Manipulated immune effector cells and their use

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 on 29 March 2018, U.S. Provisional Patent Application No. 62 / 774,052 filed on 30 November 2018, and International Application PCT / US18 / 67289 filed on 21 December 2018, the disclosures thereof incorporated herein by reference in their entirety.

[0002] References to electronically submitted sequence listings This application incorporates, by reference, a computer-readable format (CRF) of an ASCII text sequence listing, titled 13601-204-228_SEQ_LISTING.txt, created on 28 March 2018, and measuring 45,867 bytes, which was submitted together with this application.

[0003] This disclosure broadly relates to the field of ready-made immune cell products. More specifically, this disclosure relates to strategies for developing multifunctional effector cells that can deliver therapeutically appropriate properties in vivo. Cell products developed under this disclosure address significant limitations of patient-derived cell therapies. [Background technology]

[0004] The field of adoptive cell therapy currently focuses on using patient-derived and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapy and to deliver therapies to all patients who could potentially 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 anti-tumor effectors that play a crucial role in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains challenging, and there is an unmet need for improvement. Therefore, there remains a significant opportunity to maximize the potential of T cells, NK cells, or other lymphocytes in adoptive immunotherapy. [Overview of the Initiative]

[0005] Functionally improved effector cells are needed to address a variety of issues, including response rate, cell depletion, loss of infused cells (viability and / or persistence), tumor escape due to target loss or lineage change, accuracy of tumor targeting, extratarget toxicity, extratumor effects, and efficacy against solid tumors, i.e., the tumor microenvironment and associated immunosuppression, recruitment, transport, 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) clone line, wherein the iPSC line contains one or more genetic modifications in its genome. The aforementioned one or more genetic modifications include DNA insertions, deletions, and substitutions, and these modifications are retained and continue to function in subsequent induced cells after differentiation, expansion, passage, and / or transplantation.

[0007] The iPSC-derived non-pluripotent cells of this application include, but are not limited to, CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, and B cells. The iPSC-derived non-pluripotent cells of this application contain one or more genetic modifications in their genomes through differentiation from iPSCs containing the same genetic modification. The engineered clonal iPSC differentiation strategy for obtaining genetically engineered induced cells requires that the possibility of iPSC development in the directed differentiation is not adversely affected by the engineered modality of the iPSC, and that the engineered modality functions as intended in the induced cells. Furthermore, this strategy overcomes the current barriers to manipulating primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely the difficulty in manipulating such cells, which often result in cells lacking reproducibility and uniformity, exhibiting insufficient cellular persistence with high cell death and low cell proliferation. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations, which are obtained by other means using a primary cell source that is initially heterogeneous.

[0008] Several aspects of the present invention provide genome-engineered iPSCs obtained using methods comprising (I), (II), or (III), respectively, which reflect a genome engineering strategy, simultaneously with and prior to a reprogramming process:

[0009] (I): Genetically engineer iPSCs using one or both of (i) and (ii) in any order: (i) introduce one or more constructs into the iPSCs to enable targeted integration at selected sites; (ii)(a) introduce one or more double-strand breaks into the iPSCs at selected sites using one or more endonucleases capable of recognizing selected sites; (b) culture the iPSCs from step (I)(ii)(a) to enable endogenous DNA repair to generate targeted indels at selected sites; thereby obtaining genomically engineered iPSCs capable of differentiating into partially or fully differentiated cells.

[0010] (II): Genetically engineer reprogrammed non-pluripotent cells to obtain genomically engineered iPSCs: (i) Contact non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and, optionally, a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to initiate the reprogramming of the non-pluripotent cells; (ii) Introduce one or both of (a) and (b) into the reprogrammed non-pluripotent cells of step (II)(i) in any order: (a) Targeted integration at selected sites(s) (b) one or more constructs that enable the following: (a) one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, and then culturing the cells from step (II)(ii)(b) to perform endogenous DNA repair and enable the generation of targeted indels at the selected site; thus the obtained genome-engineered iPSCs contain at least one functionally targeted genome edit, and the aforementioned genome-engineered iPSCs can differentiate into partially or completely differentiated cells.

[0011] (III): Genetically engineer non-pluripotent cells for reprogramming to obtain genome-engineered iPSCs including (i) and (ii): (i) Introduce one or both of (a) and (b) into non-pluripotent cells in any order: (a) One or more constructs that enable targeted integration at selected sites; (b) Make one or more double-strand breaks at the selected sites using at least one endonuclease capable of recognizing the selected sites, and then culture the cells from step (III)(i)(b) to perform endogenous DNA repair at the selected sites (ii) the cells of step (III)(i) are contacted 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 genome-engineered iPSC having targeted editing at a selected site, thereby obtaining a genome-engineered iPSC having at least one functionally targeted genome edit at a selected site, the genome-engineered iPSC being able to differentiate into partially differentiated or fully differentiated cells.

[0012] In one embodiment of the method described above, at least one targeted genome edit 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, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of genome-engineered iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides for insertion are operably ligated 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) one or more endogenous promoters contained in a selected site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genome-safe harbor. In some embodiments, the genome-engineered iPSCs produced using the methods described above comprise one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, where, if the genome-engineered iPSC comprises two or more suicide genes, the suicide genes are incorporated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta-2 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, partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors, encoded by exogenous polynucleotides, are in the form of fusion proteins.

[0013] In some other embodiments, the genome-engineered iPSCs produced using the methods provided herein include indels in one or more endogenous genes related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response modulation and regulation, or proteins that suppress engraftment, transport, homing, viability, self-renewal, persistence, and / or viability of iPSCs or their induced 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 genes in the chromosome 6p21 region.

[0014] In some other embodiments, the genome-engineered iPSCs produced using the methods provided herein include a caspase encoding an exogenous polynucleotide at the AAVS1 locus and a thymidine kinase encoding an exogenous polynucleotide at the H11 locus.

[0015] In several other embodiments, approaches (I), (II), and / or (III) further include contacting the genetically engineered iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain the pluripotency of the genetically engineered iPSCs. In one embodiment, the obtained genetically engineered iPSCs containing at least one targeted genome edit are functional, differentiateable, and can differentiate into non-pluripotent cells containing the same functional genome edit.

[0016] The present invention also provides the following:

[0017] One aspect of this application is a cell or population thereof, wherein the cell is an induced pluripotent cell (iPSC), a cloned iPSC, or an iPS cell line, or an induced cell obtained by differentiating any of the above iPSCs; any of the above cells comprises at least one CD38 knockout or a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) that does not have an intracellular domain. In some embodiments of induced cells obtained from iPSC differentiation, the induced cells are hematopoietic cells including, but not limited to, CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, and B cells; these hematopoietic cells (i.e., induced CD34 cells, induced hematopoietic endothelial cells, induced hematopoietic stem cells and progenitor cells, induced hematopoietic pluripotent progenitor cells, induced T cell progenitor cells, induced NK cell progenitor cells, induced T cells, induced NKT cells, induced NK cells, or induced B cells) contain longer telomeres compared to their native corresponding cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue.

[0018] In some embodiments of the iPSC and its derived cells containing a CD38 knockout or a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) lacking an intracellular domain, the cells further include one or more of the following genome edits: (i) B2M null or low, (ii) CIITA (iii) null or low, (iv) introduced expression of HLA-G or uncleaved HLA-G, (iv) high affinity uncleaved CD16 (hnCD16) or its variants, (v) chimeric antigen receptor (CAR), (vi) exogenous cytokine expressed on the cell surface 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 any of the genes in the chromosome 6p21 region: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Introduced or increased expression in at least one of the following surface trigger receptors for binding to R, CAR, TCR, Fc receptors, engagers, and bispecific, multispecific, or universal engagers.

[0019] In some embodiments of the iPSC and its derived cells, which include a polynucleotide encoding an IL15 / IL15Rα fusion protein (IL15Δ) that has at least a CD38 knockout or intracellular domain, and additional genome editing as optionally described above and throughout this application, the cells may include (i) one or more exogenous polynucleotides incorporated into one safe harbor locus, or (ii) three or more exogenous polynucleotides incorporated into different safe harbor loci, or (iii) a polynucleotide encoding IL15Δ that has at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NOs: 17, 19, or 21. In some embodiments, the safe harbor locus includes at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1. In a particular embodiment, the safe harbor locus TCR is the constant region of TCR alpha.

[0020] In some embodiments of cells or populations, cells comprising at least CD38 knockout or IL15Δ and one or more of the additional genome edits described above are induced NK or induced T cells, which comprise at least one of the following features, but are not limited to: (i) improved persistence and / or viability, (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 mobilize bystander immune cells to tumor sites, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability to rescue tumor antigen escapes, and (ix) reduced fracturides, compared to its native corresponding NK or T cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue.

[0021] In one embodiment of a cell or population of cells, cells containing CD38 knockout or IL15Δ further comprise high-affinity non-cleaved CD16 (hnCD16) or a variant thereof. Some embodiments of high-affinity non-cleaved CD16 (hnCD16) or a variant thereof comprise at least one of the following: (a) F176V and S197P in the external domain of CD16, (b) a complete or partial external domain derived from CD64, (c) a non-natural (or non-CD16) transmembrane domain, (d) a non-natural (or non-CD16) intracellular domain, (e) a non-natural (or non-CD16) signaling domain, (f) a non-natural stimulating domain, and (g) transmembrane, signaling, and stimulating domains not derived from CD16 but from the same or a different polypeptide. In some embodiments, the non-native transmembrane domain is derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor (TCR) polypeptide. In some embodiments, the non-native stimulating domain is derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some other embodiments, the non-natural signaling domain is derived from the CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In some specific embodiments of the hnCD16 variant, the non-natural transmembrane domain is derived from NKG2D, the non-natural stimulating domain is derived from 2B4, and the non-natural signaling domain is derived from CD3ζ.

[0022] In one embodiment of the cells or population, the CD38 knockout or IL15Δ-containing cells further contain a chimeric antigen receptor (CAR), the CAR may be 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 partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface, either in a separate construct or a bicistronic construct as needed, (xi) co-expressed with a checkpoint inhibitor, either in a separate construct or a bicistronic construct as needed, (xii) specific to 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, antigens 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-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19).9) Specific to any one of the following: 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), mesoserin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryonic 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 the checkpoint inhibitor is co-expressed with 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 inhibitors co-expressed with the CAR may be antibodies specific to any of the checkpoint molecules described above, or humanized or Fc-modified variants or fragments, as well as their functional equivalents and biosimilars. In some embodiments, one of the CARs (i) to (ix) can be inserted into the TRAC locus. In some embodiments, one of the CARs (i) to (ix) inserted into the TRAC locus may be driven by the endogenous promoter of the TCR. In some embodiments, insertion of one of the CARs (i) to (ix) into the TRAC locus results in TCR knockout.

[0024] In one embodiment of a cell or population, a cell containing CD38 knockout further comprises a partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface, the exogenous cytokine or its receptor may comprise at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and their respective receptors, or (i) co-expression of IL15 and IL15Rα using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, or (iii) IL15 The fusion protein may include at least one of the following: (iv) an IL15 / IL15Rα fusion protein in which the intracellular domain of Rα is cleaved; (v) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and the common receptor γC, where the common receptor γC is native or modified; and (vii) a homodimer of IL15Rβ, where any one of (i) to (vii) may be co-expressed with CAR in a separate construct or in a bicistronic construct. In some embodiments, a partial or complete peptide of an exogenous cytokine or receptor on the cell surface is transiently expressed in the cells provided herein.

[0025] In another embodiment of the cell or population, the cell comprises a partial or complete peptide of an exogenous cytokine or receptor expressed on its cell surface, the cytokine may comprise IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and at least one of their respective receptors. In embodiments of cells or populations containing the IL15 cytokine or receptor, the cells may include: (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 is cleaved; (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and the common receptor γC, where the common receptor γC is native or modified; and (vii) at least one homodimer of IL15Rβ, where any one of (i) to (vii) may be co-expressed with CAR in a separate construct or in a bicistronic construct. In some embodiments, a partial or complete peptide of an exogenous cytokine or receptor on the cell surface is transiently expressed in the cells provided herein. In one embodiment, the cells or population contain a polynucleotide encoding IL15Δ which has at least 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NOs. 17, 19, or 21.In one embodiment of the cells or population, cells containing IL15Δ express B2M null or low, CIITA null or low, HLA-G or uncleaved HLA-G, high affinity uncleaved CD16 (hnCD16) or its variants, chimeric antigen receptor (CAR), exogenous cytokines or partial or complete peptides of their receptors expressed on the cell surface (cytokines are not IL15), at least one of the genotypes listed in Table 1, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and chromosomes. The embodiments may further include deletion or reduced expression of at least one of any genes in the 6p21 region, and 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 receptors, engagers, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers. In embodiments of cells or populations containing both IL15Δ and CAR, IL15Δ may be co-expressed with CAR in a separate construct or a bicistronic construct.

[0026] In one embodiment of the cells or population of cells, the CD38 knockout or IL15Δ-containing cells are induced NK cells or induced T cells, which can mobilize and / or migrate T cells to the tumor site, and which can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. In some embodiments, the checkpoint inhibitor is an antagonist to one or more checkpoint molecules, including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some other embodiments, the checkpoint inhibitor comprises (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0027] Another aspect of this application provides compositions comprising any cells or populations thereof as described above and throughout this application. In some embodiments, iPSCs or iPSC-derived cells (induced cells) may comprise any one of the genotypes listed in Table 1 of this application. In some embodiments, iPSCs or induced cells therefrom comprise CD38 knockout (CD38- / -). In some embodiments, iPSCs or induced cells therefrom comprise IL15Δ. In some embodiments, iPSCs or induced cells therefrom comprise hnCD16 and CD38 knockout. In some embodiments, iPSCs or induced cells therefrom comprise hnCD16 and IL15Δ. In some embodiments, iPSCs or induced cells therefrom comprise hnCD16, CD38 knockout, and IL15Δ. In some embodiments, iPSCs or induced cells therefrom comprise hnCD16, CD38- / -, and CAR. In some embodiments, iPSCs or induced cells therefrom comprise hnCD16, IL15Δ, and CAR. In some embodiments, iPSCs or cells derived therefrom contain hnCD16, IL15Δ, CD38- / -, and CAR. In some embodiments, iPSCs or cells derived therefrom contain hnCD16, CD38- / -, CAR, and partial or complete peptides of exogenous cytokines or their receptors expressed on the cell surface, as described above and throughout this application. In some embodiments of cells containing hnCD16, CD38- / -, and CAR, CAR is CD19 specific. In some embodiments of cells containing hnCD16, IL15Δ, and CAR, CAR is CD19 specific. In some embodiments of cells containing hnCD16, IL15Δ, CD38- / -, and CAR, CAR is CD19 specific. In other embodiments of cells containing hnCD16, CD38- / -, and CAR, CAR is CD269 (BCMA) specific. In other embodiments of cells containing hnCD16, IL15Δ, and CAR, CAR is CD269 (BCMA) specific.In some other embodiments of cells containing hnCD16, IL15Δ, CD38- / -, and CAR, CAR is specific to CD269 (BCMA). In some other embodiments, CAR is specific to 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, and antigens of cytomegalovirus (CMV)-infected cells. (For example, 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-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2) ), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion 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), mesoserine (MSLN) It is specific to any one of the following: NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryonic 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.

[0028] Accordingly, further embodiments of this application provide compositions for therapeutic use comprising one or more therapeutic agents in addition to any induced cells provided herein. In some embodiments of the compositions for therapeutic use, the therapeutic agent comprises peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory agents (IMiDs). In some embodiments of the composition for therapeutic use, the checkpoint inhibitor used with the provided cells comprises 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 composition for therapeutic use, the checkpoint inhibitor used with the provided cells comprises one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents. In some other embodiments of the composition for therapeutic use, the checkpoint inhibitor used with the provided cells comprises at least one of atezolizumab, nivolumab, and pembrolizumab. In some embodiments of the composition for therapeutic use, the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide.

[0029] In some embodiments of the composition for therapeutic use, the antibody used with the provided cells comprises one of the following: anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies. In some embodiments of the composition for therapeutic use, the antibody used with the provided cells comprises one or more of the following: rituximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cerltuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and their humanized or Fc-modified variants or fragments, as well as their functional equivalents and biosimilars. In several other embodiments of the composition for therapeutic use, the antibody used with the provided cells comprises daratumumab.

[0030] This application also provides therapeutic use of the cells or therapeutic compositions described herein by introducing the compositions into subjects suitable for adoptive cell therapy. In some embodiments, subjects suitable for and requiring adoptive cell therapy have autoimmune disorders, hematological malignancies, solid tumors, cancer, or viral infections.

[0031] Further aspects of this application provide a method for producing induced cells as described herein, comprising differentiating iPSCs containing CD38 knockout or IL15Δ and optionally one or more of the following: (i) B2M null or low, (ii) CIITA (iii) null or low, (iv) introduced expression of HLA-G or uncleaved HLA-G, (iv) high affinity uncleaved CD16 (hnCD16) or its variants, (v) chimeric antigen receptor (CAR), (vi) exogenous cytokine expressed on the cell surface 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 any of the genes in the chromosome 6p21 region: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Introduced or increased expression in at least one of the following surface trigger receptors for binding to R, CAR, TCR, Fc receptors, engagers, and bispecific, multispecific, or universal engagers.

[0032] In some embodiments of the manufacturing method, the method further comprises genomically manipulating a cloned iPSC to knock out CD38 or knock in IL15Δ, and optionally knock out B2M and CIITA, or to introduce the expression of HLA-G or uncleaved HLA-G, high-affinity uncleaved CD16 or its variants, CARs, and / or cell surface-expressed exogenous cytokines or their receptors, either partially or completely, and the CARs and cell surface-expressed exogenous cytokines or their receptors, either partially or completely, are co-expressed in separate constructs or bicistronic constructs. In some embodiments of the manufacturing method, the genomic manipulation of the iPSC includes targeted editing. In some embodiments, the targeted editing includes deletions, insertions, or indels. In some embodiments, the targeted editing is performed by CRISPR, ZFN, TALEN, homing nucleases, homologous recombination, or any other functional variation of these methods.

[0033] This application further provides CRISPR-mediated editing of cloned iPSCs, thereby generating edited cloned iPSCs containing CD38 knockout or IL15Δ knock-in, or at least one of the genotypes listed in Table 1. In some embodiments of the CRISPR-mediated editing, the obtained CD38 knockout is a biallele. In some embodiments of the CRISPR-mediated editing, the CD38 knockout is a nucleic acid cleavage between first and second target sequences, the targeting sequences containing SEQ ID NOs. 3 and SEQ ID NOs. 4, respectively. In some embodiments of the CRISPR-mediated editing, the obtained IL15Δ knock-in contains a polynucleotide encoding IL15Δ, which includes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NOs. 17, 19, or 21. In some embodiments of the CRISPR-mediated editing described above, 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] Additional embodiments of this application provide methods for improving anti-CD38 antibody therapy, comprising administering effector cells without CD38 expression to a subject under therapeutic conditions. In some embodiments of anti-CD38 antibody therapy, the anti-CD38 antibody may be daratumumab, isatuximab, or MOR202, or humanized or Fc-modified variants or fragments thereof, functional equivalents, or biosimilars thereof. In some embodiments, the effector cells provided for a method to improve anti-CD38 antibody therapy include induced hematopoietic cells, including induced NK cells or induced T cells, wherein the induced NK cells or induced T cells include CD38 knockout, high affinity uncleaved CD16 or its variants, and optionally (i) B2M and CIITA knockout, (ii) introduced expression of HLA-G or uncleaved HLA-G, CAR, and / or partially or completely peptides of exogenous cytokines or their receptors expressed on the cell surface, wherein the CAR and partially or completely peptides of exogenous cytokines or their receptors expressed on the cell surface are co-expressed in separate constructs or bicistronic constructs, and / or (iii) at least one of the genotypes listed in Table 1. In some embodiments of the method to improve anti-CD38 antibody therapy, the method reduces the decrease in anti-CD38 antibody-induced effector cells in a subject under such treatment.

[0035] A further aspect of this application provides a method for reducing or preventing allogeneic rejection of allogeneic effector cells by using a CD38-specific antagonist, wherein the allogeneic effector cells comprise CD38 knockout, and the CD38-specific antagonist can suppress activated T and B cells in the recipient of the allogeneic effector cells. In some embodiments, the CD38-specific antagonist is an anti-CD38 antibody, a CD38-specific engager, or a CD38 chimeric antigen receptor (CAR). In some other embodiments, the anti-CD38 antibody is daratumumab, isatuximab, or MOR202, or any of their humanized or Fc-modified variants or fragments, functional equivalents, and biosimilars. In yet another embodiment, the anti-CD38 antibody is daratumumab, and the method provided herein provides a novel use of daratumumab.

[0036] Further aspects of this application provide a construct for modifying cells to obtain genome-edited cells, comprising: (i) left homology arms and right homology arms targeting CD38 at selected locations of CD38 loci (LHA / CD38, RHA / CD38) in the cell genome, which are operably linked to one or more nucleic acid sequences encoding one or more exogenous proteins, wherein the genome-edited cells are induced pluripotent cells (iPSCs), cloned iPSCs, or iPS cell lines, wherein the genome-edited cells comprise targeted incorporation of one or more nucleic acid sequences encoding one or more exogenous proteins at selected locations of the CD38 locus, and CD38 knockout, and the genome-edited cells are iPSCs that can differentiate into hematopoietic cells derived from iPSCs. In one embodiment of the construct, one or more nucleic acid sequences encoding one or more exogenous proteins are operably ligated to the endogenous promoter of CD38 upon integration and are thereby driven, or a selected location of the CD38 locus is located in the exons of CD38.

[0037] In some embodiments of the construct, one or more exogenous proteins include at least one or both of the following: (i) high-affinity non-cleavable CD16 (hnCD16) or a variant thereof; or (ii) exogenous IL15 / IL15 receptor fusion protein (IL15RF) or a variant thereof expressed on the cell surface. Thus, genome-edited cells and hematopoietic cells derived therefrom contain one or more nucleic acid sequences encoding hnCD16 and IL15RF, or variants thereof. In some embodiments of the construct, hnCD16 or its variants comprises at least one of the following: (a) F176V and S197P in the external domain of CD16, (b) a complete or partial external domain derived from CD64, (c) a non-natural (or non-CD16) transmembrane domain, (d) a non-natural (or non-CD16) intracellular domain, (e) a non-natural (or non-CD16) signaling domain, (f) a non-natural stimulating domain, and (g) transmembrane, signaling, and stimulating domains not derived from CD16 but 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 is cleaved; (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 the common receptor γC, where the common receptor γC is native or modified; (g) a homodimer of IL15Rβ; (h) a polynucleotide encoding IL15RF having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 17, 19, or 21.In some embodiments of the high affinity non-cleavable CD16 (hnCD16) or its variants included in the construct, hnCD16 comprises one or more of the following: (i) CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a non-native transmembrane domain derived from a T cell receptor (TCR) polypeptide. (ii) an unnatural stimulating domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide; (iii) an unnatural signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide; or (iv) an unnatural transmembrane domain derived from NKG2D, an unnatural stimulating domain derived from 2B4, and an unnatural signaling domain derived from CD3ζ.

[0038] In some embodiments, 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-strand CAR, (vii) inducible CAR, (viii) co-expressed with another CAR, (ix) co-expressed with a cell surface-expressed exogenous cytokine or a partial or complete peptide of its receptor, either in a separate construct or a bicistronic construct as needed, (xi) co-expressed with a checkpoint inhibitor, (xii) encoded by a nucleic acid further contained in the provided constructs, the CAR being inserted at a selected position in the CD38 locus, (xiii) inserted into the TRAC locus and / or driven by an endogenous promoter of the TCR and / or the TCR being knocked out by the CAR insertion, (xii i) Specific to 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, antigens 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-α, 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 to one of the following: κ-light chain, kinase insertion 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), mesoserin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncoemetic 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 oncoprotein (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 the expression of one or more nucleic acid sequences encoding one or more exogenous proteins, wherein 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) a 3'-terminal insulator of LHA / CD38 and a 5'-terminal insulator 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 may be a 2A sequence encoding a self-cleaved 2A peptide, or the linker sequence may be an Internal Ribosome Entry Sequence (IRES).

[0040] In some embodiments of the construct, the resulting genome-edited cells or iPSC-induced hematopoietic cells, which are iPSCs, have one or more of the following phenotypes: (i) having longer telomeres compared to their native corresponding cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue; (ii) CD38 - / - CD16, (iii)CD38- / - IL15, and / or (iv)CD38 - / - CD16 IL15. In some embodiments of the construct, the resulting genome-edited cells or iPSC-induced hematopoietic cells, which are iPSCs, further include one or more of the following: (i) B2M null or low, (ii) CIITA null or low, (iii) introduced expression of HLA-G or non-cleaved HLA-G, (iv) deletion or reduced expression of at least one of any of the genes in the chromosome 6p21 region, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and (v) HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Introduced or increased expression in at least one of the following surface trigger receptors for binding to R, CAR, Fc receptors, engagers, and bispecific, multispecific, or universal engagers.

[0041] In some embodiments of the construct, hematopoietic cells derived from differentiating the resulting genome-edited iPSCs include induced CD34 cells, induced hematopoietic stem cells and progenitor cells, induced hematopoietic pluripotent progenitor cells, induced T cell progenitor cells, induced NK cell progenitor cells, induced T cells, induced NKT cells, induced NK cells, or induced B cells. In one embodiment, hematopoietic cells derived from the resulting genome-edited iPSCs include induced NK or induced T cells and have at least one feature, compared to their native corresponding cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, including: (i) improved persistence and / or viability; (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 mobilize bystander immune cells to the tumor site; (vii) enhanced ability to reduce tumor immunosuppression; (viii) improved ability to rescue tumor antigen escapes; and (ix) reduced fractorides in the presence of an anti-CD38 antibody or CD38 CAR. In some embodiments, the anti-CD38 antibody is daratumumab, isatuximab, or MOR202, or any of their humanized or Fc-modified variants or fragments, functional equivalents, and biosimilars.

[0042] Furthermore, this application provides an expression vector comprising the construct described herein. Therefore, further provided are host cells comprising the construct or the expression vector described herein. Furthermore, a composition for producing genome-edited iPSCs or induced hematopoietic cells is provided, the composition comprising the construct or expression vector described herein. Furthermore, a kit is provided herein, the kit comprising the construct, expression vector or host cell described herein, and one or more media for culturing, maintaining or differentiating iPSCs. Furthermore, a construct, expression vector, and host cell for pharmaceutical use are also provided.

[0043] Additional embodiments of this application provide a method for producing genome-edited iPSCs or hematopoietic cells derived therefrom using the construct described above, the method comprising (i) introducing the construct into iPSCs, and (ii) incubating the iPSCs for a sufficient time to obtain genome-edited iPSCs by enabling targeted incorporation 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. In some embodiments, the method may further comprise inducing differentiation of the obtained genome-edited iPSCs into hematopoietic cells, which includes targeted incorporation 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 method exhibit improved genomic stability compared to iPSCs that undergo integration and knockout in separate stepwise editing events.

[0044] The various purposes and advantages of the compositions and methods provided herein will become apparent from the following description, together with the accompanying drawings which illustrate and illustrate specific embodiments of the invention. [Brief explanation of the drawing]

[0045] [Figure 1] This is an illustration of several construct designs for cytokines expressed on the cell surface in iPSC-derived cells. IL15 is used as an exemplary example that can be replaced with other desirable cytokines. [Figure 2] This shows phenotypic profiling using flow cytometry of CD38- / - induced NK cells obtained from each of the three CD38- / - iPSC clones. [Figure 3]This study demonstrates that CD38- / -iPSC-induced NK cells exhibit the same cytotoxic capacity as wild-type iPSC-derived NK cells. [Figure 4] This graph shows flow cytometry results of mature iPSC-derived NK cells, illustrating the stepwise manipulation of hnCD16 expression, B2M knockout (loss of HLA-A2 expression), HLA-G expression, and IL-15 / IL-15ra (LNGFR) construct expression. [Figure 5] This graph shows telomere lengths determined by flow cytometry, demonstrating that iPSC-derived matured NK cells maintain longer telomeres compared to adult peripheral blood NK cells. [Figure 6] This study demonstrates 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] Figure 7 shows that the viability of CD38- / -iPSC-derived NK cells (B) is maintained for at least approximately 48 hours in culture in the presence of CD38 antibody and daratumumab, compared to iPSC-derived NK cells expressing CD38 (A). [Figure 8] Figure 7 shows that NK cells derived from CD38- / -iPSCs (lower panel) do not degranulate when stimulated in the presence of the CD38 antibody daratumumab, and produce less cytokines than iNK cells expressing CD38 (upper panel). [Figure 9] Figure 7 shows iPSC-derived NK cells with hnCD16 introduced (A) and iPSC-derived NK cells possessing both exogenous hnCD16 and CD38 knockout (B). [Figure 10] The phenotypic and functional analyses of hnCD16-CD38- / - induced NK cells are shown in comparison with those of 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 cytometry, (E) ADCC in HER2-expressing ovarian cell line SKOV3 by Incucyte® live cell imaging. [Figure 11] This shows daratumumab-mediated NK cell fracturing in induced NK cells with and without CD38 knockout, compared to peripheral blood NK cells. The specific cytotoxicity of daratumumab against different NK cell populations was measured after 4 hours of incubation of iNK cells in the presence of daratumumab, increasing in concentration. [Figure 12] CD38 null hnCD16-induced NK cells exhibit enhanced daratumumab antimyeloma activity. After incubation of these iNK cells with MM.1S myeloma target cells for 18 hours, tumor cell viability was assessed by flow cytometry using annexin V and viability / death survival markers. [Figure 13] This study demonstrates enhanced hnCD16 CD38- / -iNK cells, showing long-term antimyeloma activity and persistence with daratumumab. (A) Tumor cell clearance, measured by the number of target cells remaining at the end of a 7-day cytotoxic assay against RMPI-8226 tumor spheroids, and (B) the absence of NK cell fructoliside improved the survival rate of hnCD16 CD38- / -iNK cells over the 7-day cytotoxic assay. [Figure 14] This study demonstrates that iNK cells lacking CD38 possess more robust ADCCs, exhibiting an increased potential for serial killing in the presence of daratumumab. [Figure 15] iNK cells transduced with the full-length IL15 / IL15Rα fusion construct (black circles; positive control) or the shortened IL15 / IL15Rα fusion construct lacking the cytoplasmic signaling domain (white circles) exhibited a viability advantage in the same culture compared to untransduced or GFP-transduced cells, independently 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 having one or more transgenes driven by an exogenous promoter or a CD38 endogenous promoter (B and D vs. A and C) for generating CD38- / - transgene + pluripotent stem cells and effector cells derived therefrom (A and B vs. C and D). [Figure 17-1] The following are exemplary nucleic acid sequences contained in the exogenous promoter-driven CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct for generating CD38- / -CD16 IL15 effector cells derived from pluripotent stem cells manipulated using the construct and its variants. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above. [Figure 18-1] The following are exemplary nucleic acid sequences contained in the CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct, driven by the CD38 endogenous promoter, for generating CD38- / -CD16+IL15+ effector cells derived from pluripotent stem cells manipulated using the construct and its variants. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 19] Flow cytometry phenotyping of unmanipulated iNK cells or CD38- / -CD16 IL15 iNK cells manipulated with a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct is shown. [Figure 20] This study demonstrates that CD38- / -CD16 IL15 iNK cells derived from iPSCs engineered with a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct exhibited improved in vitro persistence compared to unengineered iNK cells (FTi250). Cells were cultured for 4 days in the absence of IL-2 or IL-15, and the percentage of introduced cells was measured on days 2 and 4. [Figure 21] This study demonstrates that CD38- / -CD16 IL15 iNK cells derived from iPSCs engineered with a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct evade daratumumab-induced fractorides compared to unengineered iNK cells. Rituximab was used as a control antibody. [Figure 22] This study demonstrates that CD38- / -CD16 IL15 iNK cells derived from iPSCs engineered with a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct exhibit superior ADCC compared to Raji cells when combined with a daratumumab antibody. [Modes for carrying out the invention]

[0046] Genome modification of iPSCs (induced pluripotent stem cells) includes polynucleotide insertions, deletions, and substitutions. Exogenous gene expression in genetically engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal proliferation of the original genetically engineered iPSCs, after cell differentiation, and in dedifferentiated cell types derived from genetically engineered iPSCs. On the other hand, directly manipulating primary immune cells such as T cells or NK cells is difficult, hindering the preparation and delivery of engineered immune cells for adoptive cell therapy. The present invention provides an efficient and reliable targeted approach for the stable incorporation of one or more exogenous genes, including suicide genes and other functional modalities, which impart improved therapeutic properties with respect to engraftment, transport, homing, migration, cytotoxicity, viability, maintenance, proliferation, lifespan, self-renewal, persistence, and / or survival rate to iPSC-inducing cells, including but not limited to HSCs (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, 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 meanings generally understood by those skilled in the art. Furthermore, unless otherwise specifically required by the context, singular terms shall include plural forms and plural terms shall include singular forms.

[0048] It should be understood that the present invention is not limited to, and is therefore subject to change, the specific methodologies, protocols, and reagents described herein. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the invention as defined solely by the claims.

[0049] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “an element” means one or more elements.

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

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

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

[0053] As used herein, the terms “substantially” or “essentially” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater, compared to the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “substantially the same” or “essentially the same” refer to a range of a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference that is substantially identical to the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0054] As used herein, the terms “substantially absent” and “essentially absent” are interchangeable and, when used to describe compositions such as cell populations or culture media, refer to compositions that do not contain a particular substance or its source, for example, 95%, 96%, 97%, 98%, 99%, or are undetectable by conventional means. The terms “absent” or “essentially absent” of a particular component or substance in a composition also mean that such component or substance is (1) not present in the composition at any concentration, or (2) present in the composition but at a low density and functionally inactive. A similar meaning may apply to the term “not present,” which refers to the absence of a particular substance or its source in a composition.

[0055] Throughout this specification, unless the context requires otherwise, “comprise,” “comprises,” and “comprising” mean to include the step or element, or group of steps or elements, described, but not to exclude any other step or element, or group of steps or elements. In certain embodiments, the terms “include,” “have,” “contain,” and “comprise” are used synonymously.

[0056] "Consisting of" means that what follows the phrase is included and limited to it. Therefore, the phrase "consisting of" indicates that the listed elements are necessary or essential, and that other elements cannot exist.

[0057] "Consisting essentially of" means including any elements listed after the phrase, but limited to other elements that do not interfere with or contribute to the activity or behavior specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that the other elements may or may not be present, not necessarily as a matter of necessity, depending on whether or not they affect the activity or behavior of the listed elements.

[0058] Throughout this specification, any reference to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “specific embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can 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 conducted in or on living tissues within an artificial environment outside of a living organism, preferably with minimal changes to natural conditions. In certain embodiments, an “ex vivo” procedure involves living cells or tissues taken from a living organism and cultured in an experimental apparatus, usually under sterile conditions, typically for several hours or up to about 24 hours, but depending on the circumstances, up to 48 hours or 72 hours or longer. In certain embodiments, such tissues or cells may be collected and frozen and subsequently thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissues that last longer than several days are typically considered “in vitro,” but in certain embodiments, this term can be used interchangeably with “ex vivo.”

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

[0061] As used herein, the terms “reprogramming,” “dedifferentiation,” “increased cellular capacity,” and “increased developmental capacity” refer to methods of increasing the capacity of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cellular capacity has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell that has not been reprogrammed. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell that has not been reprogrammed.

[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 blood cells or muscle cells. Differentiated or induced-differentiation cells are cells that occupy a more specialized (“committed”) position within a cell lineage. When applied to the process of differentiation, the term “committed” refers to cells that, under normal circumstances, continue to differentiate into a particular cell type or subset of cell types, and under normal circumstances cannot 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 cells (i.e., the embryo itself) to form all lineages of living or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency is a range of developmental capabilities, from incomplete or partial pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive and pluripotent cells (e.g., embryonic stem cells) that can produce a complete organism.

[0063] As used herein, the terms “induced pluripotent stem cells” or iPSCs mean stem cells produced from induced or modified, differentiated adult, neonatal, or fetal cells, i.e., cells that have been reprogrammed to differentiate into all tissues of all three germ layers or dermis: mesoderm, endoderm, and ectoderm. Produced iPSCs do not refer to naturally occurring cells.

[0064] As used herein, the term “embryonic stem cells” refers to the naturally occurring pluripotent stem cells in the inner cell mass of a blastocyst. Embryonic stem cells are pluripotent and give rise to derivatives of all three major germ layers: ectoderm, endoderm, and mesoderm. They are not totipotent, meaning they do not contribute to the extraembryonic membrane or placenta.

[0065] As used herein, the term “pluripotent stem cell” refers to a developmentally capable cell that can differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not into all three. Thus, pluripotent cells may also be called “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 can form many types of cells of a particular lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelet, etc.), but cannot form neurons. Thus, the term “multipotency” refers to a state of cell development that has a lower degree of developmental potential than totipotency and pluripotency.

[0066] Pluripotency can be partially determined by evaluating the pluripotent properties of cells. Pluripotent features include, but are not limited to, (i) the morphology of pluripotent stem cells, (ii) the potential for unrestricted self-renewal, (iii) the expression of pluripotent stem cell markers such as SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) the formation of teratomas consisting of all three somatic cell lineages, and (vi) the formation of embryoid bodies consisting of cells from all three somatic cell lineages.

[0067] Two types of pluripotency have been described so far: a “priming” or “metastable” state of pluripotency, similar to the epiblastocystem cells (EpiSCs) of late blastocysts, and a “naive” or “basal” state, similar to the inner cell mass of early / pre-implantation blastocysts. Both pluripotent states exhibit the characteristics described above, but the naive or basal state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and viability in single-cell culture, (iii) overall reduction in DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) decreased expression of differentiation markers compared to pluripotent cells in the priming state. 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 possess the characteristics of a pluripotent preparation state. Under standard pluripotent cell culture conditions, such cells remain in a ready state and exhibit basal state characteristics unless exogenous transgene expression is maintained.

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

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

[0070] "Pluripotency factors" or "reprogramming factors" refer to agents that can increase the developmental potential of cells, 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 cells. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0071] "Culture" or "cell culture" refers to the maintenance, proliferation, and / or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in both cases, the singular "medium"), "supplementary components," and "medium supplementary components" refer to the nutritional composition used to culture cells.

[0072] "To culture" or "to maintain" refers to sustaining, propagating, and / or differentiating tissue or extracorporeal cells, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culture" or "maintaining" can utilize the culture medium as a source of nutrients, hormones, and / or other factors that aid in cell growth and / or maintenance.

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

[0074] As used herein, the terms “secondary hematopoietic endothelium” (HE) or “secondary hematopoietic endothelium derived from pluripotent stem cells” (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem cells and progenitor cells in a process called endothelial hematopoietic transition. Hematopoietic cell development in the embryo progresses sequentially from the lateral plate mesoderm through angioblasts to secondary hematopoietic endothelial cells and hematopoietic progenitor cells.

[0075] The terms “hematopoietic stem cells 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 pluripotent hematopoietic stem cells (blood cells), myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphocyte progenitor cells. Hematopoietic stem cells and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). 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 interchangeable and refer to the primary type of leukocyte that completes maturation in the thymus and has various roles in the immune system, including the identification of certain foreign antigens in the body, as well as the activation and inactivation of other immune cells. A T cell can be any T cell, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from mammals. A T cell can be a CD3+ cell. T cells include, but are not limited to, any type of T cell and can be at any developmental stage, including 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), etc. 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) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

[0077] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are associated with cellular immune responses. They are characterized by their post-stimulation secretion profiles, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55-kD glycoprotein originally defined as a differentiation antigen for T lymphocytes, but is also found in other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as an associated recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, it defines a subset of helper / inducer T cells.

[0078] "CD8+ T cells" refer 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 in thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a relevant recognition element of major histocompatibility complex class I restricted interactions.

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

[0080] As used herein, the terms “NKT cells” or “natural killer T cells” refer to CD1d-restricted T cells that express a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire—a regular 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, exhibits a more heterogeneous use of TCRαβ. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one 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 cells or populations of cells that have been separated from their original environment; that is, the environment of isolated cells substantially does not contain at least one component found in the environment in which “unisolated” reference cells exist. This term includes, for example, cells isolated from tissue or biopsy specimens, removed from some or all components as they would be found in their natural environment. This term also includes cells removed from at least one, some or all components because cells are found in a non-natural environment, e.g., an environment in which they are isolated from a cell culture or cell suspension. Thus, isolated cells, if found in nature, or if grown, stored, or persist in a non-natural environment, are partially or completely separated from at least one component, including other substances, cells, or populations of cells. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in media that do not exist in nature. Isolated cells can be obtained by separating desired cells or populations from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0082] As used herein, terms such as “purify” mean 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 “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that function as a template for the synthesis of other polymers and macromolecules in biological processes having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties derived therefrom. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. A gene or cDNA can be said to code for a protein or other product if its nucleotide sequence is identical to the mRNA sequence and both the coding strand, which is usually listed in the sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA.

[0084] "Construct" refers to a complex of macromolecules or molecules containing polynucleotides that are delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct that can induce the delivery or transfer of foreign genetic material to a target cell and can be replicated and / or expressed in the target cell. As used herein, the term "vector" includes the construct being delivered. A vector can be a linear or cyclic molecule. A vector may or may not be incorporated. 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, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, and Sendai virus vectors.

[0085] "Integration" means that one or more nucleotides of a construct are stably inserted into the cellular genome, i.e., covalently bonded to a nucleic acid sequence within the cell's chromosomal DNA. "Targeted integration" means that nucleotides of a construct are inserted into the cell's chromosome or mitochondrial DNA at a pre-selected site or "integration site." As used herein, the term "integration" further refers to a process involving the insertion of one or more exogenous sequences or nucleotides of a construct, with or without the deletion of an endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" may further include the substitution of the deleted nucleotide in 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 reference activity is introduced into the host cell or is unnatural to the host cell. The molecule can be introduced, for example, by introducing the coding nucleic acid into the host's genetic material, for example, by incorporating it into the host's chromosomes, or as non-chromosomal genetic material, for example, as a plasmid. Therefore, as used in relation to the expression of coding nucleic acids, the term refers to the introduction of the coding nucleic acid into the cell in an expressible form. The term “endogenous” refers to a reference molecule or activity present in the host cell. Similarly, when used in relation to the expression of coding nucleic acids, this term refers to the expression of a coding nucleic acid that is contained within the cell and not introduced exogenously.

[0087] As used herein, “Gene of Interest” or “Polynucleotide Sequence of Interest” is a DNA sequence that, under the control of an appropriate regulatory sequence, is transcribed into RNA and, in some cases, translated into polypeptides in vivo. Genes of Interest or polynucleotides may include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of Interest may encode miRNA, shRNA, native polypeptides (i.e., naturally occurring polypeptides) or fragments thereof, variant polypeptides (i.e., variants of native polypeptides having less than 100% sequence identity with the native polypeptide) or fragments thereof, modified polypeptides or peptide fragments, therapeutic peptides or polypeptides, imaging markers, selectable markers, and the like.

[0088] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotide of any length, which is either a deoxyribonucleotide or a ribonucleotide, or an analogue thereof. The sequence of a polynucleotide consists of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) instead of thymine when the polynucleotide is RNA. Polynucleotides may include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to both double-stranded and single-stranded molecules.

[0089] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable 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 called peptides, oligopeptides, and oligomers in the art, and long chains, also commonly called polypeptides or proteins in the art. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0090] "Operatively linked" refers to the linking of nucleic acid sequences on a single nucleic acid fragment in which the function of one is influenced by the other. For example, a promoter is operationally linked to a coding sequence or functional RNA if it can influence 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 operationally linked to a regulatory sequence in either a sense or antisense direction.

[0091] As used herein, the term “genetic imprint” refers to genetic or epigenetic information that contributes to the preferred therapeutic attributes of a source cell or iPSC and is retainable in iPSCs derived from source cells and / or hematopoietic lineage cells derived from iPSCs. As used herein, “source cell” is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and iPSCs derived from source cells can further differentiate into specific cell types, including any hematopoietic lineage cells. iPSCs derived from source cells, and cells differentiated therefrom, may be collectively referred to as “inducible” cells or “inducible” cells, depending on the context. For example, as used throughout this application, induced effector cells, or induced NK cells or induced T cells, 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. When used herein, a genetic imprint conferring a preferred therapeutic attribute is incorporated into an iPSC by reprogramming selected source cells that are specific to a donor, disease, or therapeutic response, or by introducing a recombinant modality into the iPSC using genome editing. In the case of source cells obtained from a specific selected donor, disease, or therapeutic situation, the genetic imprint contributing to the preferred therapeutic attribute may include a context-specific genetic or epigenetic trait that represents a retainable phenotype, i.e., the preferred therapeutic attribute, which is passed on to the induced cells of the selected source cell, regardless of whether the underlying molecular event is identified. Source cells specific to a donor, disease, or therapeutic response may include genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, and these genetic imprints may include, but are not limited to, pre-configured single-specific TCRs from virus-specific T cells or invariant natural killer T (iNKT) cells, trackable desirable genetic polymorphisms, such as homozygosity of point mutations encoding high-affinity CD16 receptors in selected donors, and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting an increased haplotype in the population.When used herein, preferred therapeutic attributes include improved engraftment, transport, homing, viability, self-renewal, persistence, modulation and regulation of the immune response, survival rate, and cytotoxicity of induced cells. Preferred therapeutic attributes may also be associated with antigen-targeting receptor expression, HLA presentation or absence, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity with reduced extratumor effects, and resistance to therapies such as chemotherapy.

[0092] As used herein, the term “enhanced therapeutic properties” refers to the therapeutic properties of a cell that are enhanced compared to a typical immune cell of the same common 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, transport, homing, viability, self-renewal, persistence, modulation and regulation of the immune response, survival rate, and cytotoxicity. Therapeutic properties of immune cells can also manifest as the expression of antigen-targeting receptors, HLA presentation or absence, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity with reduced extratumor effects, and resistance to treatments such as chemotherapy.

[0093] As used herein, the term “engager” refers to a molecule, such as a fusion polypeptide, that can form a link between immune cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils, and tumor cells, and can activate immune cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), triplicate killer cell engagers, or multispecific killer cell engagers, or universal engagers that can be adapted to multiple immune cell types.

[0094] As used herein, the term “surface trigger receptor” refers to a receptor that can induce or initiate an immune response, such as a cytotoxic response. Surface trigger receptors can be manipulated and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, the surface trigger receptor facilitates the binding of bispecific or multispecific antibodies between effector cells and specific target cells, such as tumor cells, regardless of the effector cell’s innate receptor and cell type. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and differentiated into populations of various effector cell types expressing the universal surface trigger receptor. “Universal” means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind to or ligate to engagers having the same epitopes recognizable by the surface trigger receptor, regardless of the engager’s tumor-binding specificity. In some embodiments, engagers having the same tumor-targeting specificity are used to bind to the universal surface trigger receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to universal surface trigger receptors. Thus, one or more effector cell types may be used to kill one specific type of tumor cell, or two or more types of tumor cells. Surface trigger receptors generally contain a costimulatory domain for activating effector cells and an antiepitope specific to the engager's epitope. A bispecific engager is specific to the antiepitope of the surface trigger receptor at one end and specific to the tumor antigen at 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 cell therapy. In some examples, the expression of safety switch proteins is conditionally controlled to address safety concerns in transplanted engineered cells in which the gene encoding the safety switch protein is permanently incorporated into the genome. This conditional regulation may be variable and may include control by post-translational activation via small molecules and tissue-specific and / or transient transcriptional regulation. Safety switches may mediate the induction of apoptosis, inhibition of protein synthesis, arrest of DNA replication and proliferation, transcription and post-transcriptional gene regulation and / or antibody-mediated depletion. In some examples, safety switch proteins are activated by exogenous molecules, such as prodrugs, and when activated, induce apoptosis and / or cell death in therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered when an adverse event occurs is activated by a suicide gene product, killing the transduced cells.

[0096] As used herein, the term “pharmaceutically active protein or peptide” refers to a protein or peptide capable of achieving biological and / or pharmaceutical effects on an organism. A pharmaceutically active protein may have therapeutic or mitigating properties for a disease and may be administered to improve, reduce, alleviate, reverse, or reduce the severity of the disease. A pharmaceutically active protein may also have prophylactic properties and may be used to prevent the onset of a disease or, if it appears, to reduce the severity of such a disease or pathological condition. A pharmaceutically active protein may include the whole protein or peptide or a pharmaceutically active fragment thereof. It may also include a pharmaceutically active analogue of a protein or peptide or an analogue of a protein or peptide fragment. The term pharmaceutically active protein may also refer to multiple proteins or peptides that act synergistically or cooperatively to produce a therapeutic effect. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0097] As used herein, the term “signaling molecule” refers to any molecule that modulates, participates in, inhibits, activates, reduces, or increases cellular signaling. Signaling refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along pathways that ultimately lead to biochemical events within a cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Tollgate signaling, ligand-dependent ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0098] As used herein, the term “targeting modality” refers to molecules, e.g., polypeptides, that are genetically incorporated into cells and enhance the specificity of antigens and / or epitopes, including but not limited to: i) antigen specificity when associated with a specific 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 specific antigens or surface molecules.

[0099] As used herein, the terms “specific” or “specificity” can be used to refer to the ability of a molecule, such as a receptor or engager, to selectively bind to a target molecule, as opposed to nonspecific or nonselective binding.

[0100] As used herein, the term “adoptive cell therapy” refers to cell-based immunotherapy as used herein, involving the infusion of autologous or allogeneic lymphocytes, identified as T or B cells, that are proliferating ex vivo prior to infusion, whether genetically modified or not.

[0101] As used herein, “therapeutably sufficient amount” means, within its meaning, a non-toxic but sufficient and / or effective amount of the particular therapeutic and / or pharmaceutical composition it refers to that provides 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 a particular embodiment, a therapeutically sufficient amount is sufficient and / or effective to improve, alleviate, and / or improve 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, such as stimulants in the culture 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 and generate multiple lineages within a three-dimensional region. Through a differentiation process that typically lasts from a few hours to several days, simple EBs (e.g., differentiated aggregated pluripotent stem cells) continue to mature and grow into cystic EBs, which typically last from several days to several weeks at which point they are processed to continue further differentiation. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other within a three-dimensional multilayer cluster of cells, which is typically achieved by one of several methods, including settling the pluripotent cells as droplets and settling the cells into a "U" bottom well plate, or by mechanical agitation. Aggregates maintained in pluripotent culture maintenance medium do not form proper EBs, so further differentiation cues are needed for the aggregates of pluripotent stem cells to promote EB growth. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cue induction to the selected lineage. EB-based culture of pluripotent stem cells typically produces differentiated cell populations (ectoderm, mesoderm, and endoderm) with moderate proliferation within EB cell clusters. While EB has been shown to promote cell differentiation, it produces heterogeneous cells in various differentiation states due to the inconsistency of the exposure of three-dimensional cell structures to differentiation cues from the environment. Furthermore, EB is cumbersome to create and maintain. Additionally, EB-mediated cell differentiation is accompanied by moderate cell proliferation, leading to reduced differentiation efficiency.

[0103] In contrast, "aggregate formation," unlike "EB formation," can be used to proliferate populations of pluripotent stem cell-derived cells. For example, during the proliferation of pluripotent stem cells based on aggregates, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of aggregates, which form larger aggregates, and these aggregates can routinely dissociate into smaller aggregates mechanically or enzymatically to maintain cell proliferation in culture and increase cell number. Unlike EB culture, cells cultured within aggregates in maintenance culture retain markers of pluripotency. Pluripotent stem cell aggregates require a further differentiation queue to induce differentiation.

[0104] As used herein, “monolayer differentiation” refers to a differentiation method distinct from differentiation by three-dimensional multilayer clustering of cells, i.e., “EB formation.” Among the other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Because monolayer culture does not mimic embryonic development such as EB formation, differentiation into a particular lineage is considered minimal compared to the differentiation of all three germ layers in EB.

[0105] As used herein, “dissociated” cells mean cells that have been substantially separated or purified from other cells or from a surface (e.g., the surface of a culture plate). For example, cells may be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro may be dissociated from one another by enzymatic or mechanical dissociation, for example, 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 involves disrupting cell interactions with the extracellular matrix (ECM) and substrate (e.g., a culture surface), or disrupting the ECM between cells.

[0106] As used herein, “feeder cells” or “feeder” is a term that describes one type of cell that, when co-cultured with a second type of cell, provides an environment in which the second type of cell can proliferate, grow, or differentiate, and the feeder cells provide stimuli, growth factors, nutrients, and support the second cell type. Feeder cells may originate 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 proliferating more than the cells they support when co-cultured with other cells. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular type of feeder cell may be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). Generally, various feeder cells can be used to maintain pluripotency, direct differentiation into specific strains, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells.

[0107] As used herein, “feeder-free” (FF) environment refers to an environment such as culture conditions, cell cultures, or culture media that are essentially free of feeder or stromal cells and / or have not been pretreated by feeder cell culture. “Pretreated” 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. Pretreated medium contains many mediator substances, including growth factors and cytokines secreted by feeder cells cultured in the medium. In some embodiments, a feeder-free environment is free of both feeder cells and stromal cells and has not been pretreated by feeder cell culture.

[0108] 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, “functional” means (1) successful knock-in, knock-out, knock-down gene expression, transgenic or controlled gene expression at the gene level, such as inducible or transient expression at a desired cell developmental stage, achieved by direct genome editing or modification, or by “passage” through differentiation or reprogramming from the initially genome-engineered initiating cells, or (2) (i) obtained in the above cells by direct genome editing. (ii) alteration of gene expression obtained; (ii) alteration of gene expression maintained in the cells by “passing through” the initially genome-edited initiating cells via differentiation or reprogramming; (iii) downstream gene regulation of the cells as a result of alteration of gene expression that appears only in the early developmental stages of the cells or only in the initiating cells that give rise to the cells via differentiation or reprogramming; or (iv) removal, addition, or alteration of cellular function / characteristics at the cellular level by enhancement or newly achieved cellular function or attribute presented in a mature cell product that originally originated from genome editing or modification performed on iPSCs, progenitor cells, or dedifferentiated cell origins.

[0109] "HLA deficiency," including HLA class I deficiency, HLA class II deficiency, or both, refers to cells in which the surface expression level of the complete MHC complex, including the HLA class I protein heterodimer and / or HLA class II heterodimer, is insufficient, no longer maintained, or reduced, decreased, or reduced to a level lower than that naturally detectable by other cells or synthetic methods.

[0110] As used herein, “modified HLA-deficient iPSC” refers to an HLA-deficient iPSC that is further modified by introducing genes that express proteins related to, 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, resulting in improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, co-stimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3z, 41BBL, CD47, CD113, and PDL1. “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 to the most common class of antibody, IgG, are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). Classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling characteristics of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and therefore different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0112] The term "chimeric Fc receptor," abbreviated as CFcR, is used to describe engineered Fc receptors in which the native transmembrane and / or intracellular signaling domains are 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 / or signaling domains being non-native, one or more stimulating 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) that contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to Fc fragments, or the Fc region of an antibody, or to the Fc region contained in an engager or binding molecule, and activate cell function by binding to the molecule, whether or not the target cell is nearby. For example, the Fcγ receptor may be engineered to contain a selected transmembrane domain, a stimulating domain, and / or signaling domains in the intracellular region that responds to IgG binding in the extracellular domain, thereby generating a CFcR. In one example, CFcRs are produced by manipulating the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domains. To further improve the binding affinity of CD16-based CFcRs, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcRs containing the 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., non-shedding, thereby obtaining an hnCD16-based CFcR.

[0113] The FcγR receptor CD16 has been identified as having two isoforms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and promoting antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, “high affinity CD16,” “uncleaved CD16,” or “high affinity uncleaved CD16 (hnCD16)” refers to native or non-native variants of CD16. Wild-type CD16 has low affinity and, upon activation of NK cells, is subjected to external domain shedding, a proteolytic cleavage process that modulates 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) is 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 an uncleaved version of CD16. CD16 variants containing both F158V and S197P are highly affinity and uncleaved. Another exemplary high-affinity uncleaved CD16 (hnCD16) variant is an engineered CD16 containing an external domain derived from one or more of the three exons of the CD64 external domain.

[0114] I. Cells and compositions useful for adoptive cell therapy with enhanced properties Provided herein are strategies for systematically manipulating the regulatory circuits of clonal iPSCs without affecting the differentiation potential of iPSCs or the cellular developmental biology of iPSCs and their induced cells, while enhancing the therapeutic properties of the induced cells. The induced cells are functionally improved and suitable for adoptive cell therapy after the selective modality combination has been introduced into the cells at the iPSC level via genomic engineering. Prior to this invention, it was unclear whether the modified iPSCs, including one or more gene edits provided, still possessed the ability to enter cell development while retaining regulated activity, and / or the ability to mature and produce functionally differentiated cells. Unexpected failures during mediated cell differentiation from iPSCs are due to aspects including, but not limited to, specific gene expression or its absence at developmental stages, requirements for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure differentiation protocols to allow for changes in the cellular phenotype and / or function. This application demonstrates that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from the modified iPSCs possess enhanced and / or acquired therapeutic properties resulting from the individual or combined genomic modifications, which are retained in the effector cells after iPSC differentiation.

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

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

[0117] In the treatment of malignant tumors, systemic use of T cells transduced with the CD38 antigen-binding receptor leads to lysis of the CD38+ fraction of CD34+ hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells, impairing the recipient's immune effector cell function and resulting in an incomplete therapeutic response, reduced efficacy, or elimination. Furthermore, in multiple myeloma patients treated with daratumumab, a CD38-specific antibody, a decrease in NK cells was observed in both bone marrow and peripheral blood, while 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 constrained by theory, this application provides a strategy to maximize the potential of CD38-targeted cancer therapy by overcoming effector cell depletion or reduction via fractorides induced by CD38-specific antibodies and / or CD38 antigen-binding domains. Furthermore, since CD38 is upregulated in activated lymphocytes such as T cells and B cells, suppressing the activation of these lymphocytes using CD38-specific antibodies such as daratumumab in recipients of allogeneic effector cells reduces and / or prevents allogeneic rejection of these effector cells, thereby increasing the viability and persistence of effector cells. Thus, this application also provides strategies to enhance the persistence and / or viability of effector cells through the reduction or prevention of allogeneic rejection by using CD38-specific antibodies, secreted CD38-specific engagers, or CD38 CARs (chimeric antigen receptors) against the activation of recipient T cells and B cells. Specifically, the strategies provided include generating CD38 knockout iPSC lines and inducing differentiation of the manipulated iPSC lines to produce CD38 null(CD38 - / - This includes obtaining induced effector cells. Prior to this application, given that CD38 plays many important roles in cell developmental biology and cellular function as described above, it was unclear whether disrupting CD38 in iPSCs would disrupt aspects including iPSC differentiation, induced cellular phenotype, and effector cell function.

[0118] In one embodiment provided herein, CD38 knockout in an iPSC strain is a biallelic knockout. As disclosed herein, the provided CD38 null iPSC strain can differentiate as directed and produce functionally induced hematopoiesis, including but not limited to mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, when ADCC is induced using an anti-CD38 antibody or when an anti-CD38 CAR is used for targeted cell killing, CD38 - / - iPSCs and / or their induced effector cells are not eliminated by anti-CD38 antibodies or anti-CD38 CARs, thereby increasing the persistence and / or viability of iPSCs and their effector cells in the presence and / or after exposure to such therapeutic agents. In some embodiments, effector cells exhibit increased in vivo persistence and / or viability in the presence and / or after exposure to such therapeutic agents. In some embodiments, CD38 null effector cells are NK cells derived from iPSCs. In some embodiments, CD38 null effector cells are T cells derived from iPSCs. In some embodiments, CD38 null iPSCs and induced cells include one or more additional genome editing methods 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 receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and promoting antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high affinity CD16," "uncleaved CD16," or "high affinity uncleaved CD16" refers to various CD16 variants. Wild-type CD16 has low affinity and, upon activation of NK cells, is subjected to external domain 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 CD16 polymorphic variant with high affinity, while the S197P variant is a genetically engineered exemplary non-cleavable version of CD16. Engineered CD16 variants, including both F176V and S197P, are high affinity and non-cleavable, as described in detail in WO2015 / 148926, the full disclosure of which is incorporated herein by reference. Furthermore, chimeric CD16 receptors in which the external domain of CD16 is essentially replaced with at least a portion of the external domain of CD64 can also achieve the desired high affinity and non-cleavable function of a CD16 receptor capable of performing ADCC. In some embodiments, the substituted external domain of the chimeric CD16 includes one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoform or polymorphic variant).

[0120] Accordingly, in some embodiments, the high-affinity uncleaved CD16 receptor (hnCD16) comprises both F176V and S197P, and in some embodiments, comprises F176V with the cleavage region removed. In some other embodiments, hnCD16 comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, when compared to any of the exemplary sequences, SEQ ID NOs. 7, 8, and 9, each comprising at least a portion of the CD64 external domain. SEQ ID NOs. 7, 8, and 9 are coded by illustrating SEQ ID NOs. 10–12, respectively. As used herein and throughout this application, the identity percentage 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 × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of arrays and the determination of the percentage of identity between two arrays can be performed using mathematical algorithms recognized in the art.

[0121] TIFF0007927045000001.tif46167

[0122] TIFF0007927045000002.tif46167

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[0124] TIFF0007927045000004.tif93165

[0125] TIFF0007927045000005.tif94169

[0126] TIFF0007927045000006.tif90169

[0127] Accordingly, provided herein are, in particular among other edits intended and described herein, cloned iPSCs genetically engineered to contain a high-affinity non-cleavable CD16 receptor (hnCD16), and the genetically engineered iPSCs can differentiate into effector cells containing hnCD16 introduced into the iPSCs. In some embodiments, the induced effector cells containing hnCD16 are NK cells. In some embodiments, the induced effector cells containing hnCD16 are T cells. Exogenous hnCD16 expressed in iPSCs or their induced cells exhibits high affinity not only for ADCC antibodies or fragments thereof, but also for bispecific, tripspecific, or multispecific engagers or binders that recognize the extracellular binding domain of CD16 or CD64 of the hnCD16. Bispecific, tripspecific, or multispecific engagers or binders are described further below in this application (see Section I.7). Thus, this application provides inducible effector cells or a population of cells pre-loaded with one or more pre-selected ADCC antibodies via high-affinity binding to the extracellular domain of hnCD16 expressed on the inducible effector cells, in amounts sufficient for therapeutic use in the treatment of conditions, diseases, or infections as further detailed in Section V below, wherein the hnCD16 comprises the extracellular binding domain of CD64, or of CD16 having F176V and S197P.

[0128] In some other embodiments, the native CD16 transmembrane domain and / or intracellular domain of hnCD16 are further modified or replaced so that the chimeric Fc receptor (CFcR) is produced to include a non-native transmembrane domain, a non-native stimulating domain, and / or a non-native signaling domain. As used herein, the term “non-native” means that the transmembrane domain, stimulating domain, or signaling domain is derived from a different receptor other than the receptor that provides the extracellular domain. In the examples herein, CFcRs based on CD16 or a variant thereof do not have a transmembrane domain, stimulating domain, or signaling domain derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR contains unnatural transmembrane domains derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, and T cell receptor polypeptides. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native stimulating / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of hnCD16, the provided chimeric receptor comprises a transmembrane domain and a signaling domain both derived from one of the polypeptides IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D.One particular embodiment of the hnCD16-based chimeric Fc receptor comprises a transmembrane domain of NKG2D, a stimulating domain of 2B4, and a signaling domain of CD3ζ, where the extracellular domain of hnCD16 is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 includes F176V and S197P. Another embodiment of the hnCD16-based chimeric Fc receptor comprises a transmembrane domain and a signaling domain of CD3ζ, where the extracellular domain of hnCD16 is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 includes F176V and 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, triplicate, or multispecific engager or binder. Upon binding, the stimulating and / or signaling domains of the chimeric receptor enable activation and cytokine secretion of effector cells, killing tumor cells targeted by the antibody, or the bispecific, triplicate, or multispecific engager or binder having the tumor antigen-binding component and Fc region described above. Without being limited by theory, CFcRs contribute to the effector cell killing ability and increase the proliferation and / or potential of effector cells through the non-native transmembrane, stimulating, and / or signaling domains of hnCD16-based chimeric Fc receptors, or through the binding of an engager to the external domain. Antibodies and engagers can bring tumor cells expressing the antigen and effector cells expressing CFcRs into close proximity, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, tripspecific, 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, tripspecific, or multispecific engagers or binders suitable for binding effector cells expressing hnCD16-based CFcRs to attack 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, various non-cleaved versions of CD16 in induced NK cells evade CD16 shedding and maintain constant expression. In induced NK cells, non-cleaved CD16 increases the expression of TNFα and CD107a, indicators of improved cellular function. Non-cleaved CD16 also enhances antibody-dependent cell-mediated cytotoxicity (ADCC) and the binding of bi-, tri-, or multi-specific engagers. ADCC is a mechanism of NK cell-mediated lysis via the binding of CD16 to antibody-coated target cells. The additional high-affinity properties of hnCD16 introduced into induced NK cells also enable in vitro loading of hnCD16-mediated ADCC antibodies into NK cells before administering the cells to subjects requiring cell therapy. As presented, hnCD16 may comprise F176V and S197P in some embodiments, or may comprise a complete or partial external domain derived from CD64 as exemplified by SEQ ID NOs: 7, 8, or 9, or may further comprise at least one of a non-native transmembrane domain, stimulatory domain, and signaling domain. As disclosed, the application also provides induced NK cells or a population of such cells preloaded with one or more pre-selected ADCC antibodies in amounts sufficient for therapeutic use in the treatment of 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., natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing expressed exogenous, uncleaved CD16 could differentiate into functional T cells capable of performing functions through acquired ADCC mechanisms, not only expressing exogenous CD16 without compromising the developmental biology of T cells. This ADCC acquired in induced T cells can be further used as an approach to rescue antigen escape, which often occurs in dual targeting and / or CAR-T cell therapy, resulting in reduced or absent expression of CAR-T target antigens, or tumor relapse with mutated antigens that evade recognition by CAR (chimeric antigen receptor). If the above-mentioned induced T cells contain ADCC acquired 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 mitigate or prevent the relapse or recurrence of target tumors commonly seen in CAR-T therapy. Such strategies, which achieve dual targeting while reducing and / or preventing antigen escape, can also be applied to NK cells expressing one or more CARs. Various CARs that can be used in this antigen escape reduction and prevention strategy are described in detail below.

[0132] Thus, the present invention provides induced T cells containing exogenous CD16. In further provided embodiments, the induced T cells obtained herein include CD38 knockout in addition to hnCD16 expression. In some embodiments, the hnCD16 contained in the induced T cells includes F176V and S197P. In some other embodiments, the hnCD16 contained in the induced T cells includes a complete or partial exogenous domain derived from CD64, as exemplified by SEQ ID NOs. 7, 8, or 9, or may further include at least one of a non-native transmembrane domain, stimulatory domain, and signaling domain. As described, such induced T cells have an acquired mechanism for targeting tumors with monoclonal antibodies meditated by ADCC to enhance the therapeutic effect of the antibody. As disclosed, the application also provides induced T cells or a population of such cells pre-loaded with one or more pre-selected ADCC antibodies in amounts sufficient for therapeutic use in the treatment of a condition, disease, or infection, as detailed in Section V below. In some other embodiments, induced T cells expressing hnCD16 are also CD38 null, and as a result, the cells can avoid being eliminated 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 containing a CD38 binding region, such as an anti-CD38 scFV.

[0133] 3. CAR expression Applicable to genetically engineered iPSCs and their induced effector cells may be any CAR design known in the art. A CAR, or chimeric antigen receptor, is a fusion protein generally comprising an antigen-recognition region, a transmembrane domain, and an external domain including an internal domain. In some embodiments, the external domain may further include a signal peptide or a leader sequence and / or a spacer. In some embodiments, the endodomain may further include a signaling peptide that activates effector cells expressing the CAR. In some embodiments, the antigen-recognition domain may specifically bind to an antigen. In some embodiments, the antigen-recognition domain may 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 may 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 may include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or a combination thereof. In certain embodiments, the NK cells are derived from CAR-expressing iPSCs.

[0134] In certain embodiments, the antigen recognition region includes mouse antibodies, human antibodies, humanized antibodies, camel Ig, shark heavy chain-only antibodies (VNAR), Ig NAR, chimeric antibodies, recombinant antibodies, or antibody fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragments (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen-binding fragments (sdAb, nanobodies), recombinant heavy chain-only antibodies (VHH), and other antibody fragments that maintain the binding specificity of the entire antibody.Non-limiting examples of antigens that can be targeted by 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, and site Antigens of cytoplasmic megalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), Epithelial growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion 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), these include mesoserine (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryonic 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 oncoprotein (WT-1), and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that may cause disease.

[0135] In some embodiments, the transmembrane domain of CAR includes the full-length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.

[0136] In some embodiments, the signaling peptide of the internal domain (or intracellular domain) comprises the full-length or at least a portion of the polypeptide of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the signaling peptide of 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 (immune receptor tyrosine-based activation motif) of CD3ζ.

[0137] In certain embodiments, the internal domain further comprises at least one co-stimulatory signaling region. The co-stimulatory signaling region may include 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 cell-applicable CAR provided in this application comprises a co-stimulatory domain derived from CD28 and a signaling domain comprising 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 with respect to SEQ ID NO: 13. In a further embodiment, the CAR comprising a co-stimulatory domain derived from CD28 and native or modified ITAM1 of CD3ζ also comprises a hinge domain and a transmembrane domain derived from CD28, wherein the scFv may 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 with respect to SEQ ID NO: 14. Sequence ID 13 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGE RRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (CD28 co-stimulation with 153 amino acids + CD3ζITAM)

[0139] TIFF0007927045000007.tif34169

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

[0141] TIFF0007927045000008.tif44169

[0142] TIFF0007927045000009.tif51167

[0143] Non-restrictive CAR strategies include conditionally activated heterodimer CARs by dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9587020), split CARs which are homologous recombination of antigen-binding, hinge, and internal domains to generate the CAR (see, e.g., U.S. Patent Publication No. 2017 / 0183407), multi-strand CARs which enable non-shared links between two transmembrane domains linked to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Patent Publication No. 2014 / 0134142), CARs which have a bispecific antigen-binding domain (see, e.g., U.S. Patent No. 9447194), or a pair of antigen-binding domains which recognize the same or different antigens or epitopes (see, e.g., U.S. Patent No. 8409577), or tandem CARs (see, e.g., Hegde et al., J Clin). See Invest.2016;126(8):3036-3052), inductive CARs (see, for example, U.S. Patent Publication Nos. 2016 / 0046700, 2016 / 0058857, and 2017 / 0166877), switchable CARs (see, for example, U.S. Patent Publication No. 2014 / 0219975), and other designs known in the art.

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

[0145] 4. Exogenously introduced cytokines and / or cytokine signaling By avoiding clinically appropriate systemic high-dose administration of 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 soluble cytokine administration, one or more partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors are introduced into cells to enable cytokine signaling, with or without the expression of the cytokines themselves, 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 ephemeral.

[0146] Figure 1 shows several structural designs using IL15 as an example. The transmembrane (TM) domain in any of the designs in Figure 1 is either naturally occurring for the IL15 receptor or modified or substituted with a transmembrane domain from 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 excluding the cis presentation of IL15.

[0148] Design 2: IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans presentation without excluding cis presentation of IL15, and ensuring membrane binding of IL15.

[0149] Design 3: IL15Rα with a shortened intracellular domain is fused to IL15 at the C-terminus via a linker, mimicking the trans presentation of IL15, maintaining the membrane binding of IL15, and excluding cis presentation and other potential signaling pathways mediated by normal IL15R via its intracellular domain. The intracellular domain of IL15Rα is thought to be crucial for the receptor to be expressed in IL15-responsive cells and for these cells to proliferate and function. Such a shortened construct contains an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 17, which may be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 18. In one embodiment of shortened IL15 / IL15Rα, the construct does not contain the last four amino acids "KSRQ" of SEQ ID NO: 17 and contains an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 21.

[0150] TIFF0007927045000010.tif55169 Sequence ID 18 ATGGACTGGACCTGGATTCTGTTCCTGGTCGCGGCTGCAACGCGAGGTCCATAGCGGTATC CATGTTTTTATTCTTGGGTGTTTTTCTGCTGGGCTGCCTAAGACCGAGGCCAACTGGGTA AATGTCATCAGTGACCTCAAGAAAATAGAAGACCTTATACAAAGCATGCACATTGATGCT ACTCTCTACACTGAGTCAGATGTACATCCCTCATGCAAAGTGACGGCCATGAAATGTTTC CTCCTCGAACTTCAAGTCATATCTCTGGAAAGTGGCGACGCGTCCATCCACGACACGGTC GAAAACCTGATAATACTCGCTAATAATAGTCTCTCTTCAAATGGTAACGTAACCGAGTCA GGTTGCAAAGAGTGCGAAGAGTTGGAAGAAAAAAACATAAAGGAGTTCCTGCAAAGTTTC GTGCACATTGTGCAGATGTTCATTAATACCTCTAGCGGCGGAGGATCAGGTGGCGGTGGA AGCGGAGGTGGAGGCTCCGGTGGAGGAGGTAGTGGCGGAGGTTCTCTTCAAATAACTTGT CCTCCACCGATGTCCGTAGAACATGCGGATATTTGGGTAAAATCCTATAGCTTGTACAGC CGAGAGCGGTATATCTGCAACAGCGGCTTCAAGCGGAAGGCCGGCACAAGCAGCCTGACC GAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTAGCCTGAAGTGC ATCAGAGATCCCGCCCTGGTGCATCAGCGGCCTGCCCCTCCAAGCACAGTGACAACAGCT GGCGTGACCCCCCAGCCTGAGAGCCTGAGCCCTTCTGGAAAAGAGCCTGCCGCCAGCAGC CCCAGCAGCAACAATACTGCCGCCACCACAGCCGCCATCGTGCCTGGATCTCAGCTGATG CCCAGCAAGAGCCCTAGCACCGGCACCACCGAGATCAGCAGCCACGAGTCTAGCCACGGC ACCCCATCTCAGACCACCGCCAAGAACTGGGAGCTGACAGCCAGCGCCTCTCACCAGCCT CCAGGCGTGTACCCTCAGGGCCACAGCGATACCACAGTGGCCATCAGCACCTCCACCGTG CTGCTGTGTGGACTGAGCGCCGTGTCACTGCTGGCCTGCTACCTGAAGTCCAGACAGTGA (1140 nucleic acid)

[0151] TIFF0007927045000011.tif55169

[0152] Those skilled in the art will understand that the signal peptides and linker sequences described above are illustrative and in no way limit their variations to those suitable for use as signal peptides or linkers. Many suitable signal peptides or linker sequences are known and available to those skilled in the art. Those skilled in the art will understand that signal peptides and / or linker sequences can be substituted with other sequences without altering the activity of the functional peptides led by the signal peptide or linked by the linker.

[0153] Design 4: Since the construct of Design 3 has been shown to function in promoting the survival and proliferation of effector cells, it has been shown that the cytoplasmic domain of IL15Rα can be omitted in such designs without adversely affecting the autonomous function of effector cells with IL15. Design 4 is a construct that provides another working alternative to Design 3, in which essentially the entire IL15Rα is removed except for the Sushi domain, fusing IL15 at one end with the transmembrane domain at the other (mb-Sushi), and 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 via IL15Rα, including cis presentation, is eliminated, while only the desired trans presentation of IL15 is retained. In some embodiments, the component containing IL15 fused with the Sushi domain contains an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 19, which may be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 20.

[0154] TIFF0007927045000012.tif41164 Sequence ID 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 signal peptides and linker sequences described above are illustrative and in no way limit their variations to those suitable for use as signal peptides or linkers. Many suitable signal peptides or linker sequences are known and available to those skilled in the art. Those skilled in the art will understand that signal peptides and / or linker sequences can be substituted with other sequences without altering the activity of the functional peptides led by the signal peptide or linked by the linker.

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

[0157] Design 6: The native or modified common receptor γC is fused to IL15 at its C-terminus via a linker for constitutive signaling and membrane-bound trans presentation of cytokines. The common receptor γC, also known as the common gamma chain or CD132, is also known as the 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: Manipulated IL15Rβ, which forms homodimers in the absence of IL15, is useful for generating constitutive signaling of cytokines.

[0159] In some embodiments, cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or one or more of their receptors, can be introduced into iPSCs and their induced cells during iPSC differentiation using one or more designs of Figure 1. In some embodiments, cell surface expression and signaling of IL2 or IL15 are via the construct shown in any one of designs 1-7. In some embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 are via the construct shown in designs 5, 6, or 7, by using either a common receptor or a cytokine-specific receptor. In some embodiments, cell surface expression and signaling of IL7 are via the construct 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 Figure 1 may be native to the corresponding cytokine receptor or may be modified or substituted with the transmembrane domains of other membrane-bound proteins.

[0160] In iPSCs and cells derived therefrom that contain both CAR and exogenous cytokine and / or cytokine receptor signaling, CAR and IL are expressed in separate constructs or co-expressed in a bicistronic construct that contains both CAR and IL. In some further embodiments, IL15 in the form represented by any of the construct designs in Figure 1 may be ligated to either the 5' or 3' end of a CAR expression construct via an autocleaved 2A coding sequence, exemplified as CAR-2A-IL15 or IL15-2A-CAR. Thus, IL15 and CAR reside in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct contains IL15 of design 3 in Figure 1. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct contains IL15 of design 3 in Figure 1. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct includes IL15 as in design 7 of Figure 1. When CAR-2A-IL15 or IL15-2A-CAR is expressed, 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 enables coordinated expression of CAR and IL15 under the same regulatory mechanism, which can be selected to incorporate, both in terms of timing and quantity, such as an inducible promoter for the expression of a single ORF. Self-cleaving peptides are found in members of the Picornaviridae family, including aftoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tesiovirus 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)), as well as cardioviruses such as tyloviruses (e.g., Tyler mouse 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] Embodiments of the bicistronic CAR-2A-IL15 or IL15-2A-CAR disclosed herein for IL15 are also intended for the expression of any other cytokines provided herein, such as IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, cell surface expression and signaling of IL2 are via the constructs shown in any of designs 1-7. In some other embodiments, cell surface expression and signaling of IL4, IL7, IL9, or IL21 are via the constructs 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- deficiencies To avoid allogeneic rejection, multiple HLA class I and class II proteins must match for histocompatibility in allogeneic recipients. Provided herein are iPSC cell lines in which the expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or by deletion or reduced expression levels of HLA class I-related genes, including but not limited to the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for the cell surface expression of all HLA class I heterodimers. B2M null cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II-related genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through the 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 derived cells in which both B2M and CIITA are knocked out, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoiding recognition and killing by host (allogeneic) T cells.

[0163] In some cell types, the absence of class I expression leads to lysis by NK cells. To overcome this "self-loss" response, HLA-G can be knocked in as needed to evade recognition and killing by NK cells 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 induced cells further include CD38 knockout, and optionally one or more of hnCD16, CAR, and IL, without adversely affecting the differentiation potential of the iPSCs or the function of induced effector cells, including induced T cells and induced NK cells.

[0164] 6. Genetically modified iPSC lines and induced cells provided herein In light of the above, this application is CD38 - / - iPSCs, cell lines, or populations thereof (also referred to herein as "CD38 null" or CD38 knockout), and CD38 - / - The present invention provides induced functional cells, including CD38 knockout cells obtained from the differentiation of iPSCs. In some embodiments, the functional cells are hematopoietic cells and include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, the functionally induced hematopoietic cells include effector cells such as T cells, NK cells, and regulatory cells.

[0165] Further provided herein are iPSCs comprising CD38 knockout and a polynucleotide encoding high-affinity non-cleavable CD16 (hnCD16), the iPSCs capable of producing functional hematopoietic cells through directed differentiation. In some embodiments, when hnCD16-mediated enhanced ADCC is induced using an anti-CD38 antibody, the iPSCs and / or their induced effector cells can target CD38-expressing (tumor) cells without causing elimination of effector cells, i.e., a decrease 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 CAR-conjugated CD38. In some embodiments, the effector cells include T cells. iPSC-derived T cells containing CD38 null and hnCD16 experience reduced cytotoxicity in the presence of anti-CD38 antibodies or anti-CD38 CARs, acquire ADCC, and lead to further mechanisms of T cell-mediated tumor killing. In some embodiments, effector cells include NK cells. iPSC-derived NK cells containing CD38 null and hnCD16 exhibit enhanced cytotoxicity and reduced NK cell fracturing in the presence of anti-CD38 antibodies or anti-CD38 CARs.

[0166] iPSCs containing CD38 knockout and polynucleotides encoding target-specific chimeric antigen receptors (CARs) are provided herein, and the iPSCs can produce functionally induced effector cells by directed differentiation. In one embodiment, the CAR contained in the CD38 knockout iPSC and its derivative effector cells targets the tumor cell surface protein CD38, but the CD38-CAR does not result in the elimination of the CD38 knockout iPSC and / or its induced effector cells. In some embodiments, the CAR contained in the CD38 knockout iPSC and its induced effector cells does not target CD38. In some embodiments, CAR expression, CD38 null induced effector cells can be used with an anti-CD38 antibody to induce ADCC without causing the elimination of effector cells, thereby increasing the persistence and / or viability of the iPSC and its effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or viability in combination therapy.

[0167] Furthermore, by including CD38 knockout, as well as a polynucleotide encoding at least one exogenous cytokine and / or receptor (IL) that enables cytokine signaling contributing to cell viability, persistence, and / or proliferation, the iPSC line can be directed to differentiate to produce functionally induced hematopoiesis with improved viability, persistence, proliferation, and effector cell function. The exogenously introduced cytokine signaling includes the signaling of one, two, or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or complete peptides of cytokines and / or their respective 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 ephemeral. In some embodiments, transient / temporary expression of cell surface cytokines / cytokine receptors is mediated by retroviruses, Sendai viruses, adenoviruses, episomes, minicircles, or RNA, including mRNA. In some embodiments, CD38 - / - Exogenous cell surface cytokines and / or receptors contained in iPSCs or their derived cells enable IL7 signaling. In some embodiments, CD38 - / - Exogenous cell surface cytokines and / or receptors contained in 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. (See CD38 above) - / - In some embodiments of IL iPSC, IL15 expression is due to construct 3 in Figure 1. (CD38) - / -In some embodiments of IL iPSC, IL15 expression is due to construct 4 in Figure 1. The above-mentioned CD38 in the embodiments described above. - / - IL iPSCs and their derived cells can autonomously maintain or improve cell proliferation, growth, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo. In some embodiments, CD38 - / - IL iPSCs and their induced effector cells can be used with an anti-CD38 antibody to induce ADCC without causing the elimination of effector cells, thereby synergistically increasing the persistence and / or viability of the iPSCs and their effector cells.

[0168] CD38 knockout, B2M knockout, and CIITA knockout, as well as iPSCs containing polynucleotides encoding HLA-G as needed, are also provided, and the iPSCs can be directed to differentiate and produce functional induced hematopoietic cells. - / - B2 M - / - CIITA - / - iPSCs and their induced effector cells, which are deficient in both HLA-I and HLA-II, can be used with an anti-CD38 antibody to induce ADCC without causing elimination of effector cells, thereby synergistically 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.

[0169] From the above perspective, the foregoing provides for iPSCs comprising CD38 knockout, and optionally all of hnCD16, CAR, exogenous cytokine / receptor, and B2M / CIITA knockout, wherein B2M is knocked out and a polynucleotide encoding HLA-G is optionally introduced, the iPSC can be directed to differentiate and produce functional induced hematopoietic cells. This application also includes functional iPSC-induced hematopoietic cells containing CD38 knockout, and optionally all of hnCD16, B2M / CIITA knockout, CAR, and exogenous cytokines / receptors, wherein B2M is knocked out and, optionally, a polynucleotide encoding HLA-G is introduced, and the induced hematopoietic cells include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0170] Another embodiment provided herein comprises iPSCs or iPSC-derived cells containing a truncated fusion protein of IL15 and IL15Rα, the fusion protein lacking an intracellular domain. These embodiments are shown in Figure 1 as “IL15Rα(ΔICD) fusion” and “IL5 / mb-Sushi,” but these embodiments are further collectively abbreviated as IL15Δ in Table 1 and throughout this application. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NOs: 17, 19, or 21. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain contains the amino acid sequence of SEQ ID NO: 17. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain contains the amino acid sequence of SEQ ID NO: 19. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain contains the amino acid sequence of SEQ ID NO: 21. In several other embodiments, iPSCs or iPSC-induced cells containing a shortened IL15 / IL15Rα fusion protein (IL15Δ) lacking an intracellular domain further include one or more of the following: CD38 knockout, hnCD16, CAR, exogenous cytokine / receptor, and B2M / CIITA knockout, where B2M is knocked out and a polynucleotide encoding HLA-G is introduced as needed, enabling the iPSC to differentiate as directed and produce functional induced hematopoietic cells, which include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0171] Accordingly, this application provides iPSCs and their functionally induced hematopoietic cells comprising any one of the following genotypes in Table 1. Unless otherwise specified as IL15Δ, this will be described in detail as a truncated fusion protein of IL15 and IL15Rα but without an intracellular domain, but the “IL” provided in Table 1 represents one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which particular cytokine / receptor expression is selected. Furthermore, if the iPSC and its functionally induced hematopoietic cells have a genotype comprising both CAR and IL, CAR and IL are contained in a bicistronic expression cassette containing a 2A sequence. In contrast, in some other embodiments, CAR and IL are in separate expression cassettes contained in the iPSC and its functionally induced hematopoietic cells. In one particular embodiment, iPSCs expressing both CAR and IL, and their functionally induced effector cells, contain IL15 in construct 3 or 4 of Figure 1, and the IL15 construct is contained in the expression cassette together with or separately from CAR.

[0172] [Table 1]

[0173] 7. Further modifications In some embodiments, iPSCs and their induced effector cells containing any one of the genotypes in Table 1 may further include deletion or reduced expression in at least one of the following genes: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, or any of the genes in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A This may further include introduced or increased expression at at least one of the following surface trigger receptors for binding to R, TCR, Fc receptors, engagers, and bispecific, multispecific, or universal engagers.

[0174] A bispecific or multispecific engager is a fusion protein comprising 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 one other binds to a tumor cell via a tumor-specific surface molecule. Exemplary effector cell surface molecules or surface trigger receptors that can be used for bispecific or multispecific engager recognition or coupling include, but are not limited to, CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, and the chimeric Fc receptors disclosed herein. In some embodiments, the CD16 expressed on the surface of effector cells for engager recognition is hnCD16, as described in Section I.2, comprising a CD16 (including F176V and optionally S197P) or CD64 extracellular domain and native or non-native transmembrane, stimuli, and / or signaling domains. In some embodiments, the CD16 expressed on the surface of effector cells for engager recognition is an hnCD16-based chimeric Fc receptor (CFcR). In some embodiments, the hnCD16-based CFcR comprises a transmembrane domain of NKG2D, a stimulating domain of 2B4, and a signaling domain of CD3ζ, and the extracellular domain of hnCD16 is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 includes F176V and optionally S197P. Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. In one embodiment, the bispecific antibody is CD3-CD19.In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In yet 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 includes a linker between the effector cell and tumor cell antigen-binding domain, for example, a modified IL15 (referred to in some publications as TriKE, or triplicate killer enhancer) as a linker for effector NK cells that 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, surface trigger receptors for bispecific or multispecific engagers may be endogenous to 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, including those with the genotypes listed in Table 1, to direct the differentiation of the iPSCs into T cells, NK cells, or other effector cells containing the same genotype and surface trigger receptor as the source iPSC, thereby introducing one or more exogenous surface trigger receptors into the effector cells.

[0176] 8. Antibodies for immunotherapy In some embodiments, in addition to the genome-engineered effector cells provided herein, additional therapeutic agents including antibodies or antibody fragments targeting antigens associated with a condition, disease, or indicator may be used in combination therapy with these effector cells. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include, but are not limited to, anti-CD20 (rituximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), anti-HER2 (trastuzumab, partuzumab), anti-CD52 (aremtuzumab), anti-EGFR (certuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars. In some embodiments, the iPSC-derived effector cells include hematopoietic lineage cells containing the genotypes listed in Table 1. In some embodiments, the iPSC-derived effector cells include NK cells containing the genotypes listed in Table 1. In some embodiments, the iPSC-derived effector cells include T cells containing the genotypes listed in Table 1. In some embodiments of combinations useful for treating liquid or solid tumors, the combination includes iPSC-derived NK cells or T cells containing at least CD38 null, and an anti-CD38 antibody.In one embodiment, the combination comprises iPSC-derived NK cells containing CD38 null and hnCD16, and one of the anti-CD38 antibodies, daratumumab, isatuximab, or MOR202. In one embodiment, the combination comprises iPSC-derived NK cells containing CD38 null and hnCD16, and daratumumab. In some further embodiments, the iPSC-derived NK cells included in the combination with daratumumab contain CD38 null, hnCD16, IL15, and a CAR that targets at least one of CD38 or CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, and PDL1, where IL15 is co-expressed with or separately from the CAR, and IL15 is one of the forms presented in constructs 1-7 of Figure 1. In some specific embodiments, IL15 is in the form of construct 3, 4, or 7, when it is expressed with or separately from the CAR.

[0177] 9. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate the immune response if not inhibited. Tumors have been shown to select specific immune checkpoint pathways, particularly as a primary mechanism of immune resistance against tumor antigen-specific T cells. Checkpoint inhibitors (CIs) are antagonists that can block inhibitory checkpoints and restore immune system function by reducing the expression or gene product of checkpoint genes or by decreasing the activity of checkpoint molecules. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncological landscape, with these drugs leading 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 this application provides a therapeutic approach to overcome CI resistance by including genomically engineered functional inducement cells, as provided in combination therapy with CIs. In one embodiment of the combination therapy, the inducement cells are NK cells. In another embodiment of the combination therapy, the inducement cells are T cells. In addition to exhibiting direct antitumor activity, the induced NK cells provided herein have been shown to be resistant to PDL1-PD1-mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at tumor sites. Thus, T cell tumor infiltration facilitated by functionally potent genome-engineered induced NK cells suggests that these NK cells can synergistically interact with T cell-targeted immunotherapy, including checkpoint inhibitors, to alleviate local immunosuppression and reduce tumor burden.

[0178] In one embodiment, induced NK cells for combination therapy with checkpoint inhibitors include all of the following: CD38 knockout and 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, induced NK cells include any one of the genotypes listed in Table 1. In some embodiments, the induced NK cells described above further include deletion or reduced expression in at least one of the following genes in the chromosome 6p21 region: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention further includes introduced or increased expression at at least one of the following: R, CAR, TCR, Fc receptors, engagers, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers.

[0179] In another embodiment, the induced T cells for combination therapy with checkpoint inhibitors include all of the following: CD38 knockout and 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 include any one of the genotypes listed in Table 1. In some embodiments, the induced T cells described above further include deletion or reduced expression in at least one of the following genes in the chromosome 6p21 region: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2A The invention further includes introduced or increased expression at at least one of the following: R, CAR, TCR, Fc receptors, engagers, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers.

[0180] The above-mentioned induced NK cells or induced T cells are obtained by differentiating an iPSC clone containing CD38 knockout and, optionally, all of the following: 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 above-mentioned iPSC clone further includes deletion or reduced expression in at least one of the following genes: 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 The invention further includes introduced or increased expression at at least one of the following: R, CAR, TCR, Fc receptors, engagers, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers.

[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 ( This includes, but is not limited to, antagonists of 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 KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, 3DL2).

[0182] In some embodiments, the antagonist that inhibits any of the checkpoint molecules described above is an antibody. In some embodiments, the checkpoint inhibitor antibody may be a mouse antibody, a human antibody, a humanized antibody, camel Ig, shark heavy chain only antibody (VNAR), Ig NAR, a chimeric antibody, a recombinant antibody, or a 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 manufacture, easier to use, or more sensitive than the whole antibody. In some embodiments, one, two, three, or more checkpoint inhibitors include 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 at least one of their derivatives, functional equivalents, or biosimilars.

[0183] In some embodiments, many miRNAs are found to be regulators that control the expression of immune checkpoints, so antagonists that inhibit any of the above-mentioned checkpoint molecules are microRNA-based (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint antagonist 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 the provided induced NK cells or induced T cells include at least one checkpoint inhibitor targeting at least one checkpoint molecule, and the induced cells have the genotypes listed in Table 1. Some other embodiments of the combination therapy with the provided induced NK cells or induced T cells include two, three, or more checkpoint inhibitors so 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 the genotypes listed in Table 1, the checkpoint inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the checkpoint inhibitor is produced by the induced cells by expressing an exogenous polynucleotide sequence encoding the antibody, or the fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the antibody, or the fragment or variant thereof that inhibits the checkpoint, is co-expressed with the CAR in either a separate construct or a bicistronic construct containing both the CAR and the sequence encoding the antibody or fragment thereof. In some further embodiments, the sequence encoding the antibody or a fragment thereof may be ligated to either the 5' or 3' end of the CAR expression construct via an autocleaved 2A coding sequence, exemplified, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences of the checkpoint inhibitor and the CAR reside in a single open reading frame (ORF). Once the checkpoint inhibitor is delivered and expressed and secreted as a payload by induced effector cells capable of infiltrating the tumor microenvironment (TME), it activates the effector cells by binding to the TME and counteracting inhibitory checkpoint molecules, thereby activating modalities such as CARs or activating their receptors.In some embodiments, checkpoint inhibitors co-expressed with CAR inhibit at least one of the following: 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 KIRs. In some embodiments, the checkpoint inhibitors co-expressed with CAR in induced cells having the genotypes listed in Table 1 are selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is atezolizumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is nivolumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is pembrolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0185] In some other embodiments of the combination therapies provided herein, comprising at least one antibody inhibiting induced cells and checkpoint molecules, the antibody is not produced by or within the induced cells, but is administered before, concurrently with, or subsequently to the administration of induced cells having the genotypes 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 concurrent or sequential. In one embodiment of a combination therapy comprising induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitors included in the therapy are one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of combination therapy involving induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is atezolizumab, or its humanized or Fc-modified variant, fragment, and its functional equivalent or biosimilar. In some embodiments of combination therapy involving induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is nivolumab, or its humanized or Fc-modified variant, fragment, and its functional equivalent or biosimilar. In some embodiments of combination therapy involving induced NK cells or induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is pembrolizumab, or its humanized or Fc-modified variant, fragment, and its functional equivalent or biosimilar.

[0186] II. Methods for targeted genome editing at selected loci in iPSCs 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 (interchangeable as “targeted genome editing” or “targeted gene editing”) allows for insertion, deletion, and replacement at pre-selected sites within the genome. If an endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down by the sequence deletion. Thus, targeted editing can also be used to precisely disrupt the expression of endogenous genes. The term “targeted integration,” as similarly used herein, refers to a process involving the insertion of one or more exogenous sequences, with or without the deletion of an endogenous sequence at the insertion site. In contrast, randomly integrated genes are susceptible to positional effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomere regions are particularly prone to transgene silencing. Newly integrated genes can influence surrounding endogenous genes and chromatin, potentially altering cellular behavior or promoting cellular transformation. Therefore, inserting exogenous DNA into pre-selected loci, such as safe harbor loci or genome-safe harbors (GSH), is crucial for safe, efficient, copy number control, and reliable gene response regulation.

[0187] Targeted editing can be achieved through either a nuclease-independent or nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by a homologous sequence adjacent to the inserted exogenous polynucleotide via an enzymatic mechanism in the host cell.

[0188] Alternatively, targeted editing can be achieved more frequently by the specific introduction of double-strand breaks (DSBs) using specific rare-cut end nucleases. 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 result in random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast, when a donor vector containing a pair of homology arms and adjacent exogenous genetic material is present, the exogenous genetic material can be introduced into the genome during homologous recombination-directed repair (HDR), resulting in "targeted integration."

[0189] Available endonucleases capable of introducing specific, targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), and RNA-induced CRISPR (clustered, equally spaced short repeat) systems. Furthermore, DICE (dual integrase cassette exchange) systems utilizing phiC31 and Bxb1 integrases are also promising tools for targeted incorporation.

[0190] ZFNs are targeted nucleases containing 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 via one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within a zinc finger-binding domain whose structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2, C3H, and C4 zinc fingers. “Engineered” zinc finger domains are domains that do not exist naturally, and their design / construction is primarily due to reasonable criteria, e.g., the application of substitution rules and computerized algorithms to process information in databases storing information on existing ZFP designs and binding data. See, for example, U.S. Patents 6,140,081, 6,453,242, and 6,534,261. See also WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496. “Selected” zinc finger domains are non-naturally occurring domains whose production arises primarily from empirical processes such as phage display, interaction trapping, or hybrid selection. ZFNs are described in detail in U.S. Patents 7,888,121 and 7,972,854, the full disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is the fusion of a FokI nuclease with a zinc finger DNA-binding domain.

[0191] TALENs are targeted nucleases containing a nuclease fused to the 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 the TAL effector protein 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 and bind to effector-specific DNA sequences via their DNA-binding domains, activating gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA-binding domain depends on the effector variable number of incomplete 34-amino acid repeats containing polymorphisms at selected repeat positions, known as repeat variable residues (RVDs). TALENs are described in detail in U.S. Patent Application No. 2011 / 0145940, incorporated herein by reference. The most recognized example of a TALEN in the art is the fusion polypeptide of a FokI nuclease to the TAL effector DNA-binding domain.

[0192] Another example of a targeted nuclease used in the method of the present invention is a polypeptide comprising a targeted Spo11 nuclease, a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain specific to the DNA sequence of interest, such as a zinc finger DNA-binding domain or a TAL effector DNA-binding domain. See, for example, 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-related nucleases derived from families including Cas, CPF, CSE, CSY, CSN, CSD, CST, CSH, CSA, CSM, and CMR; restriction endonucleases; meganucleases; homing endonucleases, etc.

[0195] As an exemplary example, CRISPR / Cas9 requires two main components: (1) Cas9 endonuclease and (2) a crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to a target DNA sequence containing the PAM and the PAM-proximal seeding region. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA), which can guide Cas9 to the selected sequence target. These two components can be delivered to mammalian cells via transfection or transduction.

[0196] DICE-mediated insertion provides unidirectional integration of exogenous DNA, strictly limited to small attB and attP recognition sites within each enzyme itself, using, for example, a pair of recombinases such as phiC31 and Bxb1. Since these targeted att sites are not naturally present in the mammalian genome, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Patent Application Publication 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 targeted integration method comprises introducing the construct into a cell to enable site-directed homologous recombination by a cell-host enzymatic mechanism. In another embodiment, the targeted integration method 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 enable ZFN-mediated insertion. In yet another embodiment, the targeted integration method 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 enable TALEN-mediated insertion. In another embodiment, a method for targeted integration in cells includes introducing a construct containing one or more exogenous polynucleotides into cells and introducing a gRNA containing a Cas9 expression cassette and a guide sequence specific to the desired integration site into cells to enable Cas9-mediated insertion. In yet another embodiment, a method for targeted integration in cells includes introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site within the cell and introducing a construct containing one or more exogenous polynucleotides into cells and introducing an expression cassette for DICE recombinases to enable DICE-mediated targeted integration.

[0198] Promising sites for targeted integration include, but are not limited to, intragenetic or extragenetic regions of the human genome that are safe harbor loci or genome-safe harbors (GSHs) that can theoretically accommodate the predictable expression of newly integrated DNA without adverse effects on the host cell or organism. A useful safe harbor must allow sufficient transgene expression to produce the desired level of the protein or non-coding RNA encoded by the vector. A safe harbor must also not make cells more susceptible to malignant transformation or alter cellular function. For a recombination site to be a promising safe harbor locus, it must ideally meet the following conditions, but are not limited to: no disruption of regulatory elements or genes as determined by sequence annotation; being an intergeneric region within a densely populated region of genes, or a convergence site between two genes transcribed in opposite directions; maintaining distance to minimize the possibility of long-range interactions between the vector-encoded transcription activator and adjacent genes, particularly cancer-related genes and microRNA gene promoters; and possessing clearly ubiquitous transcriptional activity, as reflected by a broad spatial and temporal expression sequence tag (EST) expression pattern exhibiting ubiquitous transcriptional activity. This latter characteristic 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 regions suitable for exogenous insertion, the precise locus selected for insertion should lack repeating elements and conserved sequences, allowing for the straightforward design of primers for homology arm amplification.

[0199] Suitable sites for human genome editing, or more specifically, targeted incorporation, include, but are not limited to, human orthologues of the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the mouse ROSA26 locus. Furthermore, the human orthologue of the mouse H11 locus may also be a suitable site for insertion using the targeted incorporation compositions and methods disclosed herein. Additionally, the collagen and HTRP loci can also be used as safe harbors for targeted incorporation. However, validation of each selected site has been shown to be necessary, particularly in stem cells, for specific incorporation events, and optimization of the incorporation strategy, including promoter selection, exogenous gene sequencing and placement, and construct design, is often required.

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

[0201] Accordingly, one aspect of the present invention provides a method for targeted integration at a selected locus, including a genome-safe harbor, or at a pre-selected locus that is known or proven to be safely and adequately regulated for continuous or transient gene expression, such as the B2M, TAP1, TAP2, or tapasin locus, as provided herein. In one embodiment, the genome-safe harbor for the targeted integration method includes one or more desirable integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genome-safe harbor. In one embodiment, a method for targeted integration into cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a construct containing a pair of homologous arms specific to a desired integration site and one or more exogenous sequences into cells to enable site-directed homologous recombination by a cell-host enzyme mechanism, 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 for a genome-safe harbor.

[0202] In another embodiment, the method of targeted integration into cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a ZFN expression cassette containing a DNA-binding domain specific to a desired integration site to enable ZFN-mediated insertion, 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 for a genome-safe harbor. In yet another embodiment, the method of targeted integration into cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a TALEN expression cassette containing a DNA-binding domain specific to a desired integration site to enable TALEN-mediated insertion, 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 for a genome-safe harbor. In another embodiment, a method for targeted integration in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a gRNA containing a Cas9 expression cassette and a guide sequence specific to a desired integration site into the cells to enable Cas9-mediated insertion, 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 for a genome-safe harbor.In yet another embodiment, a method for targeted integration in cells comprises introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site within the cell, introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinases to enable DICE-mediated targeted integration, 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 for a genome-safe harbor.

[0203] Furthermore, as provided herein, the above-described methods for targeted incorporation in a safe harbor are used to insert polynucleotides of interest, such as safety switch proteins, targeted modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and polynucleotides encoding proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some other embodiments, a construct comprising one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the present invention is a suicide gene encoding a safety switch protein. Suicide gene systems suitable for 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 by the B cell molecule CD20 can be eliminated upon administration of the mAb rituximab. Additionally, modified EGFR containing an epitope recognized by cetuximab can be used to deplete genetically modified cells when they are exposed to cetuximab. Thus, one aspect of the present invention provides a method for targeted incorporation 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 incorporated by the methods herein are driven by an operablely linked exogenous promoter contained in a construct for targeted incorporation. The promoter may be inductive or constitutive and may be time-specific, tissue-specific, or cell-type-specific. Suitable constitutive promoters for the methods of the present invention include, but are not limited to, cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0205] Exogenous polynucleotides incorporated by the methods described herein may be driven at the integration site by an endogenous promoter in the host genome. In one embodiment, the method of the present invention is used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the method of the present invention is used for targeted integration at the ROSA26 locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the method of the present invention is used for targeted integration at the H11 locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous H11 promoter. In yet another embodiment, the method of the present invention is used for targeted integration at the collagen locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method of the present invention is used for targeted integration at the HTRP locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by an endogenous HTRP promoter. Theoretically, only correct insertion at the desired site would enable 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 a targeted incorporation method are driven by a single promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to enhance physical separation between the parts and maximize access to the enzymatic mechanism. The linker peptide of the linker sequence may consist of amino acids selected to make the physical separation between the parts (exogenous polynucleotides and / or proteins or peptides encoded therefrom) more flexible or more rigid, depending on the relevant function. The linker sequence may be cleavable by proteases or chemically cleavable, resulting in distinct parts. 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 may be naturally produced by the host or introduced exogenously. Alternatively, cleavage sites in the linker may be sites that can be cleaved by selected chemicals, such as cyanide bromide, hydroxylamine, or exposure to low pH. Any linker sequence may serve purposes other than providing a cleavage site. The linker sequence should allow for the effective positioning of a part relative to another adjacent part for the part to function properly. The linker may also be a simple amino acid sequence long enough to prevent any steric hindrance between parts. Furthermore, the linker sequence can provide post-translational modifications including, but not limited to, phosphorylation sites, biotinylation sites, sulfated sites, and γ-carboxylation sites. In some embodiments, the linker sequence is flexible so as not to hold a biologically active peptide in a single undesirable conformation. To provide flexibility, the linker may be predominantly composed of amino acids with small side chains, such as glycine, alanine, and serine. In some embodiments, about 80 or 90 percent or more of the linker sequence consists of glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, the G4S linker peptide separates the terminal processing and endonuclease domains of the fusion protein. In other embodiments, the 2A linker sequence allows two distinct proteins to be produced from a single translation. A suitable linker sequence can be easily identified empirically. Furthermore, 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 intra-sequence ribosome entry site (IRES). In some embodiments, any two consecutive linker sequences are distinct.

[0207] Methods for introducing constructs containing exogenous polynucleotides for targeted incorporation into cells can be achieved using methods of gene transfer into cells that are known in themselves. In one embodiment, the construct comprises a viral vector skeleton such as an adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, or Sendai virus vector. In some embodiments, plasmid vectors are used to deliver and / or express exogenous polynucleotides to target cells (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo). In some other embodiments, episomal vectors are used to deliver exogenous polynucleotides to target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV is not integrated into the host genome. Furthermore, episomal rAAV vectors mediate homologous-directed gene targeting at a much higher rate compared to transfection with conventional targeted plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the genome of iPSCs. In some embodiments, the genome-modified iPSCs and their derived cells obtained using the methods and compositions herein contain at least one genotype listed in Table 1.

[0208] III. Methods for obtaining and maintaining genetically engineered iPSCs The present invention provides a method for obtaining and maintaining genome-engineered iPSCs, which include one or more targeted edits at one or more desired sites, the targeted edits remaining intact and functional at their respective selected editing sites in the proliferated genome-engineered iPSCs or iPSC-derived non-pluripotent cells. Targeted editing introduces insertions, deletions, and / or substitutions, i.e., targeted incorporations and / or indels, at selected sites into the genomes of iPSCs and cells derived therefrom. Compared to directly manipulating patient-derived peripheral blood primary effector cells, the many advantages of obtaining genomically engineered induced cells by editing and differentiating iPSCs as provided herein 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 involved; the obtained effector cells are rejuvenated due to elongated telomeres and less depletion; and the effector cell population is homogeneous in terms of editing site, copy number, and lack of allelic mutation, random mutation, and expression diversity, primarily due to the possibility of clonal selection in the engineered iPSCs provided herein.

[0209] In certain embodiments, genome-engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and grown as single cells for extended periods in cell culture media shown in Table 2 as Fate Maintenance Medium (FMM), where the iPSCs retain the targeted edits and functional modifications at the selected sites. The components of the medium may be present in the medium in amounts within the optimal range shown in Table 2. iPSCs cultured in FMM have been shown to remain undifferentiated, maintain a basal or naive profile, retain genomic stability without requiring culture washing or selection, and readily induce in vitro differentiation through all three somatic cell lineages, embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation by teratoma formation. See, for example, U.S. Patent Application No. 61 / 947,979, whose disclosure is incorporated herein by reference.

[0210] [Table 2]

[0211] In some embodiments, genome-edited iPSCs comprising one or more targeted integrations and / or indels are maintained, passaged and expanded in a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and do not comprise or are essentially free of a TGFβ receptor / ALK5 inhibitor, wherein the iPSCs retain intact and functional targeted editing at the selected site.

[0212] Another aspect of the present invention provides a method for generating genome-edited iPSCs through targeted editing of iPSCs, or through first generating genome-edited non-pluripotent cells by targeted editing, then reprogramming the selected / isolated genome-edited non-pluripotent cells to obtain iPSCs comprising the same targeted editing as the non-pluripotent cells. A further aspect of the present invention provides genome-edited non-pluripotent cells that are simultaneously undergoing reprogramming by introducing targeted integration and / or targeted indels into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and the conditions for reprogramming comprise contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genome editing and reprogramming, the targeted integration and / or targeted indels can be introduced into non-pluripotent cells before or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally small molecules.

[0213] In some embodiments, to simultaneously manipulate and reprogram non-pluripotent cells genomes, targeted embeddings and / or indels may also be introduced into non-pluripotent cells after a 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 construct-carrying vector is introduced before the reprogrammed 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 non-pluripotent cells with at least one reprogramming factor and, optionally, a combination of TGFβ receptor / ALK inhibitors, MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors (FRM; Table 2). In some embodiments, genomically engineered iPSCs by any of the above methods are further maintained and proliferated using a mixture containing a combination of MEK inhibitors, GSK3 inhibitors, and ROCK inhibitors (FMM; Table 2).

[0215] In some embodiments of a method for generating genetically engineered iPSCs, the method comprises genomically engineering an iPSC by introducing one or more targeted embeddings and / or indels into the iPSC to obtain a genetically engineered iPSC having at least one genotype listed in Table 1. Alternatively, a method for generating genetically engineered iPSCs comprises (a) introducing one or more targeted edits into non-pluripotent cells to obtain genetically engineered non-pluripotent cells containing targeted embeddings and / or indels at selected sites, and (b) contacting the genetically engineered non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and optionally a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to obtain a genetically engineered iPSC containing targeted embeddings and / or indels at selected sites. Alternatively, a method for generating genome-engineered iPSCs comprises (a) contacting non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and, optionally, a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate the reprogramming of the non-pluripotent cells; (b) introducing one or more targeted embeddings and / or indels into the reprogrammed non-pluripotent cells for genome engineering; and (c) obtaining a clone genome-engineered iPSC containing the targeted embeddings and / or indels at a selected site.

[0216] Reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in PCT / US2015 / 018801 and PCT / US16 / 57136, the disclosures of which are incorporated herein by reference. One or more reprogramming factors may be in the form of polypeptides. Reprogramming factors may also be in the form of polynucleotides and are therefore introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by a plasmid combination. See, for example, 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 imported with multiple constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted incorporation at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNA, including but not limited to siRNA, shRNA, miRNA, 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 can be expressed under conditions that activate the promoters, for example, 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, one or more suicide genes are driven by a constitutive promoter, for example, capase-9 driven by CAG. These constructs, containing different exogenous polynucleotides and / or different promoters, can be transferred into non-pluripotent cells simultaneously or sequentially. Non-pluripotent cells subjected to targeted incorporation of multiple constructs can be simultaneously exposed to one or more reprogramming factors to initiate reprogramming concurrently with genomic manipulation, thereby obtaining genomically engineered iPSCs containing multiple targeted incorporations in the same pool of cells. Thus, this robust method allows for the induction of clonally engineered hiPSCs with multiple modalities incorporated into one or more selected target sites through a simultaneous reprogramming and manipulation strategy.In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions herein include at least one genotype listed in Table 1.

[0218] IV. Method for obtaining genetically modified effector cells by differentiating genome-modified iPSCs. Further embodiments of the present invention provide a method for in vivo differentiation of genome-engineered iPSCs by teratoma formation, wherein differentiated cells induced in vivo from genome-engineered iPSCs retain intact and functional targeted editing, including targeted incorporation and / or indels, at desired sites. In some embodiments, differentiated cells induced in vivo from genome-engineered iPSCs via teratoma contain one or more inducible suicide genes incorporated 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 for a genome-safe harbor. In some other embodiments, differentiated cells induced in vivo from genome-engineered iPSCs via teratoma contain polynucleotides encoding a targeted modality, or polynucleotides encoding proteins that promote the transport, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells. In some embodiments, differentiated cells induced in vivo from genomically engineered iPSCs via teratomas containing one or more inducible suicide genes further contain one or more indels of endogenous genes associated with the regulation and mediation of immune responses. In some embodiments, the indels contain one or more endogenous checkpoint genes. In some embodiments, the indels contain one or more endogenous T cell receptor genes. In some embodiments, the indels contain one or more endogenous MHC class I suppressor genes. In some embodiments, the indels contain one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the indels contain one or more endogenous genes including, but not limited to, B2M, PD1, TAP1, TAP2, tapasin, and TCR genes. In one embodiment, a genomically engineered iPSC containing one or more exogenous polynucleotides at a selected site further contains targeted editing in the gene encoding B2M (beta-2-microglobulin).

[0219] In certain embodiments, a genome-engineered iPSC containing one or more genetic modifications provided herein is used to induce a hematopoietic cell lineage or other specific cell type in vitro, and the induced non-pluripotent cells retain functional genetic modifications, including targeted editing, at selected sites. In one embodiment, cells derived from a genome-engineered iPSC include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, where these cells derived from a genome-engineered iPSC retain functional genetic modifications, including targeted editing, at desired sites.

[0220] Applicable differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lines include, for example, those described in International Application No. PCT / US2016 / 044122, the disclosure of which is incorporated herein by reference. As provided, the methods and compositions for generating hematopoietic cell lines involve definitive hematopoietic endothelium (HE) derived from pluripotent stem cells, including hiPSCs, in a scalable, monolayer EB-free culture platform 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 to cells of various lineages that have directly transitioned to hematopoietic fate without passing through pluripotent intermediates. Similarly, cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells, and to all intervening hematopoietic cell lineages.

[0221] A method for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture involves contacting pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from pluripotent stem cells are obtained and proliferated from the pluripotent stem cells without the formation of embryoid bodies. The mesodermal cells are then subjected to contact with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain proliferating mesodermal cells with definitive hematopoietic endothelial (HE) potential without the formation of 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 proliferated during differentiation.

[0222] The method for obtaining hematopoietic lineage cells provided herein is superior to EB-mediated pluripotent stem cell differentiation because EB formation results in moderate to minimal cell proliferation, failing to enable monolayer culture and uniform differentiation of cells within a population, which are crucial for many applications requiring uniform proliferation, making it cumbersome and less efficient.

[0223] The provided monolayer differentiation platform facilitates differentiation into definitive hematopoietic endothelium, resulting in the induction of differentiated offspring such as hematopoietic stem cells and T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale proliferation to enable the delivery of therapeutically appropriate numbers of pluripotent stem cell-derived hematopoietic cells for various therapeutic applications. Furthermore, monolayer culture using the methods provided herein results in functional hematopoietic lineage cells that enable the 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 hematopoietic endothelium, hematopoietic pluripotent progenitor cells, hematopoietic stem cells and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0224] A method for instructing the differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage, comprising: (i) contacting 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 mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally not comprising a TGFβ receptor / ALK inhibitor, to initiate differentiation and proliferation of mesodermal cells having definitive hematopoietic endothelial potential from the mesodermal cells; and (iii) contacting mesodermal cells having definitive hematopoietic endothelial potential with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator, and optionally not comprising a TGFβ receptor / ALK inhibitor, to initiate differentiation and proliferation of definitive hematopoietic endothelial cells derived from pluripotent stem cells having definitive hematopoietic endothelial potential.

[0225] In some embodiments, the method further comprises contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, and then seeding and growing the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs containing one or more genetic imprints, where the one or more genetic imprints contained in the iPSCs are retained in hematopoietic cells differentiated therefrom. In some embodiments of the method for directing the differentiation of pluripotent stem cells into hematopoietic lineage cells, the differentiation of pluripotent stem cells into hematopoietic lineage cells is in a monolayer culture form without the formation of embryoid bodies.

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

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

[0228] In some further embodiments of the above-described method for directing the differentiation of pluripotent stem cells into hematopoietic cell lineage cells, the method further comprises (i) contacting definitive hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator, to initiate the differentiation of definitive hematopoietic endothelium into pre-NK cell progenitor cells, and optionally (ii) contacting pre-NK cell progenitor cells derived from pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, but not one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate the differentiation of pre-NK cell progenitor cells into NK cell progenitor cells or NK cells. In some embodiments, pluripotent stem cell-derived NK progenitor cells are CD3-CD45+CD56+CD7+. In some embodiments, pluripotent stem cell-derived NK cells are CD3-CD45+CD56+ and may be further defined by NKp46+, CD57+, and CD16+ as needed.

[0229] Therefore, using the differentiation method described above, one or more populations of hematopoietic cells derived from iPSCs 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 hematopoietic endothelial cells (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) Definitive HSCs using one or more media selected from A2 and iNK-B2, (v) Pluripotent progenitor cells (iMPP) using iMPP-A, (v) T cell progenitor cells (ipro-T) using one or more media selected from iTC-A2 and iTC-B2, (vi) T cells (iTC) using iTC-B2, (vii) NK cell progenitor cells (ipro-NK) using one or more media selected from iNK-A2 and iNK-B2, and / or (viii) NK cells (iNK) and iNK-B2. In some embodiments, the media are: a.iCD34-C comprises one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, as well as Wnt pathway activators as optional, and does not contain TGFβ receptor / ALK inhibitors. b.iMPP-A contains a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. c.iTC-A2 contains one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors, SCF, Flt3L, TPO, and IL7, and optionally, BMP activators. d.iTC-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. e. iNK-A2 comprises a ROCK inhibitor, 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 genome-engineered iPSC-derived cells obtained from the method described above comprise one or more inducible suicide genes integrated at one or more desired integration sites comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of a genomic safe harbor. In some other embodiments, the genome-engineered iPSC-derived cells comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the trafficking, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some embodiments, the genome-engineered iPSC-derived cells comprising one or more suicide genes further comprise one or more indels contained 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 genome-engineered iPSC-derived cell comprising one or more suicide genes comprises an indel in the B2M gene, and B2M is knocked out.

[0231] Furthermore, applicable dedifferentiation methods and compositions for obtaining second-fate genome-modified hematopoietic cells from first-fate genome-modified 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 herein enable partial reprogramming of initiating non-pluripotent cells into non-pluripotent intermediate cells by restricting the expression of the endogenous Nanog gene during reprogramming, and subjecting the non-pluripotent intermediate cells to conditions for differentiation of intermediate cells into a desired cell type. In some embodiments, genome-modified iPSCs and their induced cells obtained using the methods and compositions herein include at least one genotype listed in Table 1.

[0232] V. Therapeutic use of functionally modalized inducible immune cells differentiated from genetically engineered iPSCs The present invention provides compositions comprising, in some embodiments, 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 iPSC comprises one or more targeted gene edits retainable in the iPSC-derived immune cells, and the genetically engineered iPSC and its derived cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived CD34 cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro-T cells or T cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived pro-NK cells or NK cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived immunomodulatory cells or bone marrow-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived genetically engineered immune cells are further modified ex vivo for improved therapeutic potential. In one embodiment, an isolated population or subpopulation of genetically modified immune cells derived from iPSCs includes an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In another embodiment, an isolated population or subpopulation of genetically modified immune cells derived from iPSCs includes an increased number or proportion of type I NKT cells. In yet another embodiment, an isolated population or subpopulation of genetically modified immune cells derived from iPSCs includes an increased number or proportion of adaptive NK cells. In some embodiments, an isolated population or subpopulation of genetically modified CD34 cells, HSC cells, T cells, NK cells, or bone marrow-derived suppressor cells derived from iPSCs is allogeneic. In some other embodiments, an isolated population or subpopulation of genetically modified CD34 cells, HSC cells, T cells, NK cells, or MDSCs derived from iPSCs is autologous.

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

[0234] In some embodiments, the genetic imprinting of pluripotent stem cells includes (i) one or more genetic modification modalities obtained by genomic insertions, deletions, or substitutions in the genome of pluripotent cells during or after reprogramming non-pluripotent cells into iPSCs, or (ii) one or more retainable therapeutic attributes of source-specific immune cells that are donor-specific, disease-specific, or treatment response-specific, and the pluripotent cells are reprogrammed from source-specific immune cells, and the iPSCs retain the therapeutic attributes of the therapeutic attribute source, which are also included in hematopoietic cells derived from the iPSCs.

[0235] In some embodiments, the genetically modified modality includes one or more safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, and / or viability of iPSCs or their induced cells. In some embodiments, the genetically modified iPSCs and their induced cells include the genotypes listed in Table 1. In several other embodiments, genetically modified iPSCs and their induced cells, including the genotypes listed in Table 1, further include additional genetic modification modalities, including (1) deletion or reduction of expression of one or more genes in the chromosome 6p21 region, such as TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and (2) introduced or increased expression of surface trigger receptors for coupling with HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, TCR, Fc receptors, or bispecific, multispecific, or universal engagers.

[0236] In several other embodiments, hematopoietic lineage cells include therapeutic attributes of source-specific immune cells relating to at least two combinations 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 extratumor effects, and (v) improved homing, persistence, cytotoxicity, or antigen skeletal rescue.

[0237] In some embodiments, iPSC-induced hematopoietic cells include the genotypes listed in Table 1, and these cells express at least one cytokine and / or its receptor, including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, or any modified protein thereof, and express at least CAR. In some embodiments, the engineered expression of cytokines and CAR is NK cell-specific. In some other embodiments, the engineered expression of cytokines and CAR is T cell-specific. In one embodiment, the CAR includes a CD38-binding domain. In some embodiments, the iPSC-induced 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-induced hematopoietic effector cells can rescue tumor antigen escapes.

[0238] By introducing the immune cells of the present invention into subjects suitable for adoptive cell therapy, various diseases can be improved. In some embodiments, the iPSC-induced hematopoietic cells provided are for allogeneic adoptive cell therapy. Furthermore, in some embodiments, the present invention provides the therapeutic use of the above-mentioned therapeutic composition by introducing the composition into subjects suitable for adoptive cell therapy, where the subjects have autoimmune disorders, hematological malignancies, solid tumors, or infections associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes). Examples of solid tumors include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, 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), several forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, several forms of myocarditis, and multiple sclerosis. Examples of viral infections include, but are not limited to, thyroiditis, bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, several forms of thyroiditis, several forms of uveitis, vitiligo, and granulomatous disease with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (herpes zoster virus associated with Kaposi's sarcoma), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella-zoster virus), adenovirus-, lentivirus-, and BK polyomavirus-related diseases.

[0239] Therapies using induced hematopoietic lineage cells of embodiments disclosed herein can be performed based on symptoms or to prevent relapse. Terms such as “to treat” and “therapy” are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. Effects may be prophylactic with respect to completely or partially preventing a disease, and / or therapeutic with respect to partial or complete cure of the disease and / or adverse effects resulting from the disease. As used herein, “therapy” encompasses all interventions for a disease in a subject and includes: preventing the onset of the disease in a subject susceptible to the disease but not yet diagnosed as having it; inhibiting the disease, i.e., preventing its onset; or alleviating the disease, i.e., regressing the disease. Therapeutic agents or compositions may be administered before, during, or after the onset of a disease or injury. The treatment of an ongoing disease in which the therapy stabilizes or reduces undesirable clinical symptoms in a patient is also of particular importance. In certain embodiments, the subject requiring therapy has a disease, condition, and / or injury in which at least one related symptom can be suppressed, improved, and / or improved by cell therapy. Certain embodiments are intended to include, but are not limited to, subjects requiring cell therapy, including, candidates for bone marrow or stem cell transplantation, subjects who have received chemotherapy or radiotherapy, subjects with or at risk of having hyperproliferative disorders or cancer, such as hematopoietic hyperproliferative disorders or cancer, subjects with or at risk of developing tumors, such as solid tumors, and subjects with or at risk of having viral infections or diseases associated with viral infections.

[0240] When evaluating the response to therapy including induced hematopoietic lineage cells of the embodiments disclosed herein, the response can be measured by at least one of the following: clinical benefit rate, survival to death, complete pathological response, quantitative measurement of pathological response, complete clinical remission, partial clinical remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Responsive Reference Indicators for Solid Tumors) criteria.

[0241] Therapeutic compositions containing induced hematopoietic lineage cells, as disclosed, can be administered to a subject before, during, and / or after other therapies. Therefore, combination therapy methods may include the administration or preparation of iPSC-derived immune cells before, during, and / or after the use of additional therapeutic agents. As described above, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors containing one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory agents (IMiDs). The administration of iPSC-derived immune cells can be temporally separated from the administration of additional therapeutic agents by hours, days, or weeks. Additionally or alternatively, the administration can be combined with other bioactive agents or modalities, such as antitumor agents, non-pharmacological therapies such as surgery, etc., but not limited to these.

[0242] In some embodiments of combination cell therapy, the therapeutic combination includes iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent which 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 ability. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents for administered iPSC-derived hematopoietic lineage cells include, but are not limited to, anti-CD20 (e.g., rituximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), anti-HER2 (e.g., trastuzumab, partuzumab), anti-CD52 (e.g., alemtuzumab), anti-EGFR (e.g., cerltuximab), anti-GD2 (e.g., dinutuximab), anti-PDL1 (e.g., avelumab), anti-CD38 (e.g., daratumumab, isatuximab, MOR202), anti-CD123 (e.g., 7G3, CSL362), anti-SLAMF7 (elotuzumab), and their humanized or Fc-modified variants or fragments, or their functional equivalents or biosimilars.

[0243] In some embodiments, additional therapeutic agents include one or more checkpoint inhibitors. Checkpoints refer to cellular molecules, often cell surface molecules, that can suppress or downregulate the immune response if not inhibited. Checkpoint inhibitors are antagonists 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, This list includes, but is 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 KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, 3DL2).

[0244] Some embodiments of the combination therapy, including the provided inducible effector cells, further include at least one inhibitor that targets checkpoint molecules. Some other embodiments of the combination therapy with the provided inducible effector cells include two, three, or more inhibitors so that two, three, or more checkpoint molecules are targeted. In some embodiments, the effector cells for the combination therapy described herein are inducible NK cells, as provided. In some embodiments, the effector cells for the combination therapy described herein are inducible T cells. In some embodiments, the inducible NK cells or T cells for the combination therapy are functionally enhanced as provided herein. In some embodiments, two, three, or more checkpoint inhibitors may be administered in the combination therapy concurrently with, before, or after the administration of the inducible 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 checkpoint molecules described above is an antibody. In some embodiments, the checkpoint inhibitor antibody may be a mouse antibody, a human antibody, a humanized antibody, camel Ig, shark heavy chain only antibody (VNAR), Ig NAR, a chimeric antibody, a recombinant antibody, or a 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 manufacture, easier to use, or more sensitive than the whole antibody. In some embodiments, one, two, three, or more checkpoint inhibitors include at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents.

[0246] Combination therapies including induced effector cells and one or more check-in inhibitors are effective against cutaneous T-cell lymphoma, non-Hodgkin lymphoma (NHL), mycosis fungoides, Paget's reticular disease, Sézary syndrome, granulomatous flaccid skin, lymphomatoid papulosis, chronic lichenoid pityriasis, acute lichenoid plaque, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Renat's lymphoma, subcutaneous T-cell lymphoma, vascular central lymphoma, and blastoid N1. It is applicable to the treatment of liquid and solid tumors, including but not limited to K-cell lymphoma, B-cell lymphoma, Hodgkin lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyosarcoma, Lewis lung cancer (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, small cell neuroendocrine carcinoma of the prostate (SCNC), liver cancer, glioblastoma, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal cancer.

[0247] In some embodiments other than induced effector cells as provided herein, the combination for therapeutic use includes one or more additional therapeutic agents comprising a chemotherapeutic agent or radioactive moiety. A chemotherapeutic agent refers to a cytotoxic antitumor agent, i.e., a chemical agent found to preferentially kill tumor cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and used therapeutically to prevent or reduce the proliferation of neoplastic cells. Chemotherapy agents are also sometimes called antitumor or cytotoxic drugs or agents, and are well known in the art.

[0248] In some embodiments, the chemotherapeutic agent includes anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing drugs, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, mechloretamine, mephalin, chlorambucil, hairmethylmelamine, 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, bisanthren, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, meitansine, and amsacrine.Additional medications 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, carsterone, capecitabine, celecoxib, and cetuxi. Mab, cladribine, cloflavin, cytarabine, dacarbazine, denileukin difutitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinylestradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, i Darbicin, Ifosfamide, Imatinib, Interferon alfa (2a, 2b), Irinotecan, Letrozole, Leucovorin, Leuprolide, Rebamizole, Mechloretamine, Megestrol, Melphalin, Mercaptopurine, Methotrexate, Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone, Nofetumomab, Oxaliplatin, Paclitaxel, Pamidronate, Pemetrexed, Pegademase, Pe This includes gaspargaze, pentostatin, pipobromane, plicamycin, polyfeprosan, porfimer, procarbazine, quinacrine, rituximab, salglamostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinorelbine, and zoledronate. Other suitable agents are approved for human use, including those known in the art, that are approved as chemotherapeutic or radiotherapeutic agents.Such drugs can be found in many standard physician and oncologist references (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 regularly.

[0249] Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide stimulate both NK cells and T cells. As provided herein, IMiDs may 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 may further include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable media, e.g., cell culture media), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition administered and by the specific method used to administer the therapeutic composition. Thus, a wide variety of suitable formulations of the therapeutic composition of the present invention exist (e.g., its disclosure is incorporated herein by reference in its entirety, Remington's Pharmaceutical Sciences, 17) th (See ed. 1985).

[0251] In one embodiment, the therapeutic composition comprises pluripotent T cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent NK cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent CD34+HE cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent HSCs produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent MDSCs produced by the methods and compositions disclosed herein. Therapeutic compositions comprising the population 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 to influence a desired therapeutic target.

[0252] These pharmaceutically acceptable carriers and / or diluents may be present in amounts sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffer may be as much as about 5% by weight relative to the total composition. Electrolytes, such as sodium chloride and potassium chloride, may also be included in the therapeutic composition, but are not limited to these. In one embodiment, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition includes a buffer having a pH in one of the above pH ranges. In yet 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, in part, provides the use of pharmaceutically acceptable cell culture media in certain compositions and / or cultures of the present invention. Such compositions are suitable for administration to human subjects. Generally speaking, any medium supporting the maintenance, proliferation, and / or health of iPSC-derived immune cells according to embodiments of the present invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, a pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, a serum-free medium is free of animal components and may be protein-free as needed. Optionally, the medium may contain pharmaceutically acceptable recombinant proteins. A medium free of animal components refers to a medium in which the 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, a protein-free medium is defined as substantially protein-free. Those skilled in the art will understand that the above-described examples of media are illustrative and in no way limit the formulations of media suitable for use in the present invention, and that there are many suitable media known and available to those skilled in the art.

[0254] Isolated hematopoietic cells derived from pluripotent stem cells may contain at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+HE cells, HSCs, B cells, bone marrow-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, isolated hematopoietic cells derived from pluripotent stem cells contain about 95% to about 100% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or bone marrow-derived suppressor cells (MDSCs). In some embodiments, the present invention provides therapeutic compositions containing purified T cells or NK cells, such as compositions containing an isolated population of about 95% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or bone marrow-derived suppressor cells (MDSCs) for treating subjects requiring 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 a genome-modified iPSC containing the genotypes listed in Table 1, where the induced NK cells contain 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 a genome-modified iPSC containing the genotypes listed in Table 1, where the induced T cells contain CD38 null. In some embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202 and a population of NK or T cells derived from a genome-modified iPSC containing the genotypes listed in Table 1, where the induced NK or T cells contain CD38 null and hnCD16. In several other embodiments, the combination cell therapy comprises daratumumab and a population of NK or T cells derived from genetically engineered iPSCs containing the genotypes listed in Table 1, where the induced NK or T cells include CARs targeting CD38 null, hnCD16, and CD19, BCMA, CD38, CD20, CD22, or CD123. In several additional embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202 and a population of NK or T cells derived from genetically engineered iPSCs containing the genotypes listed in Table 1, where the induced NK or T cells include CD38 null, hnCD16, and CARs, as well as one or more exogenous cytokines.

[0256] As those skilled in the art will understand, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs based on the methods and compositions herein can be used in cell therapies as described above. In the case of autologous transplantation, the isolated population of induced hematopoietic lineage cells is fully or partially HLA-matched with the patient. In another embodiment, the induced hematopoietic lineage cells are HLA-matched with the subject, where the induced hematopoietic lineage cells are NK cells or T cells having 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 × 10⁶ per dose. 5 Cells, at least 1 × 10 5 Cells, at least 5 × 10 5 Cells, at least 1 × 10 6 Cells, at least 5 × 10 6 Cells, at least 1 × 10 7 Cells, at least 5 × 10 7 Cells, at least 1 × 10 8 Cells, at least 5 × 10 8 Cells, at least 1 × 10 9 Cells, or at least 5 × 10 9 These are cells. In some embodiments, the number of induced hematopoietic lineage cells in the therapeutic composition is about 0.1 × 10⁶ per dose. 5 cells ~ approx. 1 x 10 6 Cells, approximately 0.5 × 10⁶ per dose 6 cells ~ approx. 1 x 10 7 Cells, approximately 0.5 × 10⁶ per dose 7 cells ~ approx. 1 x 10 8 Cells, approximately 0.5 × 10⁶ per dose 8 cells ~ approx. 1 x 10 9 Cells, approximately 1 × 10⁶ per dose 9 Cells ~ approx. 5 x 10 9 Cells, approximately 0.5 × 10⁶ per dose 9 cells ~ approx. 8 x 10 9 Cells, approximately 3 x 10⁶ per dose 9 cells ~ approx. 3 x 10 10 It is a cell, or any range between them. Generally, for a 60kg patient, it is 1 × 10⁻⁶ 8 The cell / dose ratio is 1.67 × 10⁻⁶. 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 portion or a single umbilical cord of blood, or at least 0.1 × 10⁶ 5 Cells / kg body weight, at least 0.5 × 10⁻⁶ 5 Cells / kg body weight, at least 1 × 10⁻⁶ 5 Cells / kg body weight, at least 5 × 10⁻⁶ 5Cells / kg body weight, at least 10 × 10 5 Cells / kg body weight, at least 0.75 × 10⁻⁶ 6 Cells / body kg body weight, at least 1.25 × 10⁻⁶ 6 Cells / kg body weight, at least 1.5 × 10⁻⁶ 6 Cells / kg body weight, at least 1.75 × 10⁻⁶ 6 Cells / kg body weight, at least 2 × 10⁻⁶ 6 Cells / kg body weight, at least 2.5 × 10⁻⁶ 6 Cells / kg body weight, at least 3 × 10⁻⁶ 6 Cells / kg body weight, at least 4 × 10⁻⁶ 6 Cells / kg body weight, at least 5 × 10⁻⁶ 6 Cells / kg body weight, at least 10 × 10 6 Cells / kg body weight, at least 15 × 10⁻⁶ 6 Cells / kg body weight, at least 20 × 10⁻⁶ 6 Cells / kg body weight, at least 25 × 10⁻⁶ 6 Cells / kg body weight, at least 30 × 10 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 Cells / kg body weight, or 1 × 10⁶ 9 It is expressed as cells per kg of body weight.

[0259] In one embodiment, a certain dose of induced hematopoietic lineage cells is delivered to the subject. In an exemplary embodiment, the effective amount of cells delivered to the subject is at least 2 × 10⁶ 6 Cells / kg, at least 3 × 10⁻⁶ 6 cells / kg, at least 4 × 10⁻⁶ 6 Cells / kg, at least 5 × 10⁻⁶ 6 Cells / kg, at least 6 × 10⁶ 6 Cells / kg, at least 7 × 10⁶ 6 Cells / kg, at least 8 × 10⁶ 6 Cells / kg, at least 9 × 10⁶ 6 cells / kg, or at least 10 × 10 6 This is cells / kg or more, and includes 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, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, or about 10×10 6 cells / kg, or more cells / kg, including all intervening cell doses.

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

[0262] In some embodiments, the therapeutic use of induced hematopoietic lineage cells is a single-dose therapy. In some embodiments, the therapeutic use of induced hematopoietic lineage cells is a multi-dose therapy. In some embodiments, the multi-dose therapy is one dose daily, 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 for any number of days in between.

[0263] A composition comprising a population of induced hematopoietic lineage cells of the present invention may be sterile, suitable for administration to human patients, and readily administered (i.e., readily administered without further processing). A readily administered cell-based composition 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 grown and / or prepared before administration of one or more agents. For induced hematopoietic lineage cells genetically engineered to express recombinant TCRs or CARs, the cells can be activated and grown using, for example, the method described in U.S. Patent No. 6,352,694.

[0264] In certain embodiments, primary stimulatory and co-stimulatory signals to 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. If bound to a surface, the agents can be bound to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent can be bound to a surface while the other agent is present in solution. In one embodiment, the agent providing the co-stimulatory signal can be bound to the cell surface, and the agent providing the primary activation signal can be in solution or bound to a surface. In certain embodiments, both agents may be in solution. In another embodiment, the agents may be in a soluble form and can then be crosslinked to a surface such as cells or antibodies expressing Fc receptors, or other binding agents that bind to the agent, as disclosed in U.S. Patent Applications Publications 2004 / 0101519 and 2006 / 0034810 for artificial antigen-presenting cells (aAPCs) intended for use in the activation and proliferation of T lymphocytes in embodiments of the present invention.

[0265] Some variations in dosage, frequency, and protocol will inevitably occur depending on the condition of the patient being treated. In any case, the person responsible for administration will determine the appropriate dosage, frequency, and protocol for each individual patient. [Examples]

[0266] The following examples are provided for illustrative purposes only and not for limitation.

[0267] Example 1 - Materials and Method Using the applicant's proprietary hiPSC platform, which enables single-cell passage and high-throughput 96-well plate-based flow cytometry sorting, we enabled the derivation of cloned hiPSCs by single or multiple gene modifications, in order to effectively select suicide systems under the control of various promoters and to test them in combination with various safe harbor locus integration strategies.

[0268] Maintenance of hiPSCs in small molecule cultures: hiPSCs were routinely passaged as single cells once the culture confluence reached 75%–90%. In the case of unicellular dissociation, hiPSCs were washed once with PBS (Mediatech), treated with Accutase (Millipore) at 37°C for 3–5 minutes, and then pipetted to ensure unicellular dissociation. The unicellular suspension was then mixed with an equal volume of conventional medium, centrifuged at 225×g for 4 minutes, resuspended in FMM, and seeded on a Matrigel-coated surface. Passaging was typically done at a ratio of 1:6–1:8, and the cells were transferred to pre-coated tissue culture plates at 37°C for 2–4 hours, with FMM supplied every 2–3 days. Cell cultures were maintained in a humidified incubator set to 37°C and 5% CO2.

[0269] Using ZFN-mediated human iPSC manipulation and CRISPR: ROSA26 targeted insertion for targeted editing of the desired modality as examples, in ZFN-mediated genome editing, 2 million iPSCs were transfected with a mixture of 2.5 μg of ZFN-L (FTV893) and 2.5 μg of ZFN-R (FTV894) and a 5 μg donor construct for AAVS1 targeted insertion. In CRISPR-mediated genome editing, 2 million iPSCs were transfected with a mixture of 5 μg of ROSA26-gRNA / Cas9 (FTV922) and a 5 μg donor construct for ROSA26 targeted insertion. Transfection was performed using a Neon transfection system (Life Technologies) with parameters 1500 V, 10 ms, and 3 pulses. On day 2 or 3 after transfection, if the plasmid contained an artificial promoter-driver GFP and / or RFP expression cassette, transfection efficiency was measured using flow cytometry. On day 4 after transfection, target cells were selected by adding puromycin to the culture medium at a concentration of 0.1 μg / ml for the first 7 days and then at a concentration of 0.2 μg / ml after 7 days. During puromycin selection, cells were passaged into fresh Matrigel-coated wells on day 10. From day 16 onward of puromycin selection, viable cells were analyzed by flow cytometry for the percentage of GFP+ iPS cells.

[0270] Batch and clone sorting of genome-edited iPSCs: iPSCs containing genome-targeted editing using ZFN or CRISPR-Cas9 were batch and clone-sorted as GFP+SSEA4+TRA181+iPSCs 20 days after puromycin selection. The single-cell dissociation-targeted iPSC pool was resuspended in a newly prepared chilled staining buffer containing Hanks equilibrium salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1× penicillin / streptomycin (MediaTech), and 10 mM Hepes (MediaTech) for optimal performance. Conjugated primary antibodies such as SSEA4-PE and TRA181-Alexa Fluor-647 (BD Biosciences) were added to the cell solution and incubated on ice for 15 minutes. All antibodies were used at a concentration of 7 μL per 1 million cells in 100 μL of staining buffer. The solution was washed once with staining buffer, spun down at 225g for 4 minutes, resuspended in staining buffer containing 10 μM thiazovib, and maintained on ice for sorting by flow cytometry. Sorting by flow cytometry was performed using a FACS Aria II (BD Biosciences). For bulk sorting, 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 directly discharged into a 96-well plate 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 overnight with 5 × Matrigel. For 5× Matrigel pre-coating, add one aliquot of Matrigel to 5 mL of DMEM / F12, incubate overnight at 4°C to allow proper resuspension, and finally add 50 μL per well to a 96-well plate and incubate overnight at 37°C. The 5× Matrigel is aspirated immediately before adding medium to each well. After sorting is complete, the 96-well plate is centrifuged at 225 g for 1-2 minutes and then incubated.The plates were left standing 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 refilled with an additional 100 μL of FMM on day 10 after sorting. Colony formation was detected as early as day 2, and most colonies proliferated between 7 and 10 days after sorting. For the first passaging, the wells were washed with PBS and dissociated with 30 μL of acetase at 37°C for approximately 10 minutes. The need for prolonged acetase treatment reflects the compactness of colonies that had not been allowed to grow for a long period. After the cells were observed to have dissociated, 200 μL of FMM was added to each well and the colonies were disrupted by pipetting several times. The dissociated colonies were transferred to another well in a 96-well plate pre-coated with 5× Matrigel and centrifuged at 225 g for 2 minutes before incubation. This 1:1 passaging is performed to spread out the early colonies before proliferation. Subsequent passaging was routinely performed with 3–5 minutes of accutase treatment and 1:4–1:8 growth at 75–90% confluence in large wells pre-coated with 1× Matrigel of FMM. Each clonal cell line was analyzed for GFP fluorescence level and TRA1-81 expression level. Clonal lines with near 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, and Alt-R® CRISPR-Cas9 tracrRNA were purchased from IDT (Coralville, Iowa) and used for iPSC-targeted editing. Table 3 lists the screened and identified target sequence pairs (1A and 1B, 2A and 2B, 3A and 3B) for gNA (i.e., gD / RNA or guide polynucleotide) design to perform bi-allele knockout of CD38 in iPSCs using Cas9 nickase.

[0272] [Table 3]

[0273] Subsequently, the genetically modified iPSCs were characterized, and the knockout of the CD38 allele was confirmed.

[0274] Example 3-CD38 - / - Validation of iPSCs and induced cells CD38 is known to be expressed at specific cellular stages and plays an important role in effector cells. During hematopoiesis, CD38 is expressed at CD34 + CD38 is expressed in stem cells, as well as in progenitor cells committed to lymphoid, erythroid, and myeloid lineages, and in the final stages of maturation of effector cells such as T cells and NK cells. Therefore, prior to this application, there was uncertainty and concern as to whether iPSCs containing CD38 knockouts would develop properly when exposed to indicated differentiation conditions, given the CD38 expression profile and functionality, and whether the resulting effector cells would function. Surprisingly, CD38 null iPSCs containing a biallele knockout of CD38 retained the ability to differentiate into induced cells. In one of the figures, three manipulated iPSC clones, gene-edited to result in biallele knockouts of the CD38 gene and hnCD16, differentiated into induced NK cells, and their phenotypes were analyzed. The flow profiles in Figure 2 show that each clone is CD56+ and CD38-negative.

[0275] To determine whether the cytotoxicity of iPSC-derived NK cells is preserved when CD38 expression is knocked out, wild-type iPSC clones and three manipulated iPSC clones with allelic disruption of the CD38 gene were differentiated into NK cells, and their ability to target and kill the tumor cell line RPMI-8266 was compared. As shown by the long-term killing assay in Figure 3, the manipulated CD38 cells were compared over 48 hours. - / - NK cells eliminated target cells in a similar manner to wild-type controls, indicating that CD38 is not essential for target cell recognition and killing. Therefore, 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 anticancer drug. It binds to CD38, which is overexpressed in multiple myeloma cells. Daratumumab-mediated cell killing of multiple myeloma cells is partially dependent on ADCC and heavily dependent on NK effector cells. However, CD38 is also expressed in NK cells, and as a result, daratumumab may induce ADCC (fratriside) against NK cells, potentially 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. CD38 - / - Figure 7 further shows that the viability of iPSC-derived NK cells (Figure 7B) is maintained for at least approximately 48 hours in culture in the presence of daratumumab, compared to iPSC-derived NK cells expressing CD38 (Figure 7A). Furthermore, as shown in Figure 7, CD38 - / - iPSC-derived NK cells, when stimulated in the presence of daratumumab, do not show degranulation and produce less cytokines than iNK cells expressing CD38.

[0277] Apart from 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 the B2M gene, loss of HLA-II by knockout of CIITA, overexpression of the non-classical HLA molecule HLA-G, and expression of the linked IL15 / IL15 receptor alpha construct. After each engineering step and before the next, iPSCs were sorted for the desired phenotype. Engineered iPSCs can be maintained in vitro or for induced cell generation. Figure 4 shows hnCD16 expression, B2M knockout, HLA-G expression, and IL15 / IL15Rα expression in iPSC-derived NK cells. Figure 7 shows introduction of hnCD16 combined with CD38 knockout in iPSC-derived NK cells. These data demonstrate that these genetically engineered modalities are maintained during hematopoietic differentiation without disrupting the directed development of cells to the desired cell fate in vitro.

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

[0279] Example 4 - Functional profiling of CD38 Null-induced NK cells The phenotype and calcium flux of hnCD16 CD38- / -iNK cells, including the expression of NKG2A, NKp46, and KIR2DL2 / 3, were evaluated, and the phenotype of hnCD16 iNK was maintained after CD38 knockout (Figures 10A-10D). The ADCC function of hnCD16 iNK with CD38 knockout was investigated against the HER2-expressing ovarian cell line SKOV3 by Incucyte live-cell imaging, and CD38 knockout did not affect the ADCC function of hnCD16 iNK (Figure 10E). Next, the specific cytotoxicity of daratumumab against various NK cell populations—peripheral blood NK cells (with CD16 shedding), hnCD16 iNK cells (with high affinity for uncleaved CD16), unmodified iNK cells (with low CD16 expression), and hnCD16 CD38- / -iNK cells—was 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 daratumumab-mediated flutorides compared to other cell populations without CD38 knockout. Therefore, CD38 loss prevents daratumumab-mediated NK cell flutorides in the presence of CD38-specific antibodies.

[0280] To evaluate the cytotoxicity of hnCD16 CD38- / -iNK cells compared to hnCD16 iNK cells, each cell population was incubated with MM.1S myeloma target cells for 18 hours, and tumor cell viability was then assessed by flow cytometry using annexin V and viability / death survival markers. As shown in Figure 12, hnCD16 iNK cells mediated potent anti-myeloma activity with daratumumab, which was further enhanced by CD38 loss. Furthermore, in a 7-day cytotoxicity assay against RMPI-8226 tumor spheroids, hnCD16 CD38- / -iNK cells showed superior tumor cell clearance, measured by the number of target cells remaining at the end of the assay, in the presence of a CD38-specific antibody and compared to hnCD16 iNK and unmodified iNK cells under the same conditions (Figure 13A). Furthermore, the absence of NK cell fructoliside improved the viability of hnCD16 CD38- / -iNK cells, as indicated by the improved persistence of these cells in a 7-day cytotoxicity assay shown in Figure 13B. Thus, hnCD16 CD38- / -iNK cells exhibit enhanced long-term antimyeloma activity and persistence with CD38-specific antibodies such as daratumumab. As shown in Figure 14, CD38-deficient hnCD16 iNK cells also demonstrate more durable ADCC with increased serial killing ability in the presence of CD38-specific antibodies. In this assay, hnCD16 or hnCD16 CD38- / -iNK cells were incubated with MM.1S myeloma target cells and daratumumab for 48 hours (stimulation round 1), and the number of MM.1S cells was quantified by Incucyte® imaging. After 48 hours, the effector cells were removed and transferred to new target cells for a second round of stimulation with daratumumab and target cell killing (stimulation round 2). (Figure 14)

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

[0282] Example 5 - Use of a CD38-specific antagonist to protect allogeneic effector cells from allogeneic rejection. Engineered iNK cells with enhanced CD16 efficacy and CD38 removal are resistant to CD38-targeted antibody-inducible fractolides and more potently mediate antimyeloma activity in combination with daratumumab. In recipients of allogeneic effector cells lacking CD38, the fact that CD38 is upregulated in activated lymphocytes such as T cells or B cells by suppressing the activation of these lymphocytes using CD38-specific antagonists containing monoclonal antibodies reduces and / or prevents allogeneic rejection of these effector cells, thereby increasing the viability and persistence of effector cells. To demonstrate the feasibility of this strategy, a mixed lymphocyte reaction (MLR, i.e., co-incubation of effector cell products and allogeneic PBMCs) is performed to test the lifespan of the induced effector cells of the present invention in an allogeneic environment in relation to CD38 knockout and in the presence or absence of an anti-CD38 monoclonal antibody (e.g., daratumumab).

[0283] hnCD16+ iNK cell populations (with and without CD38 knockout) are labeled with an intracellular dye (Celltrace Violet® or a similar Incucyte® compatible reagent) immediately before 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, unpooled) in or without daratumumab (at effective concentrations, titrated before the assay). The viability of iNK cells in each population is monitored over time by Incucyte® in long-term culture. Viability is also monitored by flow cytometry, and staining panels further track CD38 upregulation of PBMC subpopulations and clearance of PBMC subpopulations by iNK cells based on daratumumab-mediated ADCC. Total clearance of iNK cells as a control is achieved using Venetoclax (an MCL-1 inhibitor for the specific removal of NK cells).

[0284] The extended lifespan of hnCD16 CD38KO iNK cells compared to wild-type hnCD16 iNK cells in the presence of anti-CD38, and the associated clearance of CD38+ subpopulations (peripheral NK cells, activated B cells, and T cells) from PBMC samples, demonstrate the ability of CD38-specific antagonists in suppressing activated peripheral T or B cells by targeting upregulated CD38 in activated peripheral T or B cells, thereby reducing allogeneic rejection of allogeneic effector cells by these activated peripheral T or B cells in recipients of effector cells containing hnCD16 and CD38- / -, as provided herein. CD38-specific antagonists are CD38-specific antibodies, CD38-specific engagers, or CD38 chimeric antigen receptors (CARs).

[0285] Furthermore, expression of an exogenous truncated IL15 / IL15Rα fusion protein 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 lacking the intracellular domain was fused to IL15 at its C-terminus via a linker to generate a truncated IL15 / IL15Ra fusion (or, as referred to herein, "IL15Δ") construct lacking a signaling domain. Exemplary IL15Δ provided herein include those having structures such as Design 3 or 4 in Figure 1. As shown in Figure 15, iNK cells were transduced by lentiviral overexpression vectors expressing either GFP (square; negative control), the full-length IL15 / IL15Ra fusion construct (black circle; positive control; Design 2 in Figure 1), or the truncated IL15 / IL15Ra fusion construct (white circle; Design 3 in Figure 1). Neither the IL15 construct nor GFP showed enrichment in the presence of exogenous IL2 (Figure 15A), indicating that transduced cells survived at a rate comparable to non-transduced cells. In the absence of exogenous IL2, cells transduced with either IL15 / IL15Ra fusion construct enriched over time, but GFP transduced cells did not. In the absence of IL2, cells transduced with either IL15 / IL15Ra construct had a survival advantage compared to non-transduced cells under the same culture conditions (Figure 15B). Furthermore, since the intracellular domain of IL15Rα is considered important for the receptor to be expressed in IL15-responsive cells and for cells to proliferate and function in response, it is surprising that the IL15 / IL15Ra fusion construct with a shortened intracellular domain not only stably expressed in transduced iNK cells but also supported iNK cells at a higher proliferation rate than the full-length IL15 / IL15Ra fusion construct, as shown in Figure 15B. Therefore, the IL15Δ provided herein can express and maintain IL15 in a membrane-bound form and can replace full-length IL15 / IL15Ra fusion proteins to provide trans presentation of IL15 in cells.Although the underlying mechanism is not fully understood, deleting the intracellular domain of IL15R appears to have given responding cells increased viability, proliferation, and persistence, fitness, or certain advantages, possibly by completely eliminating cis-presentation and / or other potential signaling pathways mediated by normal IL15R via that intracellular domain.

[0286] Example 6 - CD38-targeted transgene knock-in construct that results in CD38 knockout and engineered cells obtained therefrom The insertion of one or more transgenes at a selected location of CD38 to simultaneously knock out CD38 was achieved by the construct design illustrated in Figure 16. With each CD38-targeting homology arm for site-selective insertion, the constructs provided herein allow the transgene(s) to be expressed under either an endogenous or exogenous CD38 promoter included in the construct (compare A-B and C-D in Figure 16). The selective insertion / knockout site within the CD38 locus conforms to the sequences of the adjacent left and right homology arms (LHA / CD38 and RHA / CD38) included in the construct. LHA / CD38 and RHA / CD38 may have varying lengths and sequences depending on the pre-selected targeting site within the CD38 locus. In some embodiments, the pre-selected targeting site is located within an exon of CD38. When two or more transgenes are inserted at a selected location of the CD38 locus, a linker sequence, e.g., a 2A linker or IRES, is positioned between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-1, and TaV (also known as "F2A," "E2A," "P2A," and "T2A," respectively), allowing for the production of distinct 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 may 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 embodiment, one or more transgenes included in a 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 containing F176V and S197P, or CD16 containing F176V but without a cleavage domain, or CD16 containing at least a portion of the CD64 extradomain. In some embodiments, as provided in Section I(4), IL15RF includes an IL15 / IL15Rα fusion protein with or without cytoplasmic domain cleavage.

[0288] Accordingly, provided herein are constructs for CD38-targeted knock-in and knock-out, the construct comprising LHA / CD38, a first transgene, and RHA / CD38, the construct knocking out CD38 expression while inserting the first transgene into CD38 at a selected site, and optionally the first transgene being driven by the endogenous promoter of CD38 by being operably linked to the endogenous promoter of CD38 at the time of incorporation. In some embodiments, the construct comprising LHA / CD38, a first transgene, and RHA / CD38 further comprises a second transgene, as well as a linker between the first and second transgenes. In some embodiments, the construct comprising LHA / CD38, one or more transgenes, and RHA / CD38 further comprises an exogenous promoter, the construct knocking out CD38 expression while inserting one or more transgenes into CD38 at a selected site, and the one or more transgenes being driven by the exogenous promoter. In one embodiment of a construct containing LHA / CD38, one or more transgenes, and RHA / CD38, the transgene expresses one or both of the hnCD16 and IL15RF proteins, as 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 hnCD16 and IL15RF (IL15RF cleaved in this particular embodiment) at selected locations of the CD38 locus, driven by a CAG promoter (Figure 17) or a CD38 endogenous promoter (Figure 18). As presented in the figures and as will be understood by those skilled in the art, some of the components included in the constructs shown in Figures 17 and 18 are optional and not essential, and the nucleic acid sequences for some of the components included may vary, and may have sequence identity of about 95%, 90%, 85%, 80%, 75%, and less than 70% but greater than 50% with respect to the exemplary nucleic acid sequences of each component presented in the figures or the construct as a whole.

[0289] (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, each construct was transduced to manipulate induced pluripotent stem cells using a CRISPR editing tool, e.g., Cas9 or Cpf1, as described in this application. The efficiency of biallele targeted insertion with biallele knockout was found to be about 2–3% in cells initially sorted by IL15 or CD16. In particular, biallele knockout efficiency was higher than that of single-allele targeted insertion (about 5%). Thus, bialleletic targeted integration with simultaneous bialleletic knockout involves sequentially (or stepwise) performing CD38 knockout (which can be 30% to 70% in this step), and then inserting CD38 null cells to include the transgene in the CD38 cells. - / - Compared to obtaining iPSCs (typically 25-80% depending on the vector used), this appears to be a rare event. However, this method of knocking out CD38 using the incorporation of a CD38-targeted transgene dramatically reduces the occurrence of karyotype abnormalities during long-term passage and maintenance of the resulting manipulated iPSCs compared to iPSCs subjected to multiple sequential genome editing events. While we do not wish to be bound by theory, such unexpected genomic stability may be due, among other unknown reasons, to a reduction in stressors associated with a reduced amount of manipulation of iPSCs from multiple genomic disturbances, including single-cell selection.

[0290] After clonal selection of the manipulated iPSCs, donor vector validation, vector clearance, sequencing confirmation of insertions and knockouts, flow cytometry of iPSC markers and transgene phenotypes, transgene copy number validation, and karyotype validation, the genome-edited clone iPSCs were differentiated into effector cells such as iNK cells in this example according to the method provided herein. 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. Neither of these is present in native / natural corresponding NK cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue. CD38 manipulated with CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct compared to unmanipulated iNK cells. - / - The flow cytometry phenotyping of CD16 IL15 iNK cells is shown in Figure 19. CD38 - / - CD16 IL15 iNK cells, when cultured for 4 days in the absence of IL-2 or IL-15, showed improved in vitro persistence compared to similarly treated unmanipulated iNK (FTi250) (see Figure 20). Furthermore, induction of flutorides by daratumumab or rituximab was observed in unmanipulated iNK or CD38 - / - Tested with CD16 IL15 iNK cells, as shown in Figure 21, CD38 - / - Only CD16 IL15 iNK cells were able to evade the fracturides induced by CD38-specific antibodies such as daratumumab, while unmanipulated iNK cells could not. Furthermore, direct cytotoxicity and ADCC, when combined with daratumumab antibodies, targeted Raji cells and CD38 - / - The test was performed on CD16 IL15 iNK cells, as shown in Figure 22. - / - CD16 IL15 iNK cells showed superior ADCC compared to unmodified iNK cells and demonstrated significantly enhanced daratumumab antibody-induced killing of CD38-expressing Raji cells.

[0291] Those skilled in the art will readily understand that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended to limit the scope of the invention. It will also 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 referenced herein represent the state of the art for those skilled in the art to which this disclosure relates. All patents and publications are incorporated herein by reference as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0293] The disclosures described herein as exemplary may be implemented without any one or more elements, limitations, or restrictions not specifically disclosed herein. For example, in each example herein, the terms “including,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms. The terms and expressions used are for illustrative purposes only, not limitation, and in the use of such terms and expressions there is no intention to exclude any equivalent of the shown and described features or any part thereof, but it is recognized that various modifications are possible within the scope of the claimed disclosure. Thus, while the disclosure is specifically disclosed by preferred embodiments and features as needed, modifications and variations of the concepts disclosed herein may often be conceived by 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 method for producing T cells, comprising differentiating iPSCs into T cells, (a) The iPSC comprises (i) targeted incorporation of one or more nucleic acid sequences encoding one or more exogenous proteins at a selected location of the CD38 locus, and (ii) CD38 knockout. (b) The iPSC is produced using a construct comprising a left homology arm and a right homology arm (LHA / CD38, RHA / CD38) that target CD38 at a selected location of the CD38 locus, which is (ii) operably ligated to one or more nucleic acid sequences encoding one or more exogenous proteins, by a method.

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

3. The one or more exogenous proteins include at least one or both of the following (i) and (ii): (i) CD16, (ii) exogenous IL15 / IL15 receptor fusion protein (IL15RF) expressed on the cell surface. The method according to claim 1, wherein the T cell comprises one or more nucleic acid sequences encoding CD16 and IL15RF.

4. (i) The CD16 is (a) F176V and S197P of the external domain of CD16, (b) All or partial external domains of CD64, (c) Non-CD16 transmembrane domain, (d) Non-CD16 intracellular domain, (e) Non-CD16 signaling domain, (f) Non-CD16 stimulating domains, and (g) Non-CD16 transmembrane domain, non-CD16 signaling domain, and non-CD16 stimulating domain, It includes at least one of the following, or (ii) The IL15RF is (a) IL15 and IL15Rα co-expressed using self-cleaving peptides, (b) A fusion protein of IL15 and IL15Rα, (c) IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα has been cleaved. (d) 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 the common receptor γC, wherein the common receptor γC is native or modified, (g) Homodimer of IL15Rβ, and (h) A polynucleotide encoding IL15RF containing the amino acid sequence of SEQ ID NO: 17, 19, or 21, The method according to claim 3, comprising at least one of the following.

5. The aforementioned CD16 (i) The non-CD16 transmembrane domain is 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, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a transmembrane domain of a T cell receptor (TCR) polypeptide. (ii) The non-CD16 stimulating domain is a stimulating domain of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. (iii) The non-CD16 signaling domain is a signaling domain of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide, or (iv) The non-CD16 transmembrane domain is the transmembrane domain of NKG2D, the non-CD16 stimulating domain is the stimulating domain of 2B4, and the non-CD16 signaling domain is the signaling domain of CD3ζ. The method according to claim 4, characterized in that it is one or more of the following.

6. The T cells further contain a chimeric antigen receptor (CAR), and the CAR has the following characteristics (i) bispecific antigen-binding CAR; (ii) Switchable CAR, (iii) Dimerized CAR, (iv) Split CAR, (v) multi-chain CAR; (vi) Inductionable CAR, (vii) Those co-expressed with other CARs, (viiii) Exogenous cytokines expressed on the cell surface or their receptors, co-expressed with partial or complete peptides, (ix) Co-expressed with checkpoint inhibitors, (x) Encoded by nucleic acids further contained in the construct, wherein the CAR is inserted into the selected position of the CD38 locus, (xi) Inserted into the TRAC locus and / or driven by the endogenous promoter of TCR and / or TCR knocked out by the CAR insertion, (xi) CD19 or BCMA specific, 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, antigens of cytomegalovirus (CMV) infected cells, epithelial glycoside 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 telomere Reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion 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), mesoserine (M SLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryo 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 the pathogen antigens that is specific to one of these. The method according to claim 1, comprising at least one of the following.

7. The aforementioned construct, (i) an exogenous promoter that drives the expression of one or more nucleic acid sequences encoding one or more exogenous proteins, wherein the exogenous promoter is constitutive, inducible, time-specific, tissue-specific, and / or cell-type specific. (ii) A linker sequence inserted between two nucleic acid sequences encoding an exogenous protein, (iii) Insulators at the 3' end of LHA / CD38 and 5' end of RHA / CD38, and (iv)polyA signal, The method according to claim 1, further comprising one or more of the above.

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 is an intra-sequence ribosome entry site (IRES) The method according to claim 7.

9. The aforementioned T cells exhibit the following phenotypes (i) Containing longer telomeres compared to its native corresponding cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, (ii)CD38 －／－ CD16、 (iii) CD38 －／－ IL15, and / or (iv)CD38 －／－ CD16 IL15 The method according to claim 1, comprising one or more of the following.

10. The T cells are as follows: (i) HLA-I deficiency, (ii) HLA-II deficiency, (iii) Expression of HLA-G or non-cleaved HLA-G, (iv) Deletion or reduced expression of at least one of the following genes in the chromosome 6p21 region: B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, (v) HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ Introduced or increased expression in at least one of the following surface trigger receptors for binding to R, CAR, Fc receptors, engagers, and bispecific, multispecific, or universal engagers. The method according to claim 1, further comprising one or more of the following.

11. The aforementioned T cells, compared to their native corresponding cells obtained from peripheral blood, umbilical cord blood, or other donor tissues, are as follows: (i) Improved persistence and / or survival rate, (ii) Increased resistance to innate immune cells, (iii) Increased cytotoxicity, (iv) Improved tumor penetration, (v) Enhanced or acquired ADCC, (vi) Enhanced ability to migrate bystander immune cells to tumor sites and / or activate or mobilize them. (vii) Enhanced ability to reduce tumor immunosuppression, (viiii) Improved ability to rescue tumor antigen escapes, and (ix) Reduced fractolides in the presence of anti-CD38 antibody or CD38 CAR, The method according to claim 1, having at least one feature including

12. The method according to claim 11, wherein the anti-CD38 antibody is daratumumab, isatuximab, or MOR202, or a humanized or Fc-modified variant thereof, or an antigen-binding fragment thereof.

13. The method according to any one of claims 1 to 12, further comprising manufacturing a pharmaceutical product comprising T cells containing CD38 knockout.

14. (a) Introducing the above construct into the iPSC, (b) The method according to any one of claims 1 to 12, further comprising incubating the iPSC with the construct into which the iPSC was introduced to generate a targeted integration at the CD38 locus, thereby knocking out CD38 in the iPSC.

15. The method according to claim 14, wherein the iPSC has improved genomic stability compared to iPSCs that undergo integration and knockout in separate stepwise editing events.

16. The method according to claim 14, further comprising introducing CRISPR-cas / gNA (guide nucleic acid), ZFN, TALEN, or homing nuclease.

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