiments of hnCD16-based chimeric Fc receptors as described above can bind with high affinity to the Fc region of an antibody or fragment thereof, or to the Fc region of a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory and / or signaling domains of the chimeric receptor allow activation of effector cells and cytokine secretion, killing tumor cells targeted by the antibody or the bispecific, trispecific, or multispecific engager or binder having a tumor antigen binding component and an 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 ectodomain, the CFcR contributes to the killing ability of the effector cells and increases the proliferation and / or growth potential of the effector cells. The antibody and the engager can bring the antigen-expressing tumor cells and the CFcR-expressing effector cells into close proximity, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, or 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 hnCD16-based CFcR in attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0130] Unlike the endogenous CD16 receptor expressed by primary NK cells, which is cleaved from the cell surface following NK cell activation, the various non-cleavable versions of CD16 on induced NK cells avoid CD16 shedding and maintain constant expression. In induced NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cellular cytotoxicity (ADCC) and the binding of bi-, tri-, or multi-specific engagers. ADCC is a mechanism of NK cell-mediated lysis via binding of CD16 to antibody-coated target cells. The additional high affinity properties of hnCD16 introduced into induced NK cells also allow in vitro loading of ADCC antibodies via hnCD16 into NK cells prior to administration of the cells to a subject in need of cell therapy. As presented, hnCD16 may in some embodiments comprise F176V and S197P, or may comprise a complete or partial ectodomain derived from CD64 as exemplified by SEQ ID NO: 7, 8, or 9, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides induced NK cells or cell populations thereof preloaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in treating a condition, disease, or infection, as detailed in Section V below. In some embodiments, the NK cells comprising hnCD16 further comprise a CD38 knockout. In some embodiments, the induced NK cells comprising hnCD16 and a CD38 knockout are preloaded with an anti-CD38 antibody. In some embodiments, the preloaded anti-CD38 antibody is daratumumab.

[0131] Unlike primary NK cells, mature T cells derived from primary sources (i.e., native / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs with expressed exogenous uncleavable CD16 could be differentiated into functional T cells that not only express exogenous CD16 but also can perform functions through acquired ADCC mechanisms without compromising the developmental biology of T cells. This acquired ADCC in induced T cells can further be used as an approach to rescue antigen escape, which often occurs in dual targeting and / or CAR-T cell therapy, where tumors relapse with reduced or lost expression of CAR-T target antigens, or mutated antigens that evade recognition by the CAR (chimeric antigen receptor). If said induced T cells contain acquired ADCC via 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 relapse or recurrence of the target tumor, which is common in CAR-T therapy. Such a strategy of reducing and / or preventing antigen escape while achieving dual targeting can be similarly applied to NK cells expressing one or more CARs. The various CARs that can be used in this strategy of reducing and preventing antigen escape are described in detail below.

[0132] 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 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 ectodomain derived from CD64, as exemplified by SEQ ID NO: 7, 8, or 9, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As described, such induced T cells have an acquired mechanism to target tumors with monoclonal antibodies meditated by ADCC to enhance the therapeutic effect of the antibody. As disclosed, the present application also provides induced T cells, or cell populations thereof, preloaded with one or more preselected 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, so that the cells can avoid being eliminated when in the presence of a therapeutic agent that targets the tumor antigen CD38. In one embodiment, the therapeutic agent that targets the tumor antigen CD38 is an anti-CD38 antibody. In another embodiment, the therapeutic agent that targets the tumor antigen CD38 is a CAR that includes a CD38 binding region, for example, an anti-CD38 scFV.

[0133] 3. CAR expression Applicable to engineered iPSCs and their derived effector cells can be any CAR design known in the art. CARs, or chimeric antigen receptors, are fusion proteins that generally include an ectodomain, including an antigen recognition region, a transmembrane domain, and an endodomain. In some embodiments, the ectodomain can further include a signal peptide or leader sequence and / or a spacer. In some embodiments, the endodomain 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 CAR. In some embodiments, the CAR is NK cell specific, including an NK-specific signaling component. In certain embodiments, the 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 NK cells are derived from CAR-expressing iPSCs.

[0134] In certain embodiments, the antigen recognition region comprises 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.Non-limiting examples of antigens that can be targeted by a CAR 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, Cytoplasmic reticulum (CRM), and CAR-associated antigens (CAR-associated antigens). Antigens of 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 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 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), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal 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 various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0135] In some embodiments, the transmembrane domain of the CAR comprises the full length or at least a portion of a native or modified transmembrane region of a CD3D, CD3E, CD3G, CD3zeta, 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.

[0136] In some embodiments, the signaling peptide of the endodomain (or intracellular domain) comprises the full length or at least a portion of a polypeptide of CD3zeta, 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 CD3zeta.

[0137] In certain embodiments, the endodomain further comprises at least one costimulatory signaling region, which can comprise the full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.

[0138] In one embodiment, a CAR applicable to the cells provided in the present application comprises a costimulatory domain derived from CD28 and a signaling domain comprising a native or modified ITAM1 of CD3ζ represented by an amino acid sequence of 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, a CAR comprising a costimulatory domain derived from CD28 and a native or modified ITAM1 of CD3ζ also comprises a hinge domain and a transmembrane domain derived from CD28, and the scFv can be connected to the transmembrane domain via the hinge, and the CAR comprises an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 14. SEQ ID NO:13 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGE RRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (153 amino acid CD28 costimulatory + CD3ζ ITAM)

[0139] TIFF2025013853000008.tif34169

[0140] In another embodiment, a CAR applicable to the cells provided in the present application comprises a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising a native or modified CD3ζ represented by an amino acid sequence of 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 comprising a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising a native or modified CD3ζ may further comprise a CD8 hinge, the amino acid sequence of such structure being at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 16.

[0141] TIFF2025013853000009.tif44169

[0142] TIFF2025013853000010.tif51167

[0143] Non-limiting CAR strategies include heterodimeric, conditionally activated CARs through dimerization of a pair of intracellular domains (see, e.g., U.S. Pat. No. 9,587,020), split CARs, which are homologous recombination of antigen-binding, hinge, and endodomains to generate a CAR (see, e.g., U.S. Pat. Publ. No. 2017 / 0183407), multi-chain CARs that allow for a non-covalent link between two transmembrane domains connected to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Pat. Publ. No. 2014 / 0134142), CARs with bispecific antigen-binding domains (see, e.g., U.S. Pat. No. 9,447,194), or with a pair of antigen-binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Pat. No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Exp 1999, 10, 143-151, 2002). Invest. 2016;126(8):3036-3052), inducible CARs (see, e.g., U.S. Patent Publication 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.

[0144] Thus, provided herein are derived cells obtained from differentiation of genomically engineered iPSCs, both the iPSCs and derived cells comprising one or more CARs with additional modified modalities including, but not limited to, CD38 knockout and / or hnCD16. In one particular embodiment, the iPSCs and derived cells comprise CD38 knockout, hnCD16, and a CAR targeting a selected tumor or viral antigen, where the derived cells are NK or T cells, and the derived cells may be used with one or more of ADCC antibodies or bispecific, trispecific, multispecific engagers that target a tumor antigen different from that targeted by the CAR, via hnCD16 binding, to avoid or reduce tumor antigen escape while achieving dual targeting of the same tumor. In a further embodiment, the CAR-containing iPSCs and derived T cells have the CAR inserted into the TCR constant region, resulting in a TCR knockout and placing CAR expression under the control of the endogenous TCR promoter. In some embodiments, induced TCR null CAR-T cells derived from engineered iPSCs further comprise an hnCD16 with an ectodomain native to CD16 (F176V and / or S197P) or derived from CD64, and native or non-native transmembrane, stimulatory, and signaling domains. In another embodiment, iPSCs containing a CAR and their derived NK cells have the CAR inserted into the NKG2A or NKG2D locus resulting in an NKG2A or NKG2D knockout, placing CAR expression under the control of the endogenous NKG2A or NKG2D promoter.

[0145] 4. Exogenously Introduced Cytokines and / or Cytokine Signaling By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicity from such actions is reduced and cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, partial or complete peptides of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors are introduced into cells to allow cytokine signaling with or without expression of the cytokine itself, thereby reducing the risk of cytokine toxicity and maintaining or improving cell growth, proliferation, expansion, and / or effector function. In some embodiments, the introduced cytokines and / or their respective native or modified receptors for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or temporary.

[0146] Figure 1 shows several construct designs using IL15 as an example. The transmembrane (TM) domain of any 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.

[0147] Design 1: IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics the trans-presentation of IL15 without eliminating the cis-presentation of IL15.

[0148] 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.

[0149] Design 3: IL15Rα with a truncated intracellular domain is fused to IL15 at the C-terminus via a linker to mimic trans-presentation of IL15, maintain membrane binding of IL15, and eliminate cis-presentation and / or other potential signaling pathways mediated by normal IL15R via its intracellular domain. The intracellular domain of IL15Rα is believed to be important for the receptor to be expressed in IL15-responsive cells and for the responsive cells to proliferate and function. Such truncated constructs comprise an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical 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 truncated IL15 / IL15Rα, the construct does not include the last four amino acids "KSRQ" of SEQ ID NO:17 and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:21.

[0150] TIFF2025013853000011.tif55169 SEQ ID NO:18 ATGGACTGGACCTGGATTCTGTTCCTGGTCGCGGCTGCAACGGCGAGTCCATAGCGGTATC CATGTTTTTATTCTTGGGTGTTTTTCTGCTGGGCTGCCTAAGACCGAGGCCAACTGGGTA AATGTCATCAGTGACCTCAAGAAAATAGAAGACCTTATACAAAGCATGCACATTGATGCT ACTCTCTACACTGAGTCAGATGTACATCCCTCATGCAAAGTGACGGCCATGAAATGTTTC CTCCTCGAACTTCAAGTCATATCTCTGGAAAGTGGCGACGCGTCCATCCACGACACGGTC GAAAACCTGATAATACTCGCTAATAATAGTCTCTCTTCAAATGGTAACGTAACCGAGTCA GGTTGCAAAGAGTGCGAAGAGTTGGAAGAAAAAAACATAAAGGAGTTCCTGCAAAGTTTC GTGCACATTGTGCAGATGTTCATTAATACCTCTAGCGGCGGAGGATCAGGTGGCGGTGGA AGCGGAGGTGGAGGCTCCGGTGGAGGAGGTAGTGGCGGAGGTTCTCTTCAAATAACTTGT CCTCCACCGATGTCCGTAGAACATGCGGATATTTGGGTAAAATCCTATAGCTTGTACAGC CGAGAGCGGTATATCTGCAACAGCGGCTTCAAGCGGAAGGCCGGCACAAGCAGCCTGACC GAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTAGCCTGAAGTGC ATCAGAGATCCCGCCCTGGTGCATCAGCGGCCTGCCCCTCCAAGCACAGTGACAACAGCT GGCGTGACCCCCCAGCCTGAGAGCCTGAGCCCTTCTGGAAAAGAGCCTGCCGCCAGCAGC CCCAGCAGCAACAATACTGCCGCCACCACAGCCGCCATCGTGCCTGGATCTCAGCTGATG CCCAGCAAGAGCCCTAGCACCGGCACCACCGAGATCAGCAGCCACGAGTCTAGCCACGGC ACCCCATCTCAGACCACCGCCAAGAACTGGGAGCTGACAGCCAGCGCCTCTCACCAGCCT CCAGGCGTGTACCCTCAGGGCCACAGCGATACCACAGTGGCCATCAGCACCTCCACCGTG CTGCTGTGTGGACTGAGCGCCGTGTCACTGCTGGCCTGCTACCTGAAGTCCAGACAGTGA (1140 nucleic acids)

[0151] TIFF2025013853000012.tif55169

[0152] Those skilled in the art will understand that the above-mentioned signal peptides and linker sequences are exemplary and in no way limit their variations suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence can be replaced with another sequence without changing the activity of the functional peptide led by the signal peptide or linked by the linker.

[0153] Design 4: Because the constructs of Design 3 have been shown to function in promoting effector cell survival and proliferation, indicating that the cytoplasmic domain of IL15Rα can be omitted in such designs without adversely affecting the autonomous function of IL15-equipped effector cells, Design 4 is a construct that offers another functioning alternative to Design 3, in which essentially the entire IL15Rα is deleted, except for the Sushi domain, and fused to IL15 at one end and the transmembrane domain at the other (mb-Sushi), optionally including 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 through IL15Rα, including cis-presentation, is eliminated when only the desired trans-presentation of IL15 is retained. In some embodiments, a component comprising IL15 fused to a Sushi domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 19, which may be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 20.

[0154] TIFF2025013853000013.tif41164 SEQ ID NO:20 ATGGACTGGACCTGGATTCTGTTCCTGGTCGCGGCTGCAACGCGAGTCCATAGCGGTATC CATGTTTTTATTCTTGGGTGTTTTTCTGCTGGGCTGCCTAAGACCGAGGCCAACTGGGTA AATGTCATCAGTGACCTCAAGAAAATAGAAGACCTTATACAAAGCATGCACATTGATGCT ACTCTCTACACTGAGTCAGATGTACATCCCTCATGCAAAGTGACGGCCATGAAATGTTTC CTCCTCGAACTTCAAGTCATATCTCTGGAAAGTGGCGACGCGTCCATCCACGACACGGTC GAAAACCTGATAATACTCGCTAATAATAGTCTCTCTTCAAATGGTAACGTAACCGAGTCA GGTTGCAAAGAGTGCGAAGAGTTGGAAGAAAAAAACATAAAGGAGTTCCTGCAAAGTTTC GTGCACATTGTGCAGATGTTCATTAATACCTCTAGCGGCGGAGGATCAGGTGGCGGTGGA AGCGGAGGTGGAGGCTCCGGTGGAGGAGGTAGTGGCGGAGGTTCTCTTCAAATAACTTGT CCTCCACCGATGTCCGTAGAACATGCGGATATTTGGGTAAAATCCTATAGCTTGTACAGC CGAGAGCGGTATATCTGCAACAGCGGCTTCAAGCGGAAGGCCGGCACAAGCAGCCTGACC GAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTAGCCTGAAGTGC ATCAGA (726 nucleic acids)

[0155] Those skilled in the art will understand that the above-mentioned signal peptides and linker sequences are exemplary and in no way limit their variations suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence can be replaced with another sequence without changing the activity of the functional peptide led by the signal peptide or linked by the linker.

[0156] Design 5: Native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker, allowing constitutive signaling and maintaining IL15 membrane binding and trans-presentation.

[0157] Design 6: Native or engineered common receptor γC is fused at the C-terminus to IL15 via a linker for constitutive signaling and membrane-bound transpresentation of the cytokine. Common receptor γC is also known as common gamma chain or CD132, and IL2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukin receptors, including but not limited to the IL2, IL4, IL7, IL9, IL15, and IL21 receptors.

[0158] Design 7: An engineered IL15Rβ that forms homodimers in the absence of IL15 is useful to generate constitutive signaling of cytokines.

[0159] In some embodiments, one or more of the cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or their receptors, may be introduced into iPSCs, and their derived cells upon iPSC differentiation, using one or more of the designs in FIG. 1. In some embodiments, cell surface expression and signaling of IL2 or IL15 is via the constructs shown in any one of designs 1-7. In some embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 is via the constructs shown in designs 5, 6, or 7, by using either a common receptor or a cytokine-specific receptor, such as the IL4 receptor. The transmembrane (TM) domains of any of the designs in FIG. 1 may be native to the corresponding cytokine receptor or have been modified or replaced with the transmembrane domains of other membrane-bound proteins.

[0160] In iPSCs and derived cells containing both 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 containing both CAR and IL. In some further embodiments, IL15 in a form represented by any of the construct designs in 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 CAR are in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises IL15 of design 3 in FIG. 1. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises IL15 of design 3 in FIG. 1. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises IL15 of design 7 in Figure 1. Upon expression of CAR-2A-IL15 or IL15-2A-CAR, the self-cleaving 2A peptide dissociates the expressed CAR and IL15, allowing the dissociated IL15 to be presented on the cell surface. The bicistronic design of CAR-2A-IL15 or IL15-2A-CAR allows for coordinated expression of CAR and IL15, both in timing and quantity, under the same control mechanism that can be chosen to incorporate, such as, for example, an inducible promoter for expression of a single ORF. Self-cleaving peptides are found in members of the Picornaviridae family, including the Aphthovirus genus, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tessiovirus 1 (PTV-I) (Donnelly, ML, et al., J. Gen. Virol., 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732 (2001)), and the Cardiovirus genus, such as Theilovirus (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.

[0161] The bicistronic CAR-2A-IL15 or IL15-2A-CAR embodiments disclosed herein for IL15 also contemplate 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 is via a construct shown in any of Designs 1-7. In some other embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 is via a construct shown in Designs 5, 6, or 7, using either a common receptor and / or a cytokine-specific receptor.

[0162] 5. HLA-I and HLA-II deficiency To avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins must be matched for histocompatibility of the allogeneic recipient. Provided herein is an iPSC cell line in which 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 by deletion or reduced expression levels of HLA class I-associated genes, including but not limited to the beta 2 microglobulin (B2M) gene, the TAP1 gene, the 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-associated 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, which is required for the expression of class II proteins. CIITA null cells are HLA-II deficient. Provided here are iPSC lines and their derivatives in which both B2M and CIITA are knocked out, and the derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching to avoid recognition and killing by host (allogeneic) T cells.

[0163] In some cell types, lack of class I expression leads to lysis by NK cells. To overcome this "loss of self" response, HLA-G can be optionally knocked in to avoid NK cell recognition and killing of 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 capacity of iPSCs and the function of induced effector cells, including induced T cells and induced NK cells.

[0164] 6. Genetically engineered iPSC lines and derived cells provided herein In light of the above, the present application provides - / - (also referred to herein as "CD38 null" or CD38 knockout) iPSCs, cell line cells, or populations thereof, and CD38 - / - Provided are functional inducer cells that contain CD38 knockout derived from differentiation of iPSC. In some embodiments, the functional inducer cells are hematopoietic cells, including but not limited to mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitors, NK cell progenitors, bone marrow cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, the functional inducer hematopoietic cells include effector cells such as T cells, NK cells, and regulatory cells.

[0165] Further provided herein are iPSCs comprising a CD38 knockout and a polynucleotide encoding high affinity non-cleavable CD16 (hnCD16), wherein the iPSCs can produce functional hematopoietic cells by directed differentiation. In some embodiments, when anti-CD38 antibodies are used to induce hnCD16-mediated enhanced ADCC, the iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing effector cell elimination, i.e., reduction or depletion of CD38-expressing effector cells, thereby increasing the persistence and / or viability of the iPSCs and their effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or viability in the presence of an anti-CD38 therapeutic agent, which may be an anti-CD38 antibody or a CAR-bound CD38. In some embodiments, the effector cells include T cells. T cells derived from iPSCs containing CD38 null and hnCD16 experience reduced cell depletion in the presence of anti-CD38 antibodies or anti-CD38 CARs, and acquire ADCC, providing an additional mechanism of T cell-mediated tumor killing. In some embodiments, the effector cells comprise NK cells. NK cells derived from iPSCs containing CD38 null and hnCD16 have enhanced cytotoxicity and reduced NK cell fratricide in the presence of anti-CD38 antibodies or anti-CD38 CARs.

[0166] Provided herein are iPSCs comprising a CD38 knockout and a polynucleotide encoding a target-specific chimeric antigen receptor (CAR), which can produce functional inducer effector cells by directed differentiation. In one embodiment, the CAR contained in the iPSCs comprising a CD38 knockout and its derivative effector cells targets the tumor cell surface protein CD38, but the CD38-CAR does not result in the elimination of the iPSCs and / or its derived effector cells with a CD38 knockout. In some embodiments, the CAR contained in the iPSCs comprising a CD38 knockout and its derived effector cells does not target CD38. In some embodiments, the CAR-expressing, CD38 null inducer effector cells can be used with an anti-CD38 antibody to induce ADCC without causing the elimination of the effector cells, thereby increasing the persistence and / or survival of the iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or survival in vivo in combination therapy.

[0167] Further comprising a CD38 knockout and a polynucleotide encoding at least one exogenous cytokine and / or its receptor (IL) that allows cytokine signaling that contributes to cell viability, persistence, and / or proliferation, the iPSC line can direct differentiation to produce functional derived hematopoietic cells with improved viability, persistence, proliferation, and effector cell function. The exogenously introduced cytokine signaling includes any one or two or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21 signaling. In some embodiments, introduced partial or complete peptides of the cytokine and / or their respective receptors for cytokine signaling are expressed on the cell surface. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, the activation of the cytokine signaling is inducible. In some embodiments, the activation of the cytokine signaling is transient and / or temporary. In some embodiments, the transient / transient expression of cell surface cytokine / cytokine receptor is via retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA, including mRNA. - / - The exogenous cell surface cytokines and / or receptors contained in the iPSCs or their derived cells enable IL7 signaling. In some embodiments, CD38 - / - The exogenous cell surface cytokines and / or receptors contained in the iPSCs or their derived cells enable IL10 signaling. In some embodiments, CD38 - / - Exogenous cell surface cytokines and / or receptors contained in iPSCs or their derived cells enable IL15 signaling. - / - In some embodiments of the IL iPSCs, IL15 expression is by construct 3 of FIG. - / -In some embodiments of the IL iPSCs, IL15 expression is according to construct 4 of FIG. - / - IL iPSCs and their derived cells can autonomously maintain or improve cell growth, proliferation, proliferation, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo. In some embodiments, CD38 - / - IL iPSCs and their derived effector cells can be used with anti-CD38 antibodies to induce ADCC without causing effector cell elimination, thereby synergistically increasing the persistence and / or viability of iPSCs and their effector cells.

[0168] Also provided are iPSCs that contain a polynucleotide encoding CD38 knockout, B2M knockout, and CIITA knockout, and optionally HLA-G, which iPSCs can direct differentiation to produce functional induced hematopoietic cells. - / - B2M - / - CIITA - / - The iPSCs and their derived effector cells are both HLA-I and HLA-II deficient and can be used with anti-CD38 antibodies to induce ADCC without causing effector cell elimination, thereby synergistically increasing the persistence and / or survival of the iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or survival in vivo.

[0169] In view of the above, provided herein include iPSCs that contain a CD38 knockout and, optionally, one, two, three, or all four of hnCD16, CAR, exogenous cytokine / receptor, and B2M / CIITA knockout, where when B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced and the iPSCs can be directed to differentiate to produce functional induced hematopoietic cells. The application also includes functional iPSC-derived hematopoietic cells comprising a CD38 knockout and, optionally, one, two, three, or all four of hnCD16, B2M / CIITA knockout, CAR, and exogenous cytokine / receptor, where B2M is knocked out and a polynucleotide encoding HLA-G is optionally introduced, the induced hematopoietic cells include, but are not limited to, mesodermal cells with definitive hemogenic 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.

[0170] Another aspect provided herein includes iPSCs or iPSC-derived cells comprising a truncated fusion protein of IL15 and IL15Rα, where the fusion protein does not include an intracellular domain. Although shown in FIG. 1 as "IL15Rα(ΔICD) fusion" and "IL5 / mb-Sushi", these embodiments are further summarized in Table 1 and throughout the application as abbreviated IL15Δ. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO:21. In yet some other embodiments, iPSCs or iPSC-derived cells comprising a truncated IL15 / IL15Rα fusion protein lacking the intracellular domain (IL15Δ) further comprise one or more of CD38 knockout, hnCD16, CAR, an exogenous cytokine / receptor, and B2M / CIITA knockout, where if B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced, and the iPSCs are capable of directed differentiation to produce functional induced hematopoietic cells, including, but not limited to, mesoderm cells with definitive hemogenic 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.

[0171] Thus, the present application provides iPSCs and their functional derived hematopoietic cells comprising any one of the following genotypes in Table 1. Unless specified as IL15Δ, which is detailed as a truncated fusion protein of IL15 and IL15Rα but without the 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. Furthermore, when the iPSCs and their functional derived hematopoietic cells have a genotype that includes both CAR and IL, the CAR and IL are included in a bicistronic expression cassette that includes the 2A sequence. In contrast, in some other embodiments, the CAR and IL are in separate expression cassettes included in the iPSCs and their functional derived hematopoietic cells. In a specific embodiment, iPSCs expressing both CAR and IL and their functionally derived effector cells include IL15 in constructs 3 or 4 of FIG. 1, where the IL15 construct is included in an expression cassette together with or separately from the CAR.

[0172] [Table 1]

[0173] 7. Further modifications In some embodiments, iPSCs and derived effector cells comprising any one of the genotypes in Table 1 may further comprise a deletion or reduced expression of 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 It may further include introduced or increased expression of at least one of a surface triggering receptor for binding to an R, a TCR, an Fc receptor, an engager, and a bispecific, multispecific or universal engager.

[0174] Bispecific or multispecific engagers are fusion proteins consisting of two or more single chain variable fragments (scFv) of different antibodies, where at least one scFv binds to an effector cell surface molecule and at least another binds to tumor cells via a tumor-specific surface molecule. Exemplary effector cell surface molecules or surface triggering 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 chimeric Fc receptors disclosed herein. In some embodiments, the CD16 expressed on the surface of the effector cell for engager recognition is hnCD16, which includes CD16 (including F176V and optionally S197P) or CD64 extracellular domains, and natural or non-natural transmembrane, stimulatory and / or signaling domains, as described in section I.2. In some embodiments, the CD16 expressed on the surface of the effector cells for engager recognition is an hnCD16-based chimeric Fc receptor (CFcR). In some embodiments, the hnCD16-based CFcR comprises 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 a partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises 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 (called TriKE, or trispecific killer engager in some publications) as a linker for effector NK cells to promote effector cell proliferation. In one embodiment, TriKE is CD16-IL15-EPCAM or CD64-IL15-EPCAM. In another embodiment, TriKE is CD16-IL15-CD33 or CD64-IL15-CD33. In yet another embodiment, TriKE is NKG2C-IL15-CD33.

[0175] In some embodiments, the surface triggering receptors for bispecific or multispecific engagers can be endogenous to the effector cells, sometimes depending on the cell type. In some other embodiments, the methods and compositions provided herein can be used to further manipulate iPSCs comprising the genotypes listed in Table 1, directing the differentiation of iPSCs into T cells, NK cells, or other effector cells that comprise the same genotype and surface triggering receptors as the source iPSCs, and introduce one or more exogenous surface triggering receptors into the effector cells.

[0176] 8. Antibodies for immunotherapy In some embodiments, in addition to the genomically engineered effector cells provided herein, additional therapeutic agents including antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication 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 capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (certuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc-engineered variants or fragments thereof, or functional equivalents and biosimilars thereof. In some embodiments, the iPSC-derived effector cells comprise hematopoietic lineage cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise NK cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise T cells comprising a genotype listed in Table 1. In some embodiments of the combination useful for treating liquid or solid tumors, the combination comprises iPSC-derived NK 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 the anti-CD38 antibodies daratumumab, isatuximab, MOR202. 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 in combination with daratumumab comprise CD38 null, hnCD16, IL15, and a CAR targeting at least one of CD38 or CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, and PDL1, where IL15 is co-expressed or separately expressed with 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 constructs 3, 4, or 7 when it is expressed together with or separately from the CAR.

[0177] 9. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate immune responses if not inhibited. It has become evident that tumors co-opt certain immune checkpoint pathways as a primary mechanism of immune resistance, particularly to T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can reduce the expression or gene products of checkpoint genes or decrease the activity of checkpoint molecules, thus blocking inhibitory checkpoints and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, and these agents have led to long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. One aspect of the present application provides a therapeutic approach to overcome CI resistance by including functional inducer cells that are genomically engineered to be provided in a combination therapy with CI. In one embodiment of the combination therapy, the inducer cells are NK cells. In another embodiment of the combination therapy, the inducer cells are T cells. In addition to exhibiting direct antitumor capabilities, the induced NK cells provided herein have been shown to have the ability to resist PDL1-PD1-mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at tumor sites. Thus, the tumor infiltration of T cells promoted by functionally potent, genomically engineered induced NK cells indicates that said NK cells can synergize with T cell-targeted immunotherapies, including checkpoint inhibitors, to alleviate local immune suppression and reduce tumor burden.

[0178] In one embodiment, the induced NK cells for checkpoint inhibitor combination therapy comprise a CD38 knockout, and one, two, three, or all four of hnCD16 expression, B2M / CIITA knockout, CAR expression, and exogenous cell surface cytokine and / or receptor expression, where if B2M is knocked out, a polynucleotide encoding HLA-G is optionally included. In some embodiments, the induced NK cells comprise any one of the genotypes listed in Table 1. In some embodiments, the induced NK cells described above further comprise a deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene of the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D ... 2A Further comprising induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific, multispecific or universal engager.

[0179] In another embodiment, the induced T cells for checkpoint inhibitor combination therapy comprise CD38 knockout, and one, two, three, or all four of hnCD16 expression, B2M / CIITA knockout, CAR expression, and exogenous cell surface cytokine and / or receptor expression, where if B2M is knocked out, a polynucleotide encoding HLA-G is optionally included. In some embodiments, the induced T cells comprise any one of the genotypes listed in Table 1. In some embodiments, the induced T cells further comprise deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene of the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D ...2A Further comprising induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific, multispecific or universal engager.

[0180] The induced NK cells or induced T cells are obtained from differentiating an iPSC clonal line comprising 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 if B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced. In some embodiments, the iPSC clonal line further comprises a deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene of the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Further comprising induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific, multispecific or universal engager.

[0181] Checkpoint inhibitors suitable for combination therapy with induced NK cells or induced T cells provided herein include 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 inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, 3DL2).

[0182] In some embodiments, the antagonist that inhibits any of the above-mentioned checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a mouse 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 may be more cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, the one or two or three or more checkpoint inhibitors comprise 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), lilimumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0183] In some embodiments, the antagonists that inhibit any of the above-mentioned checkpoint molecules are microRNA-based, as many miRNAs are found as regulators that control the expression of immune checkpoints (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.

[0184] Some embodiments of the combination therapy with induced NK cells or induced T cells provided include at least one checkpoint inhibitor that targets at least one checkpoint molecule, and the induced cells have a genotype listed in Table 1. Some other embodiments of the combination therapy with induced NK cells or induced T cells provided include two, three, or more checkpoint inhibitors, such that two, three, or more checkpoint molecules are targeted. In some embodiments of the combination therapy including at least one checkpoint inhibitor and induced cells having a genotype 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 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 as CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences for 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 counteracts the inhibitory checkpoint molecules when bound to the TME, activating modalities such as CAR or activating receptor activation, thereby activating the effector cells.In some embodiments, the checkpoint inhibitor co-expressed with the CAR inhibits at least one of the 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 the induced cells having the genotypes listed in Table 1 is selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is atezolizumab, or humanized or Fc-modified variants, fragments, or functional equivalents or biosimilars thereof. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is nivolumab, or humanized or Fc-modified variants, fragments, or functional equivalents or biosimilars thereof. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab, or a humanized, or Fc-engineered variant, fragment, or functional equivalent or biosimilar thereof.

[0185] In some other embodiments of the combination therapy comprising the inducer cells and at least one antibody that inhibits a checkpoint molecule provided herein, the antibody is not produced by or within the inducer cells and is further administered prior to, concurrently with, or after administration of the inducer cells having a genotype listed in Table 1. In some embodiments, the administration of one, two, three, or more checkpoint inhibitors in the combination therapy with the provided induced NK cells or induced T cells is simultaneous or sequential. In one embodiment of the combination therapy comprising the induced NK cells or induced T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments of combination therapies comprising induced NK cells or induced T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is atezolizumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising induced NK cells or induced T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is nivolumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising induced NK cells or induced T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is pembrolizumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof.

[0186] II. Methods for targeted genome editing at selected loci in iPSCs Genome editing, or genome editing, or gene editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and / or replaced in the genome of a target cell. Targeted genome editing (interchangeably "targeted genome editing" or "targeted gene editing") allows insertion, deletion, and replacement at preselected sites in the genome. When endogenous sequences are deleted at the insertion site during targeted editing, endogenous genes containing the affected sequences can be knocked out or knocked down by sequence deletion. Thus, targeted editing can also be used to precisely disrupt endogenous gene expression. The term "targeted integration," as also used herein, refers to a process that involves the insertion of one or more exogenous sequences, with or without the deletion of endogenous sequences at the insertion site. In contrast, randomly integrated genes are susceptible to position effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomeric regions are particularly prone to transgene silencing. Reciprocally newly integrated genes may affect surrounding endogenous genes and chromatin, altering cell behavior or promoting cell transformation. Therefore, inserting foreign DNA into preselected loci such as safe harbor loci or genomic safe harbor (GSH) is important for safety, efficiency, copy number control, and reliable gene response control.

[0187] 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 host cell's enzymatic machinery.

[0188] Alternatively, targeted editing can be achieved at higher frequencies by specific introduction of double-strand breaks (DSBs) with specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in random insertion or deletion (indels) of a small number of endogenous nucleotides. In contrast, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homology arms, exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration."

[0189] Available endonucleases that can introduce 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 repeats) systems. In addition, the DICE (dual integrase cassette exchange) system, which utilizes phiC31 and Bxb1 integrases, is also a promising tool for targeted integration.

[0190] ZFN is a targeted nuclease that comprises a nuclease fused to a zinc finger DNA binding domain. "Zinc finger DNA binding domain" or "ZFBD" refers to a polypeptide domain that binds to DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids in the zinc finger binding domain whose structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2 zinc finger, C3H zinc finger, and C4 zinc finger. A "designed" zinc finger domain is a domain that does not occur in nature, whose design / construction results primarily from rational criteria, such as the application of substitution rules and computerized algorithms to process information from databases that store 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. A "selected" zinc finger domain is a domain not found in nature, whose production is mainly derived from empirical processes such as phage display, interaction trap, or hybrid selection. ZFNs are described in detail in U.S. Patent No. 7,888,121 and U.S. Patent No. 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of FokI nuclease with zinc finger DNA binding domain.

[0191] TALENs are targeted nucleases that contain a nuclease fused to a TAL effector DNA binding domain. "Transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" refers to 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 domain, and activate gene transcription at these sequences via the transactivation domain. 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 residues (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 TALENs in the art is the fusion polypeptide of FokI nuclease to a TAL effector DNA binding domain.

[0192] Another example of a targeted nuclease for use in the methods of the invention is a targeted Spo11 nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA binding domain having specificity for a DNA sequence of interest, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. See, e.g., U.S. Patent Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0193] Further examples of targeted nucleases suitable for the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used individually or in combination.

[0194] Other non-limiting examples of targeted 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.

[0195] As an illustrative example, CRISPR / Cas9 requires two major 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 that contains a PAM and a PAM-proximal seeding region. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target a sequence of choice. These two components can be delivered into mammalian cells via transfection or transduction.

[0196] DICE-mediated insertion uses a pair of recombinases, such as phiC31 and Bxb1, to provide unidirectional integration of exogenous DNA that is tightly restricted by the small attB and attP recognition sites of each enzyme. These targeted att sites do not naturally occur in mammalian genomes and must first be 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.

[0197] 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 allow site-specific homologous recombination by a cellular host enzyme machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to allow ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to allow TALEN-mediated insertion. In another embodiment, a method of targeted integration in a cell includes 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 for a desired integration site into the cell to allow insertion via Cas9. In yet another embodiment, a method of targeted integration in a cell includes introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to allow targeted integration via DICE.

[0198] Promising sites for targeted integration include, but are not limited to, safe harbor loci, or genomic safe harbors (GSH), which are intragenic or extragenic regions of the human genome that, in theory, can accommodate predictable expression of the newly integrated DNA without adverse effects on the host cell or organism. A useful safe harbor should allow sufficient transgene expression to produce desired levels of vector-encoded protein or non-coding RNA. A safe harbor should also not predispose cells to malignant transformation or alter cellular function. For a recombination site to be a promising safe harbor locus, it should ideally meet conditions including, but not limited to, the following: be free of disruption of regulatory elements or genes, as judged by sequence annotation; be an intergenic region within a dense region of genes or be a convergent location between two genes that are transcribed in opposite directions; maintain distance to minimize the possibility of long-range interactions between vector-encoded transcriptional activators and promoters of adjacent genes, particularly cancer-associated and microRNA genes; and have apparently ubiquitous transcriptional activity, as reflected by widespread spatial and temporal expressed sequence tag (EST) expression patterns indicative of ubiquitous transcriptional activity. This latter feature is particularly important in stem cells, where chromatin remodeling during differentiation typically results in the silencing of some loci and the potential activation of others.Within a region suitable for exogenous insertion, the precise locus selected for insertion should be devoid of repetitive elements and conserved sequences, allowing straightforward design of primers for amplification of homology arms.

[0199] 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 orthologue of the mouse ROSA26 locus. In addition, the human orthologue of the mouse H11 locus may also be a suitable site for insertion using the compositions and methods of targeted integration disclosed herein. In addition, 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 necessary, especially in stem cells, for certain integration events, and optimization of the insertion strategy, including promoter selection, exogenous gene sequence and placement, and construct design, is often required.

[0200] For targeted indels, the editing site is often contained in the endogenous gene whose expression and / or function is intended to be disrupted.In one embodiment, the endogenous gene containing targeted indels is related to immune response regulation and adjustment.In some other embodiments, the endogenous gene containing targeted indels is related to targeting modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and adjustment, or proteins that suppress the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells and cells derived therefrom.

[0201] Thus, one aspect of the invention provides a method of targeted integration at a selected locus that includes a genomic safe harbor, or a pre-selected locus that is known or proven to be safe and well-regulated for continuous 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 includes one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of a genomic safe harbor. In one embodiment, a method for targeted integration into a cell includes 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 to a desired integration site and one or more exogenous sequences to enable site-specific homologous recombination by the cellular host enzymatic machinery, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of the genomic safe harbor.

[0202] In another embodiment, the method of targeted integration into a cell includes 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 to a desired integration site to allow for ZFN-mediated insertion, where the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of genomic safe harbor. In yet another embodiment, the method of targeted integration into a cell includes 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 to a desired integration site to allow for TALEN-mediated insertion, where the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of genomic safe harbor. In another embodiment, a method of targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell a gRNA comprising a Cas9 expression cassette and a guide sequence specific for a desired integration site to enable Cas9-mediated insertion, the desired integration site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other locus that meets the criteria for the Genomic Safe Harbor.In yet another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in 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, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other locus that meets the criteria for the Genomic Safe Harbor.

[0203] Furthermore, as provided herein, the above-mentioned methods for targeted integration in safe harbor are used to insert polynucleotides of interest, such as polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates, and proteins that promote stem cell and / or progenitor cell engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival. In some other embodiments, the constructs comprising one or more exogenous polynucleotides further comprise 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 induced 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, modified EGFR containing an epitope recognized by cetuximab can be used to deplete genetically engineered cells when the cells are exposed to cetuximab. Thus, one aspect of the present invention provides a method for targeted integration of one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0204] In some embodiments, one or more exogenous polynucleotides integrated by the methods herein are driven by an exogenous promoter operably linked to the construct for targeted integration. The promoter can be inducible or constitutive, and can be time-specific, tissue-specific or cell type-specific. Constitutive promoters suitable for the methods of the present invention include, but are not limited to, cytomegalovirus (CMV), elongation factor 1 alpha (EF1 alpha), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0205] The exogenous polynucleotides integrated by the methods herein may be driven by an endogenous promoter in the host genome at the integration site. In one embodiment, the methods of the present invention are 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 integrated polynucleotide is driven by an endogenous AAVS1 promoter. In another embodiment, the methods of the present invention are used for targeted integration at the ROSA26 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by an endogenous ROSA26 promoter. In yet another embodiment, the methods of the present invention are used for targeted integration at the H11 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by an endogenous H11 promoter. In another embodiment, the methods of the present invention are used for targeted integration at the collagen locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by an endogenous collagen promoter. In yet another embodiment, the methods of the present invention are used for targeted integration at the HTRP locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous HTRP promoter. In theory, only correct insertion at the desired position will allow gene expression of the exogenous gene driven by the endogenous promoter.

[0206] In some embodiments, one or more exogenous polynucleotides included in the construct for the 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 increase the physical separation between the moieties and maximize access to the enzymatic machinery. The linker peptide of the linker sequence may be composed of amino acids selected to make the physical separation between the moieties (exogenous polynucleotides and / or proteins or peptides encoded therefrom) more flexible or more rigid, depending on the function involved. The linker sequence may be cleavable by a protease or may be chemically cleavable to yield separate moieties. Examples of enzymatic cleavage sites in the linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is one that is naturally produced by the host or is exogenously introduced. Alternatively, the cleavage site in the linker may be one that can be cleaved upon exposure to a selected chemical, such as cyanogen bromide, hydroxylamine, or low pH. Any linker sequence may serve a purpose other than providing a cleavage site. The linker sequence should allow for effective positioning of a moiety relative to another adjacent moiety in order for the moiety to function properly. The linker may also be a simple amino acid sequence of sufficient length to prevent any steric hindrance between the moieties. In addition, the linker sequence may provide for post-translational modifications, including but not limited to, for example, phosphorylation sites, biotinylation sites, sulfation sites, gamma-carboxylation sites, and the like. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single undesired conformation. The linker may be composed primarily 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 includes glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, a G4S linker peptide separates the terminal processing and endonuclease domains of the fusion protein. In other embodiments, a 2A linker sequence allows two separate proteins to be produced from a single translation. A suitable linker sequence can be easily identified empirically. In addition, 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). In some embodiments, any two consecutive linker sequences are different.

[0207] The 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, or a Sendai viral vector. In some embodiments, a plasmid vector is used to deliver and / or express the exogenous polynucleotide into the target cell (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.). In some other embodiments, an episomal vector is used to deliver the exogenous polynucleotide into the target cell. In some embodiments, recombinant adeno-associated viruses (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV do not integrate into the host genome. Furthermore, episomal rAAV vectors mediate a much higher rate of homology-directed gene targeting compared to traditional targeting plasmid transfection. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions or substitutions into the target sites in the genome of iPSCs. In some embodiments, the genomically modified iPSCs and their derived cells obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0208] III. Methods for obtaining and maintaining genome-engineered iPSCs The present invention provides methods for obtaining and maintaining genomically engineered iPSCs that contain one or more targeted edits at one or more desired sites, which remain 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, i.e., targeted integrations and / or indels, at selected sites into the genome of the iPSCs and cells derived therefrom. The many advantages of obtaining genomically engineered derivative cells by editing and differentiating iPSCs as provided herein compared to directly manipulating primary effector cells derived from patient-derived peripheral blood include, but are not limited to: an unlimited source of engineered effector cells; no need to repeatedly manipulate effector cells, especially when multiple engineered modalities are included; the obtained effector cells have extended telomeres and are younger due to less attrition; and the effector cell population is homogenous in terms of editing sites, copy number, and lack of allelic mutations, random mutations, and expression diversity, primarily due to the ability to perform clonal selection on the engineered iPSCs provided herein.

[0209] In certain embodiments, genomically engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells for extended periods of time in a cell culture medium as shown in Table 2 as Fate Maintenance Medium (FMM), in which 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, maintaining a basal or naive profile, genomic stability without the need for washing or selection of the culture, and readily give rise to all three somatic lineages, in vitro differentiation via embryoid bodies or monolayers (without formation of embryoid bodies), and in vivo differentiation by teratoma formation. See, e.g., U.S. Patent Application No. 61 / 947,979, the disclosure of which is incorporated herein by reference.

[0210] [Table 2]

[0211] In some embodiments, genomically engineered iPSCs containing one or more targeted integrations and / or indels are maintained, passaged, and expanded in medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and free or essentially free of a TGFβ receptor / ALK5 inhibitor, wherein the iPSCs retain an intact and functional targeted edit at the selected site.

[0212] Another aspect of the invention provides a method for generating genomically engineered iPSCs through targeted editing of iPSCs or by first generating genomically engineered non-pluripotent cells by targeted editing and then reprogramming the selected / isolated genomically engineered non-pluripotent cells to obtain iPSCs containing the same targeted edits as the non-pluripotent cells. A further aspect of the invention provides genomically engineered non-pluripotent cells undergoing simultaneous reprogramming by introducing targeted integrations and / or targeted indels into the cells, the contacted non-pluripotent cells being under conditions sufficient for reprogramming, the reprogramming conditions comprising contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genomic engineering and reprogramming, targeted integrations and / or targeted indels can be introduced into the non-targeted pluripotent cells prior to or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally small molecules.

[0213] In some embodiments, for simultaneous genomic engineering and reprogramming of non-pluripotent cells, targeted integrations and / or indels may 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 reprogramming cells exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181 and CD30.

[0214] In some embodiments, reprogramming is initiated by contacting the 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, genomically engineered iPSCs by any of the methods described above are further maintained and expanded using a mixture comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 2).

[0215] In some embodiments of the method of generating genomically engineered iPSCs, the method comprises introducing one or more targeted integrations and / or indels into the iPSCs to genomically engineer the iPSCs to obtain genomically engineered iPSCs having at least one genotype listed in Table 1. Alternatively, the method of generating genomically engineered iPSCs comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genomically engineered non-pluripotent cell comprising a targeted integration and / or indel at a selected site, and (b) contacting the genomically engineered non-pluripotent cell 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 a genomically engineered iPSC comprising a targeted integration and / or indel at a selected site. Alternatively, a method of generating genomically engineered iPSCs includes: (a) contacting a non-pluripotent cell 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 cell; (b) introducing one or more targeted integrations and / or indels into the reprogramming non-pluripotent cell for genomic engineering; and (c) obtaining a clonal genomically engineered iPSC comprising the targeted integrations and / or indels at the selected sites.

[0216] The reprogramming factor is 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. The 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 the non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, the one or more polynucleotides are introduced by an episomal vector. In various other embodiments, the one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by a plasmid combination, see, e.g., U.S. Patent Application No. 62 / 571,105, the disclosure of which is incorporated herein by reference.

[0217] In some embodiments, non-pluripotent cells are transfected with multiple constructs, including different exogenous polynucleotides and / or different promoters, by multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNAs, including but not limited to siRNAs, shRNAs, miRNAs, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue-specific or cell type-specific promoters. Thus, the polynucleotides are expressible under conditions that activate the promoter, e.g., in the presence of an inducer, or in a particular differentiated cell type. In some embodiments, the polynucleotides are expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, the one or more suicide genes are driven by a constitutive promoter, e.g., capase-9 driven by CAG. These constructs, containing different exogenous polynucleotides and / or different promoters, can be transferred simultaneously or sequentially into non-pluripotent cells. Non-pluripotent cells subjected to targeted integration of multiple constructs can be contacted with one or more reprogramming factors simultaneously to initiate reprogramming simultaneously with genome manipulation, thereby obtaining genome-engineered iPSCs containing multiple targeted integrations in the same pool of cells. Thus, this robust method allows for the derivation of clonal genome-engineered hiPSCs with multiple modalities integrated into one or more selected target sites by simultaneous reprogramming and manipulation strategies.In some embodiments, the genomically modified iPSCs and derived cells thereof obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0218] IV. Methods for obtaining genetically engineered effector cells by differentiating genomically engineered iPSCs Further aspects of the invention provide methods of in vivo differentiation of genomically engineered iPSCs by teratoma formation, where differentiated cells derived in vivo from genomically engineered iPSCs retain intact and functional targeted edits, including targeted integrations and / or indels, at desired sites. In some embodiments, differentiated cells derived 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 that meet the criteria of the genomic safe harbor. In some other embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratomas contain polynucleotides encoding targeting modalities or polynucleotides encoding proteins that promote the trafficking, homing, viability, self-renewal, persistence, and / or survival of stem and / or progenitor cells. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratomas containing one or more inducible suicide genes further comprise one or more indels in endogenous genes associated with regulating and mediating immune responses. In some embodiments, the indels are comprised in one or more endogenous checkpoint genes. In some embodiments, the indels are comprised in one or more endogenous T cell receptor genes. In some embodiments, the indels are comprised in one or more endogenous MHC class I suppressor genes. In some embodiments, the indels are comprised in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the indels are comprised 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 selected sites further comprise targeted editing in the gene encoding B2M (beta-2-microglobulin).

[0219] In certain embodiments, genomically engineered iPSCs containing one or more 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 the functional genetic modification, including the targeted edit, at the selected site. In one embodiment, the genomically engineered iPSC-derived cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitors, NK cell progenitors, bone marrow cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, where these cells derived from the genomically engineered iPSCs retain the functional genetic modification, including the targeted edit, at the desired site.

[0220] Applicable differentiation methods and compositions for obtaining hematopoietic cell lineages derived from iPSCs include, for example, those described in International Application No. PCT / US2016 / 044122, the disclosure of which is incorporated herein by reference. As provided, methods and compositions for generating hematopoietic cell lineages through definitive hemogenic endothelium (HE) derived from pluripotent stem cells, including hiPSCs, in a culture platform that is scalable and does not require monolayer EB formation under serum-free, feeder-free, and / or stroma-free conditions. 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 cells of various lineages that have transitioned directly to hematopoietic fate without passing through a pluripotent intermediate. Similarly, cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0221] A method for differentiating and expanding cells of hematopoietic lineage from pluripotent stem cells in monolayer culture includes contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from 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 expanded mesodermal cells with definitive hemogenic endothelial (HE) potential without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF, and, optionally, with a ROCK inhibitor, and / or a WNT pathway activator, differentiates the mesodermal cells with definitive HE potential into definitive HE cells, which are also expanded during differentiation.

[0222] The methods for obtaining cells of hematopoietic lineage provided herein are superior to EB-mediated pluripotent stem cell differentiation because EB formation results in modest to minimal cell proliferation, does not allow for monolayer culture, which is important for many applications requiring uniform proliferation, and uniform differentiation of cells within a population, making it laborious and less efficient.

[0223] The provided monolayer differentiation platform promotes differentiation into definitive hemogenic endothelium resulting in the induction 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 therapeutically relevant numbers of pluripotent stem cell-derived hematopoietic cells for a variety of therapeutic applications. Furthermore, monolayer culture using the methods provided herein results in functional hematopoietic lineage cells that enable a full range of in vitro differentiation, ex vivo conditioning, 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 hemogenic endothelium, hematopoietic pluripotent progenitor cells, hematopoietic stem cells and progenitors, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0224] A method of directing differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage, comprising: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator, and optionally bFGF, to initiate 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 no TGFβ receptor / ALK inhibitor, to initiate differentiation and proliferation of mesodermal cells having definitive HE potential from the mesodermal cells; and (iii) contacting the mesodermal cells having definitive HE potential with a composition comprising one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor; bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator, and optionally no TGFβ receptor / ALK inhibitor, to initiate differentiation and proliferation of definitive hemogenic endothelial cells from pluripotent stem cell-derived mesodermal cells having definitive hemogenic endothelial potential.

[0225] 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, and seeding and expanding 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 iPSC are retained in hematopoietic cells differentiated therefrom. In some embodiments of the method for directing the differentiation of pluripotent stem cells into cells of hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of hematopoietic lineage is in monolayer culture form without the generation of embryoid bodies.

[0226] In some embodiments of the above-described methods, the obtained pluripotent stem cell-derived definitive hemogenic endothelial cells are CD34+. In some embodiments, the obtained definitive hemogenic endothelial cells are CD34+CD43-. In some embodiments, the obtained definitive hemogenic endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the definitive hemogenic endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the definitive hemogenic endothelial cells are CD34+CD43-CD93-. In some embodiments, the definitive hemogenic endothelial cells are CD34+CD93-.

[0227] In some embodiments of the above-mentioned method, the method further comprises: (i) contacting the definitive hemogenic endothelium derived from the 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 hemogenic endothelium into pre-T cell precursors; and, optionally, (ii) contacting the pre-T cell precursor with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not including one or more of VEGF, bFGF, TPO, BMP activators, and a ROCK inhibitor, to initiate differentiation of the pre-T cell precursor into T cell precursors or T cells. In some embodiments of the method, the T cell precursor derived from the pluripotent stem cells is CD34+CD45+CD7+. In some embodiments of the method, the T cell precursor derived from the pluripotent stem cells is CD45+CD7+.

[0228] In some further embodiments of the above-described methods for directing differentiation of pluripotent stem cells into cells of a hematopoietic lineage, the method further comprises: (i) contacting the definitive hemogenic endothelium derived from the 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 differentiation of the definitive hemogenic endothelium into pre-NK cell precursors, and optionally (ii) contacting the pre-NK cell precursor derived from the 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 including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to initiate differentiation of the pre-NK cell precursor into an NK cell precursor or an NK cell. In some embodiments, the pluripotent stem cell-derived NK progenitor cells are CD3-CD45+CD56+CD7+. In some embodiments, the pluripotent stem cell-derived NK cells are CD3-CD45+CD56+, and optionally further defined by NKp46+, CD57+, and CD16+.

[0229] Thus, using the above-described differentiation method, one or more populations of iPSC-derived hematopoietic cells can be obtained: (i) CD34+ HE cells (iCD34), using one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) definitive hemogenic endothelial (iHE), using one or more media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iii) iMPP-A, iTC-A2, iTC-B2, iNK- (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 is: 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 comprises 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 comprises 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 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. e. iNK-A2 comprises 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 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0230] In some embodiments, the genomically engineered iPSC-derived cells obtained from the above-described methods comprise one or more inducible suicide genes integrated into one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of the genomic safe harbor. In some other embodiments, the genomically engineered iPSC-derived cells comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutical active proteins and peptides, drug target candidates, or proteins that promote stem cell and / or progenitor cell trafficking, homing, viability, self-renewal, persistence, and / or survival. In some embodiments, the genomically engineered iPSC-derived cells comprising one or more suicide genes further comprise one or more indels in one or more endogenous genes associated with immune response regulation and mediation, including, but not limited to, checkpoint genes, endogenous T cell receptor genes, and MHC class I suppressor genes. In one embodiment, the genomically engineered iPSC-derived cells containing one or more suicide genes contain an indel in the B2M gene, resulting in a knockout of B2M.

[0231] Further, applicable dedifferentiation methods and compositions for obtaining second fate genomically engineered hematopoietic cells from first fate genomically engineered hematopoietic cells include, for example, those shown in International Publication No. WO2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided therein allow for partial reprogramming of starting non-pluripotent cells into non-pluripotent intermediate cells by limiting the expression of the endogenous Nanog gene during reprogramming, and subjecting the non-pluripotent intermediate cells to conditions for differentiating the intermediate cells into a desired cell type. In some embodiments, the genomically modified iPSCs and derived cells thereof obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0232] V. Therapeutic Use of Induced Immune Cells with Functional Modalities Differentiated from Genetically Engineered iPSCs The invention provides, in some embodiments, compositions comprising an isolated population or subpopulation of functionally enhanced derived 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 are retainable in the iPSC-derived immune cells, and the engineered iPSCs and their derived cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises CD34 cells derived from iPSCs. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises HSC cells derived from iPSCs. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises pro-T cells or T cells derived from iPSCs. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises pro-NK cells or NK cells derived from iPSCs. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises immune regulatory cells or myeloid-derived suppressor cells (MDSCs) derived from iPSCs. In some embodiments, the engineered immune cells derived from iPSCs are further conditioned ex vivo for improved therapeutic potential. In one embodiment, the isolated population or subpopulation of engineered immune cells derived from iPSCs comprises an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of engineered immune cells derived from iPSCs comprises an increased number or proportion of type I NKT cells. In another embodiment, the isolated population or subpopulation of engineered immune cells derived from iPSCs comprises an increased number or proportion of adaptive NK cells. In some embodiments, the isolated population or subpopulation of engineered CD34 cells, HSC cells, T cells, NK cells, or myeloid-derived suppressor cells derived from iPSCs is allogeneic. In some other embodiments, the isolated population or subpopulation of engineered CD34 cells, HSC cells, T cells, NK cells, or MDSC derived from iPSCs is autologous.

[0233] In some embodiments, the iPSCs for differentiation contain genetic imprints selected to convey desirable therapeutic attributes in effector cells, provided that cellular developmental biology is not disrupted during differentiation and that the genetic imprints are retained and functional in differentiated hematopoietic cells derived from said iPSCs.

[0234] In some embodiments, the genetic imprint of the pluripotent stem cells comprises (i) one or more genetic modification modalities obtained by genomic insertion, deletion, or substitution in the genome of the pluripotent cells during or after reprogramming of the 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 therapeutic response-specific, where the pluripotent cells are reprogrammed from source-specific immune cells and the iPSCs retain the therapeutic attributes of the source that are also contained in the hematopoietic lineage cells derived from the iPSCs.

[0235] In some embodiments, the genetic modification modalities include one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and coordination, and / or survival of iPSCs or their derived cells. In some embodiments, the genetically modified iPSCs and their derived cells comprise the genotypes listed in Table 1. In some other embodiments, genetically modified iPSCs and derived cells thereof comprising the genotypes listed in Table 1 further comprise additional genetic modification modalities including (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 of the chromosome 6p21 region, and (2) introduced or increased expression of HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptor, or surface triggering receptor for coupling with a bispecific or multispecific or universal engager.

[0236] In yet some other embodiments, the hematopoietic lineage cells comprise therapeutic attributes of source-specific immune cells relating to a combination of at least two of the following: (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) resistance to the tumor microenvironment, (iv) recruitment and immunomodulation of bystander immune cells, (iv) improved target specificity with reduced extratumoral effects, (v) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0237] In some embodiments, the iPSC-derived hematopoietic cells comprise the genotypes listed in Table 1, and the cells express at least one cytokine and / or its receptor, or any modified protein thereof, including 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 CAR is NK cell specific. In some other embodiments, the engineered expression of the cytokine and 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.

[0238] By introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy, various diseases can be improved. In some embodiments, the iPSC-derived hematopoietic cells provided are for allogeneic adoptive cell therapy. Furthermore, the present invention provides, in some embodiments, a therapeutic use of the above-mentioned 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 infection 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 lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes. Examples of solid cancers include, but are not limited to, cancer 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 alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, and rheumatoid arthritis. Examples of viral infections include, but are not limited to, pemphigoid / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpes virus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella zoster virus), adenovirus-, lentivirus-, and BK polyomavirus-associated diseases.

[0239] Treatment using the induced hematopoietic lineage cells of the embodiments disclosed herein can be on a symptomatic basis or to prevent relapse. The terms "treating", "treatment" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of completely or partially preventing the disease, and / or therapeutic, in terms of partially or completely curing the disease and / or adverse effects resulting from the disease. As used herein, "treatment" encompasses any intervention of a disease in a subject, including: preventing the disease from arising, inhibiting the disease, i.e. arresting its development, or alleviating the disease, i.e. reversing the disease, in a subject susceptible to the disease but not yet diagnosed as having it. Therapeutic agents or compositions can be administered before, during, or after the onset of the disease or injury. Treatment of ongoing diseases, where the treatment stabilizes or reduces the patient's undesirable clinical symptoms, is also of particular interest. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury in which at least one associated symptom can be inhibited, ameliorated, and / or ameliorated by cell therapy. Certain embodiments contemplate that subjects in need of cell therapy include, but are not limited to, bone marrow or stem cell transplant candidates, subjects undergoing chemotherapy or radiation therapy, subjects having or at risk of having a hyperproliferative disorder or cancer, e.g., a hyperproliferative disorder or cancer of the hematopoietic system, subjects having or at risk of developing a tumor, e.g., a solid tumor, subjects having or at risk of having a viral infection or a disease associated with a viral infection.

[0240] When assessing responsiveness to a treatment comprising the induced hematopoietic lineage cells of the embodiments disclosed herein, response can be measured by at least one of clinical benefit rate, survival to death, pathologic complete response, quantitative measurement of pathologic response, clinical complete response, clinical partial response, clinical stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Response Evaluation Criteria in Solid Tumors) criteria.

[0241] Therapeutic compositions 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 the use of additional therapeutic agents. As described above, the one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or supplements thereof, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). Administration of iPSC-derived immune cells can be separated in time by hours, days, or weeks from administration of the additional therapeutic agent. Additionally or alternatively, administration can be combined with other bioactive agents or modalities, such as, but not limited to, anti-tumor agents, non-pharmacologic therapies such as surgery, etc.

[0242] In some embodiments of the combined cell therapy, the therapeutic combination comprises the iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent that is an antibody or 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 capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived hematopoietic lineage cells include, but are not limited to, anti-CD20 (e.g., rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-HER2 (e.g., trastuzumab, pertuzumab), anti-CD52 (e.g., alemtuzumab), anti-EGFR (e.g., certuximab), 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 humanized or Fc-engineered variants or fragments thereof, or functional equivalents or biosimilars thereof.

[0243] In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. A checkpoint refers to a cellular molecule, often a cell surface molecule, that can suppress or downregulate an immune response if not inhibited. A checkpoint inhibitor is an antagonist that can reduce the gene expression or gene product of a checkpoint or reduce the activity of a checkpoint molecule. Checkpoint inhibitors suitable for combination therapy with induced effector cells, including NK cells or T cells, provided herein include 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, These include, but are not limited to, antagonists of 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 inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, 3DL2).

[0244] Some embodiments of the combination therapy including the induced effector cells provided further include at least one inhibitor targeting a checkpoint molecule. Some other embodiments of the combination therapy with the induced effector cells provided 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 therapy described herein are induced NK cells as provided. In some embodiments, the effector cells for the combination therapy described herein are induced T cells. In some embodiments, the induced NK cells or T cells for the combination therapy are functionally enhanced as provided herein. In some embodiments, two, three, or more checkpoint inhibitors can be administered in the combination therapy simultaneously, before, or after administration of the induced effector cells. In some embodiments, two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

[0245] In some embodiments, the antagonist that inhibits any of the above-mentioned checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a mouse 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 may be more cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, the one or two or three or more checkpoint inhibitors comprise at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0246] Combination therapy containing inducible effector cells and one or more check-in inhibitors is being investigated for the treatment of 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 NHL, and leukemia. It can be applied to the treatment of liquid and solid cancers, including but not limited to K-cell 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.

[0247] 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.

[0248] In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethylenimines, methylmelamine, 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, mephalin, chlorambucil, heparinmethylmelamine, 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 amine glutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calcitabine, capecitabine, celecoxib, and cetaxel. Mab, cladribine, cloflavine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, ibuprofen Darubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegadome, pemetazepam, These include gaspargase, pentostatin, pipobroman, plicamycin, porifeprosan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those approved as chemotherapeutic or radiotherapeutic agents and known in the art.Such agents can be found in many standard physician or oncologist reference works (e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, NY, 1995) or on the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated from time to time.

[0249] Immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, stimulate both NK cells and T cells. As provided herein, IMiDs can be used in conjunction with iPSC-derived therapeutic immune cells for cancer treatment.

[0250] In addition to the isolated population of iPSC-derived hematopoietic lineage cells contained in the therapeutic composition, a composition suitable for administration to a patient can further include one or more pharma- ceutically acceptable carriers (additives) and / or diluents (e.g., a pharma- ceutically acceptable vehicle, e.g., cell culture medium), or other pharma- ceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents will be determined, in part, by the particular composition being administered, as well as by the particular method used to administer the therapeutic composition. Thus, there are a wide variety of suitable formulations of the therapeutic compositions of the invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., the disclosure of which is incorporated herein by reference in its entirety). th (see ed. 1985).

[0251] In one embodiment, the therapeutic composition comprises T cells derived from the pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises NK cells derived from the pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises CD34+HE cells derived from the pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises HSCs derived from the pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises MDSCs derived from the pluripotent cells generated by the methods and compositions disclosed herein. The therapeutic compositions comprising 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.

[0252] These pharma- ceutically 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% on a weight-to-weight basis of the total composition. Electrolytes such as, but not limited to, 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, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH in one of the above 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.

[0253] The present invention also provides, in part, the use of pharma- ceutically acceptable cell culture media in certain compositions and / or cultures of the invention. Such compositions are suitable for administration to a human subject. Generally speaking, any medium that supports the maintenance, growth, and / or health of iPSC-derived immune cells according to embodiments of the invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharma- ceutical acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, the serum-free medium is free of animal components and can be optionally protein-free. Optionally, the medium can include pharma- ceutical acceptable recombinant proteins. Animal component-free medium refers to a medium in which components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, protein-free medium is defined as being substantially free of proteins. Those skilled in the art will appreciate 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 there are many suitable media known and available to those skilled in the art.

[0254] The isolated pluripotent stem cell-derived hematopoietic lineage 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, the isolated pluripotent stem cell-derived hematopoietic lineage cells have 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 invention provides therapeutic compositions having purified T cells or NK cells, such as compositions 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.

[0255] In one embodiment, the combination cell therapy comprises an anti-CD38 therapeutic protein or peptide and a population of NK cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, where the induced NK cells comprise CD38 null. In another embodiment, the combination cell therapy comprises an anti-CD38 therapeutic protein or peptide and a population of T cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, where 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 a genotype listed in Table 1, where the induced NK or T cells comprise CD38 null and hnCD16. In still some other embodiments, the combination cell therapy comprises daratumumab and a population of NK or T cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, where 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 a genotype listed in Table 1, where the induced NK or T cells comprise CD38 null, hnCD16, and a CAR and one or more exogenous cytokines.

[0256] As those skilled in the art will understand, both autologous and allogeneic hematopoietic lineage cells derived from iPSC based on the methods and compositions herein can be used in cell therapy as described above.In the case of autologous transplantation, the isolated population of induced hematopoietic lineage cells is HLA-matched completely or partially with the patient.In another embodiment, the induced hematopoietic lineage cells are HLA-mismatched with the subject, where the induced hematopoietic lineage cells are NK cells or T cells with HLAI null and HLAII null.

[0257] In some embodiments, the number of induced hematopoietic lineage cells in the therapeutic composition is at least 0.1 x 10 per dose. 5 Cells, at least 1 x 10 5 Cells, at least 5 x 10 5 Cells, at least 1 x 10 6 Cells, at least 5 x 10 6 Cells, at least 1 x 10 7 Cells, at least 5 x 10 7 Cells, at least 1 x 10 8 Cells, at least 5 x 10 8 Cells, at least 1 x 10 9 cells, or at least 5 × 10 9 In some embodiments, the number of induced hematopoietic lineage cells in the therapeutic composition is about 0.1 x 10 per dose. 5 cells ~ approx. 1×10 6 Cells, approximately 0.5 x 10 per dose 6 cells ~ approx. 1×10 7 Cells, approximately 0.5 x 10 per dose 7 cells ~ approx. 1×10 8 Cells, approximately 0.5 x 10 per dose 8 cells ~ approx. 1×10 9 Cells, approximately 1 x 10 per dose 9 Cells ~ approx. 5 x 10 9 Cells, approximately 0.5 x 10 per dose 9 cells ~ approx. 8 x 10 9 Cells, approximately 3 x 10 per dose 9 cells ~ approx. 3 x 10 10 cells, or any range in between. Generally, for a 60 kg patient, 1 × 10 8 Cells / dose is 1.67 x 10 6 Converted to cells / kg.

[0258] In one embodiment, the number of induced hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a fraction of blood or a single umbilical cord, or at least 0.1 x 10 5 At least 0.5 × 10 cells / kg body weight 5 Cells / kg body weight, at least 1 x 10 5 Cells / kg body weight, at least 5 × 10 5Cells / kg body weight, at least 10 x 10 5 At least 0.75 x 10 cells / kg body weight 6 At least 1.25 x 10 cells / kg body weight 6 Cells / kg body weight, at least 1.5 x 10 6 Cells / kg body weight, at least 1.75 x 10 6 Cells / kg body weight, at least 2 x 10 6 At least 2.5 x 10 cells / kg body weight 6 Cells / kg body weight, at least 3 × 10 6 Cells / kg body weight, at least 4 x 10 6 Cells / kg body weight, at least 5 × 10 6 Cells / kg body weight, at least 10 x 10 6 Cells / kg body weight, at least 15 x 10 6 Cells / kg body weight, at least 20 x 10 6 Cells / kg body weight, at least 25 x 10 6 Cells / kg body weight, at least 30 x 10 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 cells / kg body weight, or 1 x 10 9 cells / kg body weight.

[0259] In one embodiment, a dose of induced hematopoietic lineage cells is delivered to the 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 x 10 6 Cells / kg, at least 4 x 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 x 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 doses.

[0260] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, or approximately 10 x 10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0261] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 4×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 5×10 6 cells / kg ~ approx. 10×10 6 cells / kg, 2×10 6 cells / kg~about 6×10 6 cells / kg, 2×10 6 cells / kg~about 7×10 6 cells / kg, 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 3×10 6 cells / kg~about 6×10 6 cells / kg, 3×10 6 cells / kg~about 7×10 6 cells / kg, 3×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 4×10 6 cells / kg~about 6×10 6 cells / kg, 4×10 6 cells / kg~about 7×10 6 cells / kg, 4×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 5×10 6 cells / kg~about 6×106 cells / kg, 5×10 6 cells / kg~about 7×10 6 cells / kg, 5×10 6 cells / kg ~ approx. 8×10 6 cells / kg, or 6 x 10 6 cells / kg ~ approx. 8×10 6 Cells / kg and includes all intervening cell doses.

[0262] In some embodiments, the therapeutic use of the induced hematopoietic lineage cells is a single dose treatment. In some embodiments, the therapeutic use of the induced hematopoietic lineage cells is a multiple dose treatment. In some embodiments, the multiple dose treatment is a single dose every 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 doses therebetween.

[0263] The composition comprising the population of induced hematopoietic lineage cells of the present invention may be sterile, suitable for administration to a human patient, and may be administered immediately (i.e., without further processing). A cell-based composition that is ready to be administered means that the composition does not require any further processing or manipulation before 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 conditioned prior to administration of one or more agents. For induced hematopoietic lineage cells that are genetically engineered to express a recombinant TCR or CAR, the cells may be activated and expanded using, for example, the methods described in U.S. Patent No. 6,352,694.

[0264] In certain embodiments, the primary and costimulatory signals for induced hematopoietic lineage cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" formation) or to separate surfaces (i.e., in a "trans" formation). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal can be bound to a 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 soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor or an antibody, or other binding agent that binds the agent, as disclosed in US Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen presenting cells (aAPCs) contemplated for use in the activation and proliferation of T lymphocytes in embodiments of the present invention.

[0265] Some variation in dosage, frequency and protocol will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose, frequency and protocol for the individual subject. EXAMPLES

[0266] The following examples are offered by way of illustration and not by way of limitation.

[0267] Example 1 - Materials and Methods To effectively select suicide systems under the control of various promoters and test them in combination with various safe harbor locus integration strategies, Applicant's proprietary hiPSC platform, which allows single cell passaging and high-throughput 96-well plate-based flow cytometry sorting, was used, allowing the derivation of clonal hiPSCs with single or multiple gene modulations.

[0268] Maintenance of hiPSCs in small cell cultures: hiPSCs were routinely passaged as single cells once the cultures reached 75%–90% confluency. For single cell dissociation, hiPSCs were washed once with PBS (Mediatech) and treated with Accutase (Millipore) for 3–5 min at 37°C, followed by pipetting to ensure single cell dissociation. The single cell suspension was then mixed with equal volumes of conventional medium, centrifuged at 225 × g for 4 min, resuspended in FMM, and seeded onto matrigel-coated surfaces. Passages were typically 1:6–1:8, transferred to tissue culture plates precoated with matrigel for 2–4 h at 37°C, and fed with FMM every 2–3 days. Cell cultures were maintained in a humidified incubator set at 37°C and 5% CO2.

[0269] Using human iPSC manipulation with ZFNs, CRISPR for targeted editing of the desired modality:ROSA26 targeted insertion as an example, for ZFN-mediated genome editing, 2 million iPSCs were transfected with a mixture of 2.5 μg ZFN-L (FTV893), 2.5 μg ZFN-R (FTV894) and 5 μg donor construct for AAVS1 targeted insertion. For CRISPR-mediated genome editing, 2 million iPSCs were transfected with a mixture of 5 μg ROSA26-gRNA / Cas9 (FTV922) and 5 μg donor construct for ROSA26 targeted insertion. Transfection was performed using a Neon transfection system (Life Technologies) using the parameters 1500V, 10 ms, 3 pulses. On the second or third day after transfection, transfection efficiency was measured using flow cytometry if the plasmid contained an artificial promoter-driver GFP and / or RFP expression cassette. 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 0.2 μg / ml after 7 days to select target cells. During puromycin selection, cells were passaged into fresh Matrigel-coated wells on day 10. From day 16 of puromycin selection onwards, viable cells were analysed by flow cytometry for the percentage of GFP+ iPS cells.

[0270] Bulk and clonal sorting of genome-edited iPSCs: iPSCs containing genome-targeted edits using ZFNs or CRISPR-Cas9 were bulk- and clonal-sorted for GFP+SSEA4+TRA181+ iPSCs after 20 days of puromycin selection. Single-cell dissociated targeted iPSC pools were resuspended in chilled staining buffer containing Hanks' balanced salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1x penicillin / streptomycin (Mediatech), and 10 mM Hepes (Mediatech), freshly made 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 min. All antibodies were used at 7 μL in 100 μL of staining buffer per million cells. The solution was washed once with staining buffer, spun down at 225g for 4 min, resuspended in staining buffer containing 10 μM thiazobib, and kept on ice for flow cytometric sorting. Flow cytometric sorting was performed on a FACS Aria II (BD Biosciences). For bulk sorting, GFP+SSEA4+TRA181+ cells were gated and sorted into 15 ml standard tubes filled with 7 ml of FMM. For clonal sorting, sorted cells were ejected directly into 96-well plates using a 100 μM nozzle 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 5x Matrigel pre-coating, add one aliquot of Matrigel to 5 mL of DMEM / F12, incubate overnight at 4 °C, resuspend appropriately, and finally add to the 96-well plate at 50 µL per well and incubate overnight at 37 °C. The 5x Matrigel is aspirated immediately before adding medium to each well. Once sorting is complete, the 96-well plate is centrifuged at 225 g for 1-2 min before incubation.The plates were left undisturbed for 7 days. On day 7, 150 μL of medium was removed from each well and replaced with 100 μL of FMM. The wells were re-fed with an additional 100 μL of FMM on day 10 after sorting. Colony formation was detected as early as day 2, and most colonies grew between 7 and 10 days after sorting. For the first passage, the wells were washed with PBS and dissociated with 30 μL of Accutase for approximately 10 min at 37°C. The need for long-term Accutase treatment reflects the compactness of the colonies that did not remain in culture for long periods. After the cells were observed to be dissociated, 200 μL of FMM was added to each well and pipetted several times to disrupt the colonies. The dissociated colonies were transferred to another well of a 96-well plate previously coated with 5× Matrigel and centrifuged at 225 g for 2 min before incubation. This one-to-one passage is performed to expand the initial colonies before they grow. Subsequent passaging was routinely performed with 3–5 min of Accutase treatment and 1:4–1:8 expansion at 75–90% confluency into large wells previously coated with 1× Matrigel in FMM. Each clonal cell line was analyzed for GFP fluorescence and TRA1-81 expression levels. Clonal lines close to 100% GFP+ and TRA1-81+ 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).

[0271] Example 2 - CD38 knockout in iPSCs using CRISPR / Cas9-mediated genome editing Alt-R® SpCas9 D10A Nickase 3NLS, 100 μg, Alt-R® CRISPR-Cas9 tracrRNA were purchased from IDT (Coralville, Iowa) and used for iPSC targeted editing. To perform biallelic knockout of CD38 in iPSCs using Cas9 nickase, the screened and identified targeting sequence pairs (1A and 1B, 2A and 2B, 3A and 3B) for gNA (i.e., gD / RNA or guide polynucleotide) design are listed in Table 3.

[0272] [Table 3]

[0273] Genome-engineered iPSCs were then characterized, and the biallelic CD38 knockout was confirmed.

[0274] Example 3 - CD38 - / - Validation of iPSCs and derived cells CD38 is known to be expressed at certain cell stages and plays an important role in effector cells. During hematopoiesis, CD38 is expressed in combination with CD34 + It is expressed in stem cells and progenitor cells committed to lymphoid, erythroid, and myeloid lineages, as well as in the final stages of maturation of effector cells such as T cells and NK cells. Thus, prior to this application, there was uncertainty and concern as to whether iPSCs containing CD38 knockout would develop properly when exposed to the indicated differentiation conditions, and whether the generated effector cells would be functional, given the CD38 expression profile and functionality. CD38 null iPSCs containing a biallelic 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 biallelic 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.

[0275] 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 that were genetically edited to cause an allelic disruption of the CD38 gene were differentiated into NK cells and compared for their ability to target and kill the tumor cell line RPMI-8266. As shown by the long-term killing assay in Figure 3, over a 48-hour period, 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 induction or the cytotoxicity of induced effector cells.

[0276] Daratumumab (Darzalex) is an anti-cancer drug. It binds to CD38, which is overexpressed on multiple myeloma cells. Daratumumab-mediated multiple myeloma cell killing depends in part on ADCC and largely on NK effector cells. However, CD38 is also expressed on 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 ADCC function when stimulated by the tumor cell line RPMI-8266 in the presence of daratumumab. - / - It is further shown in Figure 7 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). - / - iPSC-derived NK cells, when stimulated in the presence of daratumumab, do not exhibit degranulation and show less cytokine production than CD38-expressing iNK cells.

[0277] Besides CD38 null, induced pluripotent stem cells were also sequentially engineered to obtain expression of high affinity non-cleavable CD16, loss of HLA-I by knockout of B2M gene, loss of HLA-II by knockout of CIITA, overexpression of non-classical HLA molecule HLA-G, and expression of linked IL15 / IL15 receptor alpha constructs. After each manipulation step, iPSCs were sorted for the desired phenotype before the next manipulation step. Engineered iPSCs can be maintained in vitro or for induced cell generation. Figure 4 showed hnCD16 expression, B2M knockout, HLA-G expression, and IL15 / IL15Rα expression in iPSC-derived NK cells. Figure 7 shows introduction of hnCD16 in combination with CD38 knockout in iPSC-derived NK cells. These data indicate that these genetically engineered modalities are maintained during hematopoietic differentiation without perturbing the directed progression of cells to the desired cell fate in vitro.

[0278] Telomere shortening occurs with cellular senescence and is associated with stem cell dysfunction and cellular senescence. Here, we show that mature iNK cells maintain longer telomeres compared with adult peripheral bold NK cells. Telomere length was measured by flow cytometry in iPSCs, adult peripheral blood NK cells, and iPSC-derived NK cells in the G 0 / 1 The DNA index of the cells was determined using the 1301 T cell leukemia line as a corrected control (100%). As shown in Figure 5, iPSC-derived NK cells maintained significantly longer telomere length compared to adult peripheral blood NK cells (p=.105, ANOVA), indicating a higher likelihood of proliferation, viability, and persistence of iPSC-derived NK cells.

[0279] Example 4 - Functional profiling of CD38 Null-induced NK cells The phenotype of hnCD16 CD38 iNK cells, including expression of NKG2A, NKp46, and KIR2DL2 / 3, as well as calcium flux, were evaluated, and the phenotype of hnCD16 iNK is maintained after CD38 knockout (Figure 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 does not affect the ADCC function of hnCD16 iNK (Figure 10E). The specific cytotoxicity of daratumumab against various NK cell populations: peripheral blood NK cells (with CD16 shedding), hnCD16 iNK cells (with high avidity non-cleavable CD16), non-modified iNK cells (with low CD16 expression) and hnCD16 CD38 iNK cells was then 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.

[0280] To assess 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, after which tumor cell viability was assessed by flow cytometry with Annexin V and live / dead viability markers. As shown in Figure 12, hnCD16 iNK cells mediate potent antimyeloma activity with daratumumab, which is further enhanced by CD38 loss. Furthermore, in a cytotoxicity assay against RMPI-8226 tumor spheroids over a 7-day period, hnCD16 CD38- / - iNK cells demonstrated 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 non-modified iNK cells under the same conditions (Figure 13A). Furthermore, the lack of NK cell fratricide improved the viability of hnCD16 CD38- / - iNK cells, as shown by the improved persistence of these cells in the cytotoxicity assay over 7 days shown in Figure 13B. Thus, hnCD16 CD38- / - iNK cells show enhanced long-term anti-myeloma activity and persistence with CD38-specific antibodies such as daratumumab. As shown in Figure 14, hnCD16 iNK cells lacking CD38 also demonstrate more durable ADCC with increased sequential killing capacity in the presence of CD38-specific antibodies. In this assay, hnCD16 or hnCD16 CD38- / - iNK cells were incubated with MM.1S myeloma target cells with daratumumab for 48 hours (stimulation round 1) and MM.1S cell numbers were quantified by Incucyte™ imaging. After 48 hours, effector cells were removed and transferred to new target cells for a second round of stimulation and target cell killing with daratumumab (stimulation round 2) (Figure 14).

[0281] In light of the above, it was found that targeted knockout of CD38 does not affect either the phenotype of induced NK cells or general cell function, and the resulting CD38-deficient induced NK cells are protected from CD38-specific antibody, daratumumab, for example, mediated fratricide. The synergistic effect of hnCD16 and CD38- / - provides enhanced antimyeloma activity and durable ADCC of induced NK cells in combination with CD38-specific antagonists, including monoclonal antibodies such as daratumumab. Based on these findings, a clinical strategy is proposed to overcome the NK cell-depleting effect of CD38-targeted drugs and improve outcomes for myeloma patients by combining off-the-shelf hnCD16CD38- / - iNK cells with daratumumab.

[0282] Example 5 - Use of CD38-specific antagonists to protect allogeneic effector cells from allogeneic rejection Engineered iNK cells with enhanced CD16 efficacy and CD38 ablation are resistant to CD38-targeted antibody-induced fratricide and more potently mediate antimyeloma activity in combination with daratumumab. In recipients of allogeneic effector cells lacking CD38, the fact that CD38 is upregulated on activated lymphocytes such as T or B cells reduces and / or prevents allorejection of these effector cells, thereby increasing the survival and persistence of effector cells. To demonstrate the feasibility of this strategy, mixed lymphocyte reactions (MLRs, i.e., co-incubation of effector cell products with allogeneic PBMCs) are performed to test the longevity of the induced effector cells of the present invention in an allogeneic environment in the context of CD38 knockout and in the presence or absence of anti-CD38 monoclonal antibodies (e.g., daratumumab).

[0283] hnCD16+ iNK cell populations (with and without CD38KO) are labeled with an intracellular 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 effective concentrations, titrated prior to assay). Viability of iNK cells from each population is monitored over time by Incucyte™ in long-term cultures. 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 daratumumab-mediated ADCC. As a control, total clearance of iNK cells is achieved using Venetoclax (an MCL-1 inhibitor, for specific depletion of NK cells).

[0284] The extended life span 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 indicates the ability of CD38-specific antagonists in suppressing activated peripheral T or B cells by targeting upregulated CD38 on activated peripheral T or B cells, thereby reducing allorejection of allogeneic effector cells by these activated peripheral T or B cells in recipients of effector cells containing 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).

[0285] Furthermore, expression of exogenous truncated IL15 / IL15Rα fusion proteins lacking the intracellular domain of IL15Rα was shown to support the viability of iPSC-derived NK cells in vitro, regardless of the addition of soluble exogenous IL2. IL15Rα, which does not have the intracellular domain, was fused to IL15 at the C-terminus via a linker to generate a truncated IL15 / IL15Ra fusion (or referred to in this application as "IL15Δ") construct lacking the signaling domain. Exemplary IL15Δ provided herein include those having structures such as design 3 or 4 in FIG. 1. As shown in FIG. 15, iNK cells were transduced with lentiviral overexpression vectors expressing either GFP (squares; negative control), a full-length IL15 / IL15Ra fusion construct (filled circles; positive control; design 2 in FIG. 1), or a truncated IL15 / IL15Ra fusion construct (open circles; design 3 in FIG. 1). Neither the IL15 construct nor the GFP showed enrichment in the presence of exogenous IL2 (Figure 15A), indicating that the transduced cells survived at rates comparable to non-transduced cells. In the absence of exogenous IL2, cells transduced with either IL15 / IL15Ra fusion construct were enriched over time, whereas GFP-transduced cells were not, and in the absence of IL2, cells transduced with either IL15 / IL15Ra construct had a survival advantage compared to non-transduced cells in the same culture (Figure 15B). Furthermore, since the intracellular domain of IL15Rα is considered important for the receptor to be expressed in IL15-responsive cells and for cells to proliferate and function in response, it is surprising that an IL15 / IL15Ra fusion construct with a truncated intracellular domain is not only stably expressed in transduced iNK cells, but also supports 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 is capable of expressing and maintaining IL15 in a membrane-bound form and can replace the full-length IL15 / IL15Ra fusion protein to provide trans-presentation of IL15 within cells.Although we do not fully understand the underlying mechanisms, it appears that deleting the intracellular domain of IL15R conferred additional vigor, fitness, or a specific advantage in survival, proliferation, and persistence to the responding cells, presumably by completely eliminating cis-presentation and / or other potential signaling pathways mediated by normal IL15R via its intracellular domain.

[0286] Example 6 - CD38-targeted transgene knock-in constructs resulting in knockout of CD38 and engineered cells derived therefrom Inserting one or more transgenes into selected locations of CD38 to simultaneously knock out CD38 was achieved by construct design exemplified in FIG. 16. With respective CD38 targeting homology arms for site-selective insertion, the constructs provided herein allow transgene(s) to be expressed under CD38 endogenous or exogenous promoters included in the construct (compare A-B and C-D in FIG. 16). The selective insertion / knockout location within the CD38 locus is compatible with the sequence of the adjacent left and right homology arms (LHA / CD38 and RHA / CD38) included in the construct. LHA / CD38 and RHA / CD38 may have various lengths and sequences depending on the preselected targeting site within the CD38 locus. In some embodiments, the preselected targeting site is within an exon of CD38. When more than one transgene is inserted into selected locations of the CD38 locus, a linker sequence, e.g., 2A linker or IRES, is placed between any two transgenes. The 2A linker encodes self-cleaving peptides (also called "F2A", "E2A", "P2A" and "T2A", respectively) derived from FMDV, ERAV, PTV-1 and TaV, allowing the production of separate proteins from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter can be CAG or other constitutive, inducible, time-specific, tissue-specific or cell type-specific promoters, including but not limited to CMV, EF1α, PGK and UBC.

[0287] As an example of this example, the one or more transgenes included in the CD38-targeted knock-in / knock-out (KI / KO) construct include one or both of hnCD16 and an IL15 / IL15R fusion protein (IL15RF). In some embodiments, as provided in Section I(2), hnCD16 includes CD16 including F176V and S197P, or includes CD16 including F176V but without a cleavage domain, or includes CD16 including at least a portion of the CD64 ectodomain. In some embodiments, as provided in Section I(4), IL15RF includes an IL15 / IL15Rα fusion protein with or without cytoplasmic domain cleavage.

[0288] Thus, provided herein are constructs for CD38 targeted knock-in and knock-out, the constructs comprising LHA / CD38, a first transgene, and RHA / CD38, the constructs knocking out CD38 expression while inserting the first transgene into CD38 at a selected location, and optionally the first transgene is operably linked to the endogenous promoter of CD38 upon integration, thereby being driven by the endogenous promoter of CD38. In some embodiments, the constructs comprising LHA / CD38, a first transgene, and RHA / CD38 further comprise a second transgene, and a linker between the first and second transgenes. In some embodiments, the constructs comprising LHA / CD38, one or more transgenes, and RHA / CD38 further comprise an exogenous promoter, the constructs knocking out CD38 expression while inserting the one or more transgenes into CD38 at a selected location, and the one or more transgenes are driven by the exogenous promoter. In one embodiment of a construct comprising LHA / CD38, one or more transgenes, and RHA / CD38, the transgenes express one or both of the hnCD16 and IL15RF proteins, and are detailed in Figure 1 and sections I(2) and I(4), respectively. Figures 17 and 18 show exemplary sequences of constructs designed to knock out CD38 expression while inserting at a selected location in the CD38 locus hnCD16 and IL15RF (IL15RF truncated in this particular example) driven by the CAG promoter (Figure 17) or by the CD38 endogenous promoter (Figure 18). As presented in the figures and as would be understood by one of skill in the art, some of the components included in the constructs shown in Figures 17 and 18 are not essential as they are optional, and the nucleic acid sequences for some of the components included may vary and may have less than about 95%, 90%, 85%, 80%, 75%, 70% but more than 50% sequence identity to the exemplary nucleic acid sequence of each component or the entire construct presented in the figures.

[0289] Constructs containing (i) LHA / CD38, hnCD16, and RHA / CD38, or (2) LHA / CD38, IL15RF hnCD16, and RHA / CD38, or (3) LHA / CD38, IL15RF, hnCD16, and RHA / CD38, respectively, were transduced to engineer induced pluripotent stem cells using CRISPR editing tools, e.g., Cas9 or Cpf1, as described in this application. The efficiency of biallelic targeted insertion with biallelic knockout was found to be about 2-3% in cells initially sorted by IL15 or CD16. Notably, the biallelic knockout efficiency is higher among monoallelic targeted integrations (about 5%). Thus, biallelic targeted integration with simultaneous biallelic knockout would involve sequential (or stepwise) CD38 knockout (which could be 30% to 70% in this step) followed by insertion into CD38 null cells to induce CD38 knockout containing the transgene. - / - It appears to be a rare event compared to obtaining iPSCs (usually 25%-80%, depending on the vector used). However, this method of knocking out CD38 using CD38-targeted transgene integration surprisingly reduces the occurrence of karyotypic abnormalities during long-term passaging and maintenance of the resulting engineered iPSCs compared to iPSCs that have been subjected to multiple sequential genome editing events. Without wishing to be bound by theory, such unexpected genomic stability could be attributed to reduced stress factors associated with the manipulation of iPSCs from multiple genomic disturbances, including single cell selection, among other unknown reasons.

[0290] After clonal selection of engineered iPSCs, donor vector validation, vector clearance, sequencing confirmation of insertions and knockouts, flow cytometry of iPSC markers and transgene phenotype, transgene copy number validation and karyotype validation, genome-edited clonal iPSCs were differentiated according to the methods provided herein into effector cells, such as iNK cells in this example. Depending on the construct used, the obtained iPSC-derived NK cells have at least one of the following genotypes: CD38 - / - CD16;CD38 - / - IL15; and CD38 - / - CD16 IL15. None of these are present in natural / natural counterpart NK cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue. CD38 engineered with CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct compared to non-engineered iNK cells. - / - Flow cytometric phenotyping of CD16IL15 iNK cells is shown in Figure 19. CD38 - / - CD16 IL15 iNK cells showed improved in vitro persistence when cultured for 4 days in the absence of IL-2 or IL-15 compared to similarly treated non-manipulated iNK (FTi250) (see FIG. 20). Furthermore, induction of fratricide by daratumumab or rituximab was not observed in non-manipulated iNK or CD38 - / - As tested on CD16 IL15 iNK cells and shown in Figure 21, - / - Only CD16IL15iNK cells, but not non-manipulated iNK cells, were able to avoid fratricide induced by CD38-specific antibodies such as daratumumab. Furthermore, direct cytotoxicity and ADCC were demonstrated in combination with daratumumab antibody to target Raji cells and inhibit CD38 - / - As shown in Figure 22, CD38 - / - CD16IL15 iNK cells exhibited superior ADCC compared with unmodified iNK cells and demonstrated significantly enhanced daratumumab antibody-induced killing of CD38-expressing Raji cells.

[0291] Those skilled 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 those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0292] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0293] The disclosure illustratively described herein can be implemented without any element or elements, limitations, or limitations not specifically disclosed herein. Thus, for example, in each example herein, the terms "comprise", "essentially consist of" and "consist of" can be replaced with either of the other two terms. The terms and expressions used are used as terms of description, not of limitation, and there is no intention in using such terms and expressions to exclude any equivalent of the features shown and described or any part thereof, but it is recognized that various modifications are possible within the scope of the disclosure as claimed. Thus, although the disclosure is specifically disclosed by preferred embodiments and necessary features, modifications and variations of the concepts disclosed herein may often occur to those skilled in the art, and such modifications and variations are within the scope of the invention as defined by the appended claims.

Claims

1. A construct for engineering a cell to obtain a genome-edited cell, comprising: (i) a left homology arm and a right homology arm (LHA / CD38, RHA / CD38) that target CD38 at a selected location of the CD38 locus in the genome of the cell, which (ii) is operably linked to one or more nucleic acid sequences encoding one or more exogenous proteins; the genome-edited cell is an induced pluripotent cell (iPSC), a clonal iPSC, or an iPS cell line cell; the genome edited cell comprises targeted integration of the one or more nucleic acid sequences encoding one or more exogenous proteins at the selected positions of the CD38 locus, and a CD38 knockout; The construct, wherein the genome edited cell is an iPSC capable of differentiating into an iPSC-derived hematopoietic cell.

2. The construct of claim 1, wherein the one or more nucleic acid sequences encoding one or more exogenous proteins are operably linked to and driven by the endogenous promoter of CD38 upon integration, or the selected position of the CD38 locus is in an exon of CD38.

3. 2. The construct of claim 1 , wherein the one or more exogenous proteins comprise at least one or both of the following: (i) high affinity non-cleavable CD16 (hnCD16) or a variant thereof, (ii) exogenous IL15 / IL15 receptor fusion protein (IL15RF) or a variant thereof expressed on the cell surface, wherein the genome edited cell and the derived hematopoietic cells therefrom comprise the one or more nucleic acid sequences encoding hnCD16 and IL15RF, or variants thereof.

4. (i) the 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) transmembrane, signaling, and stimulatory domains not derived from CD16 and derived from the same or a different polypeptide; or or (ii) the IL15RF or variant thereof is (a) IL15 and IL15Rα co-expressed by using a self-cleaving peptide; (b) a fusion protein of IL15 and IL15Rα; (c) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα has been truncated; (d) a fusion protein of the membrane-bound Sushi domain of IL15 and IL15Rα; (e) a fusion protein of IL15 and IL15Rβ; (f) a fusion protein of IL15 and common receptor γC, said common receptor γC being native or modified; (g) IL15Rβ homodimer, and (h) a polynucleotide encoding IL15RF comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21; The construct of claim 3, comprising at least one of:

5. The hnCD16 or a variant thereof (i) a non-native transmembrane domain 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; (ii) a non-native stimulatory domain derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide; (iii) a non-native signaling domain derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide; or (iv) 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 construct of claim 4, comprising one or more of:

6. The genome edited cells and the derived hematopoietic cells therefrom further comprise a chimeric antigen receptor (CAR), the CAR having the following characteristics: (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 a partial or complete peptide of a cell surface-expressed exogenous cytokine or its receptor; (xi) co-expressed with a checkpoint inhibitor; (xii) encoded by a nucleic acid further comprised in the construct, wherein the CAR is inserted at the selected position in the CD38 locus; (xiii) inserted into the TRAC locus and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by the CAR insertion; (xiii) specific for CD19 or BCMA, and / or (xiv) 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 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), 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 (M SLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal 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; The construct of claim 1 , comprising at least one of:

7. (i) an exogenous promoter driving expression of said one or more nucleic acid sequences encoding one or more exogenous proteins, wherein said exogenous promoter is constitutive, inducible, time-specific, tissue-specific, and / or cell-type specific; (ii) a linker sequence inserted between the two nucleic acid sequences encoding the exogenous proteins; (iii) an insulator at the 3' end of LHA / CD38 and an insulator at the 5' end of RHA / CD38; and (iv) polyA signal, The construct of claim 1 , further comprising one or more of:

8. (i) the exogenous promoter is CMV, EF1α, PGK, CAG, or UBC; (ii) the linker sequence is a 2A sequence encoding a self-cleaving 2A peptide; and / or (iii) the linker sequence comprises an internal ribosome entry site (IRES). The construct of claim 7,

9. The genome-edited cells or the iPSC-derived hematopoietic cells are iPSCs, and have the following phenotype: (i) contain longer telomeres compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue; (ii)CD38 －／－ CD16、 (iii) CD38 －／－ IL15, and / or (iv)CD38 －／－ CD16 IL15 2. The construct of claim 1, comprising one or more of:

10. The genome-edited cells or the iPSC-derived hematopoietic cells are iPSCs, (i) B2M null or low; (ii) CIITA null or low; (iii) induced expression of HLA-G or non-cleavable HLA-G; (iv) deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region; (v) HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ Induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, an Fc receptor, an engager, and a bispecific or multispecific or universal engager; 2. The construct of claim 1, further comprising one or more of:

11. The iPSC-derived hematopoietic 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, or the iPSC-derived hematopoietic cells comprise induced NK cells or induced T cells, and are 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; (viii) improved ability to rescue tumor antigen escape; and (ix) reduced fratricide in the presence of an anti-CD38 antibody or a CD38 CAR; 2. The construct of claim 1 having at least one feature comprising:

12. 12. The construct of claim 11, 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.

13. An expression vector comprising the construct according to any one of claims 1 to 12.

14. A host cell comprising a construct according to any one of claims 1 to 12, or an expression vector according to claim 13.

15. A composition comprising the construct of any one of claims 1 to 12 or the expression vector of claim 13 for producing genome-edited iPSCs or induced hematopoietic cells differentiated therefrom.

16. A kit comprising a construct according to any one of claims 1 to 12, an expression vector according to claim 13, or a host cell according to claim 14, and one or more media for iPSC culture or differentiation.

17. A construct according to any one of claims 1 to 12, an expression vector according to claim 13 or a host cell according to claim 14 for use in medicine.

18. A method for producing genome-edited iPSCs or hematopoietic cells derived therefrom using the construct according to any one of claims 1 to 12, comprising: (i) introducing the construct into iPSCs; (ii) incubating the iPSCs for a sufficient period of time to allow targeted integration of one or more nucleic acid sequences encoding one or more exogenous proteins into the CD38 locus, thereby knocking out the expression of CD38 in the iPSCs to obtain genome-edited iPSCs.

19. 20. The method of Claim 18, further comprising: (iii) inducing differentiation of the genome edited iPSCs from step (ii) into hematopoietic cells comprising targeted integration of one or more nucleic acid sequences encoding one or more exogenous proteins at the same CD38 locus as the genome edited iPSCs and CD38 knockout.

20. 20. The method of claim 18, wherein the genome-edited iPSCs have improved genomic stability over iPSCs that undergo integration and knockout in separate, stepwise editing events.

21. 20. The method of claim 18, wherein the introducing step further comprises introducing a CRISPR-cas / gNA (guide nucleic acid), a ZFN, a TALEN, a homing nuclease, or any other functional mutation thereof.

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