CD3 reconstitution in engineered iPSCs and immune effector cells

Abstract

Methods and compositions are provided for obtaining functionally enhanced derived effector cells obtained from directed differentiation of genomically engineered iPSCs. The iPSC-derived cells provided herein have stable, functional genome editing that provides improved or enhanced therapeutic effects. Therapeutic compositions and uses thereof are also provided, comprising functionally enhanced derived effector cells alone or in combination with antibodies or checkpoint inhibitors.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 832,622, filed April 11, 2019, which is incorporated herein by reference in its entirety.

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

[0003] Reference to an electronically submitted sequence listing This application incorporates by reference the computer readable form (CRF) of the Sequence Listing in ASCII text format that was filed herewith, named 056932-518001WO_SEQUENCE_LISTING_ST25.TXT, created on April 2, 2020, and is 76,442 bytes in size. [Background technology]

[0004] The field of adoptive cell therapy currently focuses on the use of patient- and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapies and provide treatment to all patients who may benefit. There is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. Lymphocytes, such as T cells and natural killer (NK) cells, are potent antitumor effectors that play a key role in innate and adaptive immunity. However, the use of these immune cells for adoptive cell therapy remains challenging, and there is an unmet need for improvement. Therefore, significant opportunities remain to fully utilize the potential of T cells and NK cells, or other lymphocytes, in adoptive immunotherapy. Summary of the Invention

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

[0006] The object of the present invention is to provide methods and compositions for generating differentiated derivative non-pluripotent cells from a single-cell-derived clonal iPSC (induced pluripotent stem cell) line, where the iPSC line contains one or more genetic modifications in its genome, including DNA insertions, deletions, and substitutions, that remain retained and functional in subsequent derived cells after differentiation, expansion, passaging, and / or transplantation.

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

[0008] Some embodiments of the present invention provide genomically engineered iPSCs obtained using methods including (I), (II), or (III), which reflect strategies of genomic engineering after, simultaneously with, and before the reprogramming process, respectively.

[0009] (I): Genetically engineer iPSCs by one or both of (i) and (ii), in any order: (i) introducing one or more constructs into iPSCs to enable targeted integration at selected sites; (ii) (a) introducing one or more double-strand breaks at selected sites into iPSCs using one or more endonucleases capable of recognizing the selected sites; and (b) culturing the iPSCs of step (I)(ii)(a) to allow endogenous DNA repair to simultaneously or sequentially generate targeted in / dels at the selected sites, thereby obtaining genomically engineered iPSCs capable of differentiation into partially or fully differentiated cells.

[0010] (II): Genetically engineering the reprogrammed non-pluripotent cells to obtain genomically engineered iPSCs, which includes (i) contacting the non-pluripotent cells with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells; and (ii) introducing into the reprogrammed non-pluripotent cells of step (II)(i) one or both of (a) and (b): (a) one or more constructs enabling targeted integration at a selected site; (b) one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, in any order; and then culturing the cells of step (II)(ii)(b) to allow endogenous DNA repair to generate targeted in / dels at the selected site; thus, the resulting genomically engineered iPSCs contain at least one functional targeted genome edit, and the genomically engineered iPSCs are capable of differentiating into partially or fully differentiated cells.

[0011] (III): Genetically engineering non-pluripotent cells for reprogramming to obtain genomically engineered iPSCs. This involves (i) introducing into the non-pluripotent cells one or both of (a) and (b), in any order: (a) one or more constructs that allow targeted integration at selected sites, and (b) one or more double-strand breaks at selected sites using at least one endonuclease capable of recognizing the selected sites. The cells of step (III)(i)(b) are then cultured to allow for the generation of targeted indels at the selected sites by endogenous DNA repair. and (ii) contacting the cells of step (III)(i) with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, to obtain genomically engineered iPSCs comprising a targeted edit at the selected site, thereby obtaining genomically engineered iPSCs comprising at least one functional targeted genome edit, wherein the genomically engineered iPSCs are capable of differentiating into partially or fully differentiated cells.

[0012] In one embodiment of the above method, at least one targeted genome edit at one or more selected sites comprises 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, trafficking, homing, viability, self-renewal, persistence, and / or survival of the genomically engineered iPSCs or their derivative cells. In some embodiments, the exogenous polynucleotides for insertion are operably linked to one or more exogenous promoters, including CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, or one or more endogenous promoters contained in the selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for genomic safe harbor. In some embodiments, the genomically engineered iPSCs generated using the above methods contain one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, and when the genomically engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into different safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, CD38, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotides encode partial or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors. In some embodiments, the partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors encoded by the exogenous polynucleotide are in the form of a fusion protein.

[0013] In some other embodiments, the genomically engineered iPSCs generated using the methods provided herein comprise in / dels in one or more endogenous genes associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response control and regulation, or proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells. In some embodiments, the endogenous genes for disruption include at least one of CD38, B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region.

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

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

[0016] The present invention also provides the following:

[0017] One aspect of the present application provides a cell or population thereof, wherein the cell is an induced pluripotent cell (iPSC), a clonal iPSC, or a clonal iPS cell line cell, or a derived cell obtained by differentiation of any of the iPSCs described above, any of the cells described above comprising at least a TCR negand one or more polynucleotides encoding one or more exogenous proteins that, upon expression, provide a cell-surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3). In some embodiments of derivative cells obtained from iPSC differentiation, the derivative cells are hematopoietic cells, including, but not limited to, CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells, and the derived hematopoietic cells (i.e., derived CD34 cells, derived hemogenic endothelial cells, derived hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell progenitors, derived NK cell progenitors, derived T cells, derived NKT cells, derived NK cells, or derived B cells) comprise longer telomeres compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or other donor tissue. In some embodiments of derivative cells obtained from iPSC differentiation, the derivative cells are T cell progenitors or T cells. In some embodiments of derivative cells obtained from iPSC differentiation, the derivative cells are NK cell precursors or NK cells.

[0018] TCR negand one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), the cells have one or more of the following genome edits: (i) B2M negative or low B2M, (ii) CIITA negative or low CIITA, (iii) introduced expression of HLA-G or uncleavable HLA-G, (iv) high affinity uncleavable CD16 (hnCD16) or a variant thereof, (v) chimeric antigen receptor (CAR) ), (vi) cell surface expression of exogenous cytokines or partial or full-length peptides of their receptors, (vii) CD38 negative, (viii) at least one of the genotypes listed in Table 1, (ix) deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region, (x) HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Further comprising one or more of introduced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, an Fc receptor, an engager, and a bi- or multispecific or universal engager.

[0019] At least TCR negIn some embodiments of the iPSCs and their derivatives, the iPSCs and their derivatives comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and optional additional genome editing as described above and throughout the present application. The cells may contain (i) one or more exogenous polynucleotides integrated into one safe harbor locus, or (ii) two or more exogenous polynucleotides integrated into different safe harbor loci. In some embodiments, the safe harbor loci include at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR, or RUNX1. In one specific embodiment, the safe harbor locus is H11. In one specific embodiment, the safe harbor locus TCR is the constant region of TCR alpha or TCR beta. In some embodiments, insertion of one or more exogenous polynucleotides at a safe harbor locus results in the disruption or knockout of an endogenous gene at the safe harbor, e.g., insertion at B2M, CD38, or TCR alpha / beta results in the knockout of the B2M, CD38, or TCR alpha / beta gene.

[0020] In some embodiments of the cells or populations thereof, TCR negand one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and one or more of the additional genome edits described above, are in some embodiments derived T cells, and in some other embodiments derived NK cells obtained from iPSCs containing the genome edits, wherein the derived T cells or NK cells have the following characteristics: (i) persistence, compared to their native counterpart primary T cells or NK cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue; and / or improved survival, (ii) increased tolerance to natural (i.e., recipient) immune cells, (iii) increased cytotoxicity, (iv) improved tumor penetration, (v) enhanced or acquisition of ADCC, (vi) enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability to rescue tumor antigen escape, and (ix) reduced fratricide. neg TCR when compared with T cells or NK cells (primary or derived) neg Derived T cells or NK cells that contain both the CD3 complex and one or more exogenous proteins that enable the surface to present the CD3 complex or a subunit or subdomain thereof have at least one of the following phenotypic and functional characteristics, further including, but not limited to, a cell surface that presents a complete or partial endogenous or exogenous cell surface CD3 complex, and the ability to respond to molecules that bind to a CD3-associated cell surface triggering receptor, including, but not limited to, a CD3-binding antibody or functional variant thereof, an scFV, and / or various CD3 engagers.

[0021] In one embodiment of the cell or population thereof, a TCR negand one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), further comprise a high-affinity, non-cleavable CD16 or variant thereof (hnCD16). Some embodiments of the high-affinity, non-cleavable CD16 or variant thereof (hnCD16) comprise at least one of the following: (a) F176V and S197P in the ectodomain of CD16, (b) a complete or partial ectodomain derived from CD64, (c) a non-native (or non-CD16) transmembrane domain, (d) a non-native (or non-CD16) intracellular domain, (e) a non-native (or non-CD16) signaling domain, (f) a non-native stimulatory domain, and (g) transmembrane, signaling, and stimulatory domains not derived from CD16 but derived from the same or a different polypeptide. In some embodiments, the non-native transmembrane domain is derived from a CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide. In some embodiments, the non-native stimulatory domain is derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some other embodiments, the non-native signaling domain is derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In some particular embodiments of hnCD16 or a variant thereof, the non-native transmembrane domain is derived from NKG2D, the non-native stimulatory domain is derived from 2B4, and the non-native signaling domain is derived from CD3ζ or DAP10.

[0022] In one embodiment of the cell or population thereof, a TCR negand one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex, or one or more subunits or subdomains thereof (cs-CD3), further comprise at least one chimeric antigen receptor (CAR), wherein the CAR is: (i) T cell-specific or NK cell-specific, (ii) a bispecific antigen-binding CAR, (iii) a switchable CAR, (iv) a dimerized CAR, (v) a split CAR, (vi) a multi-chain CAR, (vii) an inducible CAR, (viii) co-expressed with another CAR, (ix) co-expressed with a partial or full-length peptide of a cell surface-expressed exogenous cytokine or its receptor, optionally in a separate construct or in a bicistronic or polycistronic construct, (xi) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic or polycistronic construct, (xii) specific for CD19 or BCMA, and / or (xiii) specific for ADGRE2, carbonic anhydrase IX (CA). IX), CCRI, 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, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine kinase inhibitor (TKI). 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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19).9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigens. In some embodiments, any one of the above CARs may be inserted into the constant region of a TCR locus, such as TCRα or TCRβ. In some embodiments, the CAR inserted into the TRAC or TRBC locus can be driven by the endogenous promoter of the TCR. In some embodiments, insertion of the CAR at the TRAC or TRBC locus results in a TCR-negative or knockout. In some embodiments, the TCR-negative cells are also CD3-negative.

[0023] In some embodiments in which a checkpoint inhibitor is co-expressed with the CAR, the checkpoint inhibitor is an antagonist to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. The checkpoint inhibitor co-expressed with the CAR can be an antibody, or a humanized or Fc-modified variant or fragment thereof, as well as functional equivalents and biosimilars, specific for any of the above checkpoint molecules.

[0024] In one embodiment of the cell or population thereof, a TCR negand one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), further comprising a partial or full-length peptide of a cell surface-expressed exogenous cytokine or its receptor, wherein the exogenous cytokine or its receptor may include at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (i) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (ii) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (iii) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (iv) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (v) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (vi) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (vii) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (viii ... The CAR may comprise at least one of: (i) a fusion protein of IL15 and IL15Rα; (ii) an IL15 / IL15Rα fusion protein having a truncated intracellular domain of IL15Rα (IL15Δ); (iii) a fusion protein of IL15 and the membrane-binding Sushi domain of IL15Rα; (iv) a fusion protein of IL15 and IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and common receptor γC (wherein common receptor γC is native or modified); and (vii) an IL15Rβ homodimer, wherein any one of (i)-(vii) may be co-expressed with the CAR in a separate construct or in a bicistronic or polycistronic construct. In some embodiments, a partial or complete peptide of a cell-surface exogenous cytokine or receptor is transiently expressed in the cells provided herein. In one embodiment, the cell or population thereof comprises a polynucleotide encoding IL15Δ comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21.

[0025] In one embodiment of the cell or population thereof, a TCR negand one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and IL15 or a variant thereof, including those embodiments (i) to (vii) disclosed above, may be B2M-negative or low B2M; CIITA-negative or low CIITA; introduced expression of HLA-G or uncleavable HLA-G; high affinity uncleavable CD16 or a variant thereof (hnCD16); chimeric antigen receptor (CAR); cell surface-expressed additional exogenous cytokine or a partial or full-length peptide of its receptor (wherein the cytokine is not IL15). at least one of the genotypes listed in Table 1; deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region; and introduced or increased expression of at least one of HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, Fc receptor, engager, and surface triggering receptor for binding to a bi- or multispecific or universal engager. In embodiments of cells or populations thereof comprising both IL15Δ and a CAR, IL15Δ may be co-expressed with the CAR in a separate construct or in a bicistronic or polycistronic construct.

[0026] In one embodiment of the cell or population thereof, a TCR negand one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell-surface CD3 complex, or one or more subunits or subdomains thereof (cs-CD3), are derived NK cells or derived T cells, and the derived NK cells or derived T cells are capable of reducing tumor immunosuppression in the presence of one or more checkpoint inhibitors. In some embodiments, the presence of the one or more checkpoint inhibitors is by administering the inhibitors to the subject before, during, or after receiving the cells. In some other embodiments, the presence of the one or more checkpoint inhibitors is by expressing the inhibitors by the cells by introducing checkpoint inhibitor expression into the cells using polynucleotides encoding the selected 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 either (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0027] Another aspect of the present application provides compositions comprising any of the cells or populations thereof described above and throughout this application. In some embodiments, the iPSCs or iPSC-derived cells (also referred to herein as "derived cells") may comprise any one of the genotypes listed in Table 1 of the present application. In some embodiments, the TCR neg The iPSCs or derived cells thereof comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3). In some embodiments, the TCR neg The iPSCs or derived cells thereof comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and hnCD16. In some embodiments, TCR neg The iPSCs or derived cells thereof comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), hnCD16, and IL15 or a variant thereof. In some embodiments, the TCR neg The iPSCs or derived cells therefrom comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), hnCD16, and a CAR. In some embodiments, a TCR neg The iPSCs or derived cells thereof comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), hnCD16, IL15 or a variant thereof, and a CAR. In some embodiments, a TCR negThe iPSCs or derived cells therefrom comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), hnCD16, CD38, a CAR, and a partial or full-length peptide of a surface-expressed exogenous cytokine or its receptor as described above and throughout this application.In some other embodiments, the CAR is selected from the group consisting of ADGRE2, carbonic anhydrase IX (CAIX), CCRI, 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, CDS, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine kinase (RTK), tyrosine kinase inhibitor (TKI ... Ribosomal 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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and any one of various pathogen antigens known in the art. negIn some further embodiments of the cell comprising, the cell comprises one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), hnCD16, and a CAR, wherein the CAR is specific for CD19 or CD269 (BCMA). neg In some embodiments of the cell comprising a TCR, the cell comprises a surface CD3 complex or a subunit or subdomain thereof, hnCD16, IL15 or a variant thereof, and a CAR when expressed on the cell, wherein the CAR is specific for CD19 or CD269 (BCMA). neg In some other embodiments of the cell comprising, when expressed on the cell, the cell comprises a surface CD3 complex or a subunit or subdomain thereof, hnCD16, IL15 or a variant thereof, CD38 negative, and a CAR, wherein the CAR is specific for CD19 or CD269 (BCMA).

[0028] Thus, a further aspect of the present application provides compositions for therapeutic use that include, in addition to any of the derivative cells provided herein, one or more therapeutic agents. In some embodiments of compositions for therapeutic use, the therapeutic agent comprises a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or radioactive moiety, or an immunomodulatory drug (IMiD). In some embodiments of compositions for therapeutic use, the checkpoint inhibitors used in conjunction with the provided cells comprise one or more antagonist checkpoint molecules, including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, 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 an inhibitory KIR. In some embodiments of the compositions 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 derivatives or functional equivalents thereof. In some other embodiments of the compositions 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 compositions for therapeutic use, the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide.

[0029] In some embodiments of compositions for therapeutic use, the antibody used in conjunction with the provided cells comprises any one of anti-CD20, anti-CD22, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies. In some embodiments of the compositions for therapeutic use, the antibody used with the provided cells comprises one or more of rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, ibritumomab, ocrelizumab, inotuzumab, moxetumomab, epratuzumab, trastuzumab, pertuzumab, alemtuzumab, certuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof. In still other embodiments of the compositions for therapeutic use, the antibody used with the provided cells comprises daratumumab.

[0030] The present application also provides therapeutic uses of the cells or therapeutic compositions described herein by introducing the compositions into a subject suitable for adoptive cell therapy, hi some embodiments, the subject suitable for and in need of adoptive cell therapy has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection.

[0031] A further aspect of the application provides a method of producing a derivative cell as described herein, the method comprising: combining one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and, optionally, (i) B2M negative or low B2M, (ii) CIITA negative or low CIITA, (iii) introduced expression of HLA-G or uncleavable HLA-G, (iv) high affinity uncleavable CD16 (hnCD16) or a variant thereof, (v) chimeric antigen receptor (CA) R), (vi) cell surface expression of exogenous cytokines or partial or full-length peptides of their receptors, (vii) CD38 negativity, (viii) at least one of the genotypes listed in Table 1, (ix) deletion or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region, and (x) HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A a TCR comprising one or more of an R, a CAR, an Fc receptor, an engager, and introduced or increased expression of at least one of a surface triggering receptor for binding to a bi- or multispecific or universal engager. neg This involves differentiating iPSCs.

[0032] In some embodiments of the manufacturing methods, the methods include (1) knocking out an endogenous TCR, and (2) providing a cell with a surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3) when expressed on the cell, including tgTRAC, tgTRBC, tgTCRα, tgTCRβ, tgpTCRα, tgCD3(ε-δ)-TRAC, tgCD3(ε-γ)-TRBC, tgCD3(ε-γ)-TRAC, tgCD3(ε-δ)-TRBC, tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, and / or tgCD3(ε-γ / δ)-(28-BB)ζ. The method further comprises genomic engineering the clonal iPSCs to introduce one or more polynucleotides encoding at least one exogenous protein, including B2M and CIITA, and optionally to knock out B2M and CIITA, or to introduce expression of HLA-G or uncleavable HLA-G, high-affinity uncleavable CD16 or a variant thereof, a CAR, and / or a partial or full-length peptide of a cell surface-expressed exogenous cytokine or its receptor, wherein the CAR and the partial or full-length peptide of the cell surface-expressed exogenous cytokine or its receptor are co-expressed in separate constructs or in a bicistronic or polycistronic construct. In some embodiments of the manufacturing method, the genomic engineering of the iPSCs comprises targeted editing. In some embodiments, the targeted editing comprises a deletion, an insertion, or an in / del. In some embodiments, the targeted deletion / knockout and the targeted insertion are performed simultaneously, and the insertion is at the position where the deletion is to be made. In some embodiments, the targeted deletion / knockout and the targeted insertion are performed sequentially in any order, and the insertion and deletion may or may not be at the same position. In some embodiments, the targeted editing tool comprises a CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional alteration of these methods and compositions.

[0033] The present application further provides for editing of clonal iPSCs via a targeted editing tool, thereby producing cells that lack endogenous TCRα or TCRβ and that, when expressed in cells, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), or at least one of the genotypes listed in Table 1, including tgTRAC, tgTRBC, tgTCRα, tgTCRβ, tgpTCRα, tgCD3(ε-δ)-TRAC, Edited clonal iPSCs are produced, comprising one or more introduced polynucleotides encoding at least one exogenous protein, including tgCD3(ε-γ)-TRBC, tgCD3(ε-γ)-TRAC, tgCD3(ε-δ)-TRBC, tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, and / or tgCD3(ε-γ / δ)-(28-BB)ζ. In some embodiments of genome-targeting tool-mediated editing, the resulting TCR knockout is due to targeted disruption of the endogenous TCR α constant region (TRAC). In some embodiments of genome-targeting tool-mediated editing, the resulting TCR knockout is due to targeted disruption of the endogenous TCR β constant region (TRBC). In some embodiments of editing via the above genome targeting tools, the editing further comprises insertion of a CAR at the endogenous TRAC or TRBC locus, and / or the CAR is driven by the endogenous promoter of TCR alpha or TCR beta, and / or the TCR is knocked out by the CAR insertion. In some embodiments of editing via the genome-targeting tools described above, the editing further comprises insertion at the endogenous TRAC locus of a polynucleotide encoding an exogenous protein comprising tgTRAC, tgTCRα, tgpTCRα, tgCD3(ε-δ)-TRAC, tgCD3(ε-γ)-TRAC, tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, or tgCD3(ε-γ / δ)-(28-BB)ζ, which, when expressed in a cell, provides a surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3).In some embodiments of editing via the above-described genome-targeting tools, the editing further comprises insertion at the endogenous TRBC locus of a polynucleotide encoding an exogenous protein, including tgTRBC, tgTCRβ, tgCD3(ε-γ)-TRBC, tgCD3(ε-δ)-TRBC, tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, and / or tgCD3(ε-γ / δ)-(28-BB)ζ, which, when expressed in a cell, provides a surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3).

[0034] In one embodiment of the provided cells or populations thereof, the cells are induced pluripotent cells (iPSCs), clonal iPSCs, or clonal iPS cell line cells, or derivative cells obtained by differentiating the iPSCs, and the cells are TCR negand comprises one or more polynucleotides encoding one or more exogenous proteins that, upon expression, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3). In some embodiments, the cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3) is associated with a nonbinding recombinant TCR (nb-rTCR), a defined recombinant TCR (d-rTCR), a recombinant pre-TCR (p-rTCR), or is fixed to a nonbinding recombinant TCR (nb-rTCR-CD3), or a CD3 chimeric chain (ccCD3). In one embodiment, the nb-rTCR comprises one or both of a tgTRAC and a tgTRBC. In one embodiment, the d-rTCR comprises a tgTCRα and, optionally, a tgTCRβ, each of the tgTCRα and tgTCRβ comprising a respective defined variable region. In one embodiment, the p-rTCR comprises tgpTCRα and optionally tgTRBC or tgTCRβ, where the tgTCRβ comprises a defined variable region. In one embodiment, the nb-rTCR-CD3 comprises one or more of the following fusion proteins: (1) tgCD3(ε-δ)-TRAC, (2) tgCD3(ε-γ)-TRBC, (3) tgCD3(ε-γ)-TRAC, and / or (4) tgCD3(ε-δ)-TRBC, where the fusion proteins comprise full-length or partial-length ectodomains of CD3ε, CD3δ, CD3γ, and / or full-length or partial-length TRAC or TRBC. In one embodiment, the ccCD3 comprises at least one of the fusion proteins: tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, and tgCD3(ε-γ / δ)-(28-BB)ζ, wherein the fusion protein comprising a full-length or partial-length ectodomain of a CD3ε, CD3δ, and / or CD3γ protein further comprises a cytoplasmic domain comprising a full-length or partial-length endodomain of a CDζ protein, and optionally one or both of a CD28 signaling domain and a 41BB signaling domain.In yet another embodiment, the nb-rTCR-CD3 comprising the fusion protein tgCD3(ε-δ)-TRAC further comprises tgTRBC or tgTCRβ. In yet another embodiment, the nb-rTCR-CD3 comprising the fusion protein tgCD3(ε-γ)-TRBC further comprises tgTRAC or tgTCRα. Alternatively, in yet another embodiment, the d-rTCR comprising tgTCRα and tgTCRβ comprises the TCRα and TCRβ of invariant NKT cells.

[0035] In view of the above embodiments, the provided cells, in some aspects, comprise one of a recombinant TCR: nb-rTCR, d-rTCR, or p-rTCR, where the recombinant TCR forms a complex with an endogenous CD3 subunit, thereby enabling cell surface presentation of the endogenous CD3 subunit and its signal transduction. In some other embodiments, the cells comprise nb-rTCR-CD3 or ccCD3, thereby enabling cell surface presentation of an exogenous CD3 subunit and its signal transduction.

[0036] One aspect of the present application provides a clonal master cell bank comprising the clonal iPSC cell line cells described herein. Another aspect of the present application provides compositions comprising cells or populations thereof, including various embodiments of iPSCs and derivative cells differentiated from the iPSCs, as described herein. In some embodiments, the compositions comprising the derivative cells are suitable for therapeutic applications. In some embodiments of compositions for therapeutic applications, the compositions optionally further comprise one or more therapeutic agents in addition to the derivative cells. In some embodiments, the therapeutic agent comprises a peptide, cytokine, checkpoint inhibitor, mitogen, growth factor, small RNA, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or radioactive moiety, or an immunomodulatory drug (IMiD).

[0037] An additional aspect of the present application provides therapeutic uses of the therapeutic compositions in various embodiments by introducing the composition into a subject suitable for adoptive cell therapy, the subject having an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection.

[0038] A further aspect of the present application is a TCR neg Provided is a method for producing derivative effector cells by differentiating iPSCs, wherein the iPSCs comprise one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and optionally the iPSCs are (i) B2M-negative or B2M-low, (ii) CIITA-negative or CIITA-low, (iii) introduced expression of HLA-G or uncleavable HLA-G, (iv) high-affinity uncleavable CD16 (hnCD16) or a variant thereof, (v) a chimeric antibody. (vi) a cell surface-expressed exogenous cytokine or a partial or full-length 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 CD38, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region; (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A In some embodiments of the methods of producing cells of the present application, the method further comprises introducing or increasing expression of at least one of a TCR, a CAR, a TCR, an Fc receptor, an engager, and a surface triggering receptor for binding to a bi- or multispecific or universal engager. negThe method further comprises the steps of providing a cell-surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3) by obtaining iPSCs and simultaneously or subsequently by knocking in one or more polynucleotides encoding one or more exogenous proteins, or by reprogramming invariant NKT cells into iPSCs to provide clonal iPSCs comprising iTCRαβ but not T cell-TCR, and the optional steps of genomic engineering of TCR-negative cells by knocking out B2M and CIITA, or introducing expression of HLA-G or uncleavable HLA-G, high-affinity uncleavable CD16 or a variant thereof, a CAR, and / or a partial or full-length peptide of a cell-surface-expressed exogenous cytokine or its receptor, wherein the CAR and the partial or full-length peptide of the cell-surface-expressed exogenous cytokine or its receptor are co-expressed in separate or bicistronic constructs.

[0039] In one embodiment of the method for producing the cells, the step of knocking in one or more polynucleotides encoding one or more exogenous proteins to provide cs-CD3 includes knocking in one or both of: (i) polynucleotides encoding one or more exogenous proteins that, when expressed, provide a nb-rTCR; (ii) polynucleotides encoding one or both of tgTRAC and tgTRBC that, when expressed, provide a d-rTCR; (iii) polynucleotides encoding one or more exogenous proteins that, when expressed, provide a d-rTCR; and optionally tgTCRβ (each of tgTCRα and tgTCRβ comprising a respective defined variable region); (iv) fusion proteins that provide nb-rTCR-CD3: (1) tgCD3(ε-δ)-TRAC; (2) tgCD3(ε-γ)-TRBC; (3) tgCD3(ε-γ)-TRAC; and / or (4) tgCD3(ε-δ)-TRB. one or more polynucleotides encoding one or more of the following fusion proteins (the fusion proteins include full-length or partial-length ectodomains of CD3ε, CD3δ, CD3γ, and / or full-length or partial-length TRAC or TRBC), or (v) ccCD3, providing fusion proteins: (1) tgCD3(ε-γ)-ζ, (2) tgCD3(ε-δ)-ζ, (3) tgCD3(ε-γ / δ)-28ζ, (4) tgCD3(ε-γ / δ)-BBζ, and (5) tgCD3 The method further comprises introducing into the iPSCs one or more polynucleotides encoding one or more of (ε-γ / δ)-(28-BB)ζ (wherein the fusion protein comprises a full-length or partial-length ectodomain of a CD3ε, CD3δ, and / or CD3γ protein, a cytoplasmic domain comprising a full-length or partial-length endodomain of a CDζ protein, and optionally one or both of a CD28 signaling domain and a 41BB signaling domain). In some embodiments of the described methods for producing cells, the step of introducing into the iPSCs one or more polynucleotides encoding the fusion protein tgCD3(ε-δ)-TRAC further comprises introducing a polynucleotide encoding tgTRBC or tgTCRβ, which provide nb-rTCR-CD3.In some embodiments, the step of introducing one or more polynucleotides encoding the fusion protein tgCD3(ε-γ)-TRBC into iPSCs further comprises tgTRAC or tgTCRα, which provide nb-rTCR-CD3. In some embodiments, the step of introducing one or more polynucleotides encoding the fusion protein tgCD3(ε-γ)-TRBC into iPSCs further comprises tgTRAC or tgTCRα, which provide nb-rTCR-CD3. In some other embodiments, the step of introducing a polynucleotide encoding tgTCRα, and optionally a polynucleotide encoding tgTCRβ, which provide a d-rTCR, into iPSCs, wherein tgTCRα and tgTCRβ comprise the TCRα and TCRβ, respectively, of invariant NKT cells.

[0040] In various embodiments of the cell manufacturing method provided, genome manipulation is targeted editing, including deletion and / or insertion.Such targeted editing can be performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional modification of these tools.One aspect of the present invention provides the CRISPR-mediated editing of the clonal iPSCs of various embodiments provided herein, thereby enabling TCR neg and obtaining edited iPSCs comprising one or more polynucleotides encoding one or more exogenous proteins that, upon expression, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), wherein the edited clonal iPSCs comprise at least one of the genotypes listed in Table 1. In some embodiments, the CRISPR-mediated editing further comprises insertion of a CAR at the TRAC or TRBC 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.

[0041] Another aspect of the present application provides a method of combination therapy, the method comprising: negand providing effector cells to the subject being treated, the effector cells comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell-surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and a selected multispecific engager, wherein the effector cells comprise the provided derivative cells. In some embodiments, the selected multispecific engager in the method is at least one of: (i) a T cell engager, (ii) an NK cell engager, (iii) a bispecific T cell engager (BiTE), (iv) a bispecific killer cell engager (BiKE), (v) a trispecific killer cell engager (TriKE), (vi) a CD3 engager, or (vii) a CD16 engager. In some embodiments, the selected multispecific engager is a CD3 engager, the CD3 engager comprising a first variable segment that binds to cs-CD3 and a second variable segment that binds to one of the following: (i) ADGRE2, carbonic anhydrase IX (CAIX), CCRI, 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, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-4 ... 0), 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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2,Melanoma antigen family A 1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor and a second variable segment that binds to an antigen comprising at least one of (i) BCMA, CD19, CD20, CD33, CD38, CD52, CD123, CEA, EGFR, EpCAM, GD2, GPA33, HER2, MICA / B, PDL1, and / or PSMA, or (ii) CD19, CD33, CD123, CEA, EpCAM, GPA33, HER2, and / or PSMA. In some embodiments, the CD3 engager comprises at least one of blinatumomab, catumaxomab, ertumaxomab, RO6958688, AFM11, MT110 / AMG110, MT111 / AMG211 / MEDI-565, AMG330, MT112 / BAY2010112, MOR209 / ES414, MGD006 / S80880, MGD007, and / or FBTA05. In some embodiments, the CD3 engager is administered to the subject simultaneously with or subsequent to the effector cells. In some embodiments, the effector cells comprise derived hematopoietic cells, including derived NK cells or derived T cells, wherein the derived NK cells or derived T cells comprise a CD38 knockout, a high affinity non-cleavable CD16 or a variant thereof, and optionally (i) a B2M and CIITA knockout, (ii) introduced expression of HLA-G or non-cleavable HLA-G, a CAR, and / or a partial or full-length peptide of a cell surface-expressed exogenous cytokine or its receptor (the CAR and the partial or full-length peptide of a cell surface-expressed exogenous cytokine or its receptor are co-expressed in separate constructs or a bicistronic construct);and / or (iii) at least one of the genotypes listed in Table 1.

[0042] Another aspect of the present application provides a method for reducing or preventing allorejection by recipient T cells of effector cells in adoptive cell therapy, the method comprising administering to a subject being treated: (i) an anti-CD3 agent; and (ii) a TCR negand a cell-surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3) preloaded with a CD3 engager, wherein the effector cell comprises a derivative cell provided herein. In some embodiments, the anti-CD3 agent is a CD3 antibody or a CD3-CAR, wherein the CD3-CAR is comprised on an NK cell, and the anti-CD3 agent inactivates recipient T cells, thereby reducing or preventing allorejection. In some embodiments, the CD3 engager comprising a first variable segment that binds to cs-CD3 on the effector cell comprises a second variable segment that binds to an antigen associated with an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection. In some embodiments of a CD3 engager comprising a second variable segment that binds an antigen, the antigen is selected from the group consisting of (i) ADGRE2, carbonic anhydrase IX (CAIX), CCRI, 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, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP4), and CLEC12A. -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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19).9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R 2), Wilms tumor protein (WT-1), and / or pathogen antigens, or (ii) BCMA, CD19, CD20, CD33, CD38, CD52, CD123, CEA, EGFR, EpCAM, GD2, GPA33, HER2, MICA / B, PDL1, and / or PSMA, or (iii) at least one of CD19, CD33, CD123, CEA, EpCAM, GPA33, HER2, and / or PSMA. In yet some other embodiments, the CD3 engager comprises at least one of blinatumomab, catumaxomab, ertumaxomab, RO6958688, AFM11, MT110 / AMG110, MT111 / AMG211 / MEDI-565, AMG330, MT112 / BAY2010112, MOR209 / ES414, MGD006 / S80880, MGD007, and / or FBTA05.

[0043] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description, taken in conjunction with the accompanying drawings, in which are set forth, by way of illustration and example, certain embodiments of the invention. [Brief explanation of the drawings]

[0044] [Figure 1A]Exemplary designs for generating a cell surface-displayed CD3 complex or subunit or subdomain thereof (cs-CD3) that associates with a recombinant TCR complex by disrupting the endogenous TCR in a cell are shown: (1) nb-rTCR (non-binding recombinant TCR), (2) d-rTCR (defined recombinant TCR), (3) p-rTCR (recombinant pre-TCR alpha with optional non-binding TCR beta), (4) nb-rTCR-CD3 (non-binding recombinant TCR-anchored CD3), (5) ccCD3 (CD3 chimeric chain). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2] 1 is a graphical representation of several construct designs for cell surface expression cytokines in iPSC-derived cells. IL15 is used as an illustrative example and can be substituted with other desired cytokines. [Figure 3] Figure 1 shows the reprogramming and engineering of αβ T cells into single-cell-derived TRAC-targeted CAR TiPSC clones. A: Phase-contrast images of cultures at different stages as indicated. B: Flow cytometry profiles of αβ T cells before reprogramming (left panel), the reprogrammed and engineered cell pool before sorting, and the clonal TiPSC clones (right panel). [Figure 4] 1 is a graphical representation of flow cytometry of mature TCR iPSC-derived NK cells showing stepwise manipulation of hnCD16 expression, B2M knockout (denoted as "B2M"; loss of HLA-A2 expression), HLA-G expression, and IL-15 / IL-15ra (LNGFR) construct expression. [Figure 5] Graphical representation of telomere length determined by flow cytometry, showing that mature derived NK cells from iPSCs maintain longer telomeres compared to adult peripheral blood NK cells. [Figure 6]iNK cells transduced with a full-length IL15 / IL15Rα fusion construct (filled circles; positive control) or a truncated IL15 / IL15Rα fusion construct lacking the cytoplasmic signaling domain (open circles) had a survival advantage compared to untransduced or GFP-transduced cells in the same culture, independent of exogenous soluble IL2. A: In the presence of exogenous IL2; B: In the absence of exogenous IL2. [Figure 7] Exemplary constructs for transducing invariant NKT TCRα with or without TCRβ are shown. [Figure 8A] Figure 1 shows that CD3 is not expressed by invariant NKT TCR-transduced iPSCs or iPSC-derived iCD34 progenitor cells. [Figure 8B] Same as above. [Figure 9A] Figure 9A-B shows the flow cytometry results of CD3 and TCR expression at various time points. Figure 9C shows the MFI of CD3 at 25 days after iCD34 differentiation. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 10] We show that stimulation by cell surface CD3 binding with tetramers results in aggregation of iPSC-derived T cells containing NKT TCRα with or without NKT TCRβ. [Figure 11] 1 shows that iPSC-derived T cells containing NKT TCRα with or without NKT TCRβ increased CD25 expression upon stimulation with CD3 tetramer. [Figure 12A]Figure 12A shows representative FACS plots demonstrating enhanced target cell killing in the presence of CD19xCD3 and CD20xCD3 BiTEs at an effector-to-target ratio of 10:1. Figure 12B shows the percentage of specific cytotoxicity against iPSC-derived effector cells containing NKT TCRα with or without NKT TCRβ. [Figure 12B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0045] Genome modifications of iPSCs (induced pluripotent stem cells) include polynucleotide insertion, deletion, and substitution. Exogenous gene expression in genomically engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genomically engineered iPSCs, after cell differentiation, and in dedifferentiated cell types derived from genomically engineered iPSCs. Meanwhile, direct manipulation of primary immune cells, such as T cells or NK cells, isolated from peripheral blood, umbilical cord blood, or any other donor tissue is challenging, presenting obstacles to the preparation and delivery of engineered immune cells for adoptive cell therapy. The present invention provides an efficient, reliable, and targeted approach for stably incorporating one or more exogenous genes, including suicide genes and other functional modalities, into derived cells differentiated from iPSCs, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T cells, NKT cells, and NK cells, that provide improved therapeutic properties related to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, expansion, longevity, self-renewal, persistence, and / or survival.

[0046] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular.

[0047] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0048] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0049] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0050] The term "and / or" should be understood to mean either one or both of the alternatives.

[0051] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0052] As used herein, the term "substantially" or "essentially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about the same as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0053] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that does not contain a particular substance or source thereof, e.g., is 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance (1) is not included in the composition at any concentration, or (2) is functionally inactive but is included in the composition at low concentrations. A similar meaning can be applied to the term "absent," which refers to the absence of a particular substance or source of a composition.

[0054] Throughout this specification, unless the context requires otherwise, the terms "comprise," "comprises," and "comprising" are understood to mean the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include," "having," "containing," and "comprise" are used synonymously.

[0055] "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0056] "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or operation specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but other elements are not optional and may or may not be present depending on whether they affect the activity or operation of the listed elements.

[0057] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of these phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0058] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of a living organism, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures involve live cells or tissues taken from an organism and cultured in a laboratory setting, usually under sterile conditions, typically for a few hours or up to about 24 hours (although up to 48 or 72 hours or longer, depending on the circumstances). In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using live cells or tissues are typically considered to be "in vitro," although in certain embodiments, the term may be used interchangeably with ex vivo.

[0059] The term "in vivo" generally refers to activities that take place inside a living organism.

[0060] As used herein, the term "reprogramming" or "dedifferentiation" or "increased differentiation potential" or "increased developmental potential" refers to a method of increasing the differentiation potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased differentiation potential has greater developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an unreprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in an unreprogrammed state.

[0061] 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 differentiation-induced cells are cells that have adopted a more specialized ("committed") position within a cellular lineage. The term "committed," when applied to the process of differentiation, refers to cells that, under normal circumstances, have progressed along a differentiation pathway to the point where they continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, are unable to differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells that cannot give rise to a complete organism (e.g., epiblast stem cells or EpiSCs), to more primitive, more pluripotent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0062] As used herein, the term "induced pluripotent stem cells" or iPSCs means that stem cells are produced in vitro from induced or altered differentiated adult, neonatal, or fetal cells using reprogramming factors and / or small molecule chemical-driven methods, i.e., cells that can differentiate into tissues of all three germ layers (mesoderm, endoderm, and ectoderm) or all dermal layers. The iPSCs produced do not refer to cells found in nature.

[0063] As used herein, the term "embryonic stem cells" refers to naturally occurring pluripotent stem cells in the inner cell mass of blastocysts. Embryonic stem cells are pluripotent and give rise to all derivatives of the three major germ layers of ectoderm, endoderm, and mesoderm during development. They do not contribute to extraembryonic membranes or placenta, i.e., they are not totipotent.

[0064] As used herein, the term "multipotent stem cells" refers to cells that have the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Therefore, multipotent cells are also called "partially differentiated cells." Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Multipotency" indicates that a cell has the potential to form many cell types in a given lineage but not cells of other lineages. For example, multipotent hematopoietic cells can form many different blood cell types (red, white, platelets, etc.), but cannot form neurons. Therefore, the term "multipotency" refers to a state of a cell that has a degree of developmental potential lower than totipotency and pluripotency.

[0065] Pluripotency can be determined, in part, by assessing cellular pluripotency characteristics, including, but not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers, including, but not limited to, 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 composed of the three somatic cell lineages, and (vi) the formation of embryoid bodies composed of cells from the three somatic cell lineages.

[0066] Two types of pluripotency have previously been described: a pluripotent "primed" or "metastable" state, similar to the epiblast stem cells (EpiSCs) of late blastocysts, and a pluripotent "naive" or "ground" state, similar to the cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or ground state additionally exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival during single-cell culture, (iii) globally reduced DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental control gene promoters, and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard cell reprogramming methodologies, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or removed from the resulting pluripotent cells, are generally considered to possess the characteristics of the primed pluripotent state. Under standard pluripotent cell culture conditions, such cells will remain in a primed state and exhibit ground state characteristics unless expression of an exogenous transgene is maintained, at which point ground state characteristics will be observed.

[0067] As used herein, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a small, round shape with a high nucleus-to-cytoplasm ratio, prominent nucleoli, and typical intercellular spacing.

[0068] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domestic animal.

[0069] "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.

[0070] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in each case singular "medium"), "supplement," and "medium supplement" refer to nutritional compositions in which cell cultures are cultivated.

[0071] "Culturing" or "maintaining" refers to maintaining, expanding (growing), and / or differentiating cells outside of a tissue or outside the body, for example, in sterile plastic (or coated plastic) cell culture dishes or flasks. "Culturing" or "maintaining" may utilize culture medium as a source of nutrients, hormones, and / or other factors that help to grow and / or maintain the cells.

[0072] As used herein, the term "mesoderm" refers to one of three germ layers that emerge during early embryonic development and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0073] As used herein, the terms "secondary hemogenic endothelial cells" (HE) or "pluripotent stem cell-derived secondary hemogenic endothelial 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 conversion. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm to hemangioblasts to secondary hemogenic endothelial cells and hematopoietic progenitor cells.

[0074] The terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including multipotent hematopoietic stem cells (hemocyte blasts), myeloid progenitors, megakaryocytic progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). 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.

[0075] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cell that completes maturation in the thymus and has various roles in the immune system, including identifying specific foreign antigens in the body and activating and deactivating other immune cells. T cells can be any T cell, such as 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 a mammal. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any stage of development, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), 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), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0076] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are involved in cell-mediated immune responses. They are characterized by a secretory profile after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55 kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as the relevant recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, they define helper / inducer subsets.

[0077] "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 on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the relevant recognition element in major histocompatibility complex class I restricted interactions.

[0078] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56+, express at least one of NKG2C and CD57, and optionally CD16, but lack expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, an isolated subpopulation of CD56+ NK cells contains expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. CD56+ may be dim or bright.

[0079] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the non-classical MHC molecule, CD1d. Two types of NKT cells have been recognized. Invariant or type I NKT cells express a very limited TCR repertoire, i.e., a standard α chain (Vα24-Jα18 in humans) associated with a limited spectrum of β chains (Vβ11 in humans). A second population of NKT cells, termed non-classical or non-invariant type II NKT cells, exhibits more heterogeneous TCRαβ usage. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0080] As used herein, the term "isolated" or similar refers to a cell or population of cells that has been separated from its original environment, i.e., the environment of the isolated cell is substantially free of at least one component found in the environment of the "non-isolated" reference cell. This term includes cells that have been removed from some or all components found in their natural environment, e.g., isolated from a tissue or biopsy sample. This term also includes cells that have been removed from at least one, some, or all components found in a non-naturally occurring environment, e.g., isolated from a cell culture or cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells, or cell populations, as found in nature or when grown, stored, or subsisting in a non-naturally occurring environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a non-naturally occurring medium. Isolated cells can be obtained by separating a desired cell or population of cells from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0081] As used herein, the term "purify" or the like refers to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0082] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0083] A "construct" refers to a macromolecule or complex of molecules containing a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell, where it can replicate and / or express in the target cell. As used herein, the term "vector" includes the delivered construct. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. Major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, etc.

[0084] "Integration" means that one or more nucleotides of a construct are stably inserted into a cell genome, i.e., covalently linked to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that nucleotides of a construct are inserted into the chromosome or mitochondrial DNA of a cell at a preselected site or "integration site." As used herein, the term "integration" also refers to a process involving the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of the endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" can further include replacing the deleted endogenous sequence or nucleotide with one or more inserted nucleotides.

[0085] As used herein, the term "exogenous" is intended to mean that the referenced molecule or referenced activity is introduced into the host cell or is non-native to the host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the host's genetic material, e.g., by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to introducing the encoding nucleic acid into a cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity that is present in the host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0086] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of an appropriate regulatory sequence, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo.A gene of interest or polynucleotide may include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence.For example, a gene of interest may encode miRNA, shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (i.e., a variant of a natural polypeptide that has less than 100% sequence identity with the natural polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0087] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) for thymine. Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotide also refers to both double-stranded and single-stranded molecules.

[0088] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as polypeptides or proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0089] As used herein, the term "subunit" refers to each individual polypeptide chain of a protein complex, where each individual polypeptide chain can form a stable folded structure by itself. Many protein molecules are composed of two or more subunits, and the amino acid sequences of each subunit can be identical, similar, or completely different. For example, the CD3 complex is composed of CD3α, CD3ε, CD3δ, CD3γ, and CD3ζ subunits, which form CD3ε / CD3γ, CD3ε / CD3δ, and CD3ζ / CD3ζ dimers. Within a single subunit, adjacent portions of the polypeptide chain frequently fold into compact, localized, semi-independent units called "domains." Many protein domains further contain independent "structural subunits," also called subdomains, which may contribute to the domain's common function. Thus, as used herein, the term "subdomain" refers to a protein domain within a larger domain, such as a binding domain within the ectodomain of a cell surface receptor, or a stimulatory or signaling domain within the endodomain of a cell surface receptor.

[0090] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it can affect the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operably linked to a regulatory sequence in sense or antisense orientation.

[0091] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to preferential therapeutic attributes in source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" refers to a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can be further differentiated into specific cell types, including any hematopoietic lineage cell. iPSCs derived from source cells and cells differentiated therefrom are sometimes collectively referred to as "derived" or "derived" cells, depending on the context. For example, as used throughout this specification, derived effector cells, or derived NK cells or derived T cells are cells differentiated from iPSCs, compared to their primary counterparts obtained from a natural / natural source, such as peripheral blood, umbilical cord blood, or other donor tissue. As used herein, genetic imprints that confer preferential therapeutic attributes are incorporated into iPSCs either by reprogramming donor-, disease-, or treatment-response-specific selected source cells, or by using genome editing to introduce genetically modified modalities into iPSCs. In embodiments of source cells obtained from specifically selected donors, diseases, or treatment situations, genetic imprints that contribute to preferential therapeutic attributes can include any situation-specific genetic or epigenetic modifications that convey a retainable phenotype, i.e., preferential therapeutic attributes, that are transmitted to iPSC-derived cells of the selected source cells, regardless of whether the underlying molecular events have been identified. Donor-specific, disease-specific, or treatment response-specific source cells may contain genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including, but not limited to, pre-positioned monospecific TCRs, for example, from virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, for example, homozygosity for a point mutation encoding the high-affinity CD16 receptor of the selected donor; predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting population-enriched haplotypes.As used herein, preferential therapeutic attributes include improved engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and modulation, survival, and cytotoxicity of the derived cells. Preferential therapeutic attributes may also be related to the expression of antigen-targeting receptors, HLA presentation or lack thereof, tolerance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity due to reduced extratumoral effects, and resistance to treatments such as chemotherapy.

[0092] As used herein, the term "enhanced therapeutic properties" refers to enhanced therapeutic properties of cells compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response control and regulation, survival, and cytotoxicity. Therapeutic properties of immune cells may also be demonstrated by the expression of antigen-targeting receptors, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity due to reduced extratumoral effects, and resistance to treatments such as chemotherapy.

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

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

[0095] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of cell therapy. In some cases, the expression of a safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells that have permanently incorporated a gene encoding the safety switch protein into their genome. This conditional control can be variable and can include post-translational activation via small molecules and tissue-specific and / or temporal transcriptional regulation. Safety switches can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some cases, safety switch proteins are activated by exogenous molecules, e.g., prodrugs, and upon activation, trigger apoptosis and / or cell death of 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 in the event of an adverse event is activated by the suicide gene product and kills the transduced cells.

[0096] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative, curative, or palliative properties for disease and can be administered to ameliorate, alleviate, reduce, reverse, or mitigate the severity of disease. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the onset of disease or to lessen the severity of such disease or pathological conditions once they appear. Pharmaceutically active proteins include whole proteins or peptides or pharmaceutically active fragments thereof. They also include pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides. The term pharmaceutically active protein also refers to multiple proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0097] As used herein, the term "signaling molecule" refers to any molecule that regulates, is involved in, inhibits, activates, reduces, or increases cell signaling. Signal transduction refers to the transmission of a molecular signal in the form of a chemical modification through the recruitment of protein complexes along a pathway that ultimately leads to biochemical events within the cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, toll gate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

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

[0099] As used herein, the terms "specific" or "specificity" may be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0100] As used herein, the term "adoptive cell therapy," as used herein, refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, specified as T cells or B cells, whether genetically modified or not, that have been expanded in vivo prior to infusion.

[0101] As used herein, a "therapeutically sufficient amount" includes, within its meaning, a non-toxic, but sufficient and / or effective amount of the particular treatment and / or pharmaceutical composition to which it refers to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's general health, the patient's age, and the stage and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, reduce, 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 the addition of stimuli in the culture medium or changes in the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a general and important intermediate for initiating lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development because they give rise to multiple lineages within a three-dimensional area. Throughout the differentiation process, which typically takes hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point (typically days to weeks) they are further processed to continue differentiation. EB formation is initiated by placing pluripotent stem cells in close proximity to each other in a three-dimensional, multilayered cell cluster; this is typically achieved by one of several methods, including sedimenting pluripotent cells into droplets, settling cells into "U"-bottom well plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require further differentiation cues to promote EB development. Therefore, pluripotent stem cell aggregates must be transferred to a differentiation medium that provides cues for induction toward a selected lineage. EB-based culture of pluripotent stem cells typically results in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm germ layers) with moderate proliferation within the EB cell clusters. Although proven to promote cell differentiation, EBs give rise to heterogeneous cells with different differentiation states because the three-dimensional structure of cells is not consistently exposed to differentiation cues from the environment. In addition, EBs are difficult to generate and maintain. Furthermore, EB-mediated cell differentiation involves moderate cell expansion, which also contributes to reduced differentiation efficiency.

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

[0104] As used herein, "monolayer differentiation" refers to a differentiation method that differs from differentiation through three-dimensional multilayered clusters of cells, i.e., "EB formation." Among 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 specific lineages is considered minimal compared to differentiation into all three germ layers in EBs.

[0105] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be enzymatically or mechanically dissociated from each other, such as by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture plate or other surface. Thus, dissociation can involve disruption of cellular interactions with the extracellular matrix (ECM) and the substrate (e.g., the culture surface), or disruption of the ECM between cells.

[0106] As used herein, "feeder cells" or "feeders" are terms used to describe cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimuli, growth factors, and nutrients to support the second cell type. Feeder cells are optionally 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 expansion and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with mitotic inhibitors such as mitomycin to prevent them from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one specific feeder cell type can be human feeder, such as human skin fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEF). Generally, various feeder cells can be partially used to maintain pluripotency, direct differentiation into a specific lineage, enhance proliferation capacity, and promote maturation into specialized cell types, such as effector cells.

[0107] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, that is essentially free of feeder cells or stromal cells and / or is not preconditioned by culturing feeder cells. A "preconditioned" medium refers to a medium that is harvested after feeder cells have been cultured in the medium for a period of time, such as at least one day. A preconditioned medium contains many mediator substances, including growth factors and cytokines, secreted by feeder cells cultured in the medium. In some embodiments, the feeder-free environment does not contain either feeder cells or stromal cells and is not preconditioned by culturing feeder cells.

[0108] When 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" refers to (1) successful knock-in, knock-out, knock-down gene expression, transgenic or controlled gene expression at the genetic level, e.g., inducible or transient expression at a desired developmental stage of the cell, achieved by direct genome editing or modification, or by "transfer" via differentiation or reprogramming from an initially genomically engineered starting cell, or (2) (i) the genetic modification or modification of the resulting gene in the cell by direct genome editing. (ii) modifications of gene expression that are maintained in a cell by "transfer" from an initially genomically engineered starting cell through differentiation or reprogramming thereof; (iii) downstream gene regulation in a cell as a result of modifications of gene expression that are only apparent in the cell's early developmental stages or only in the starting cell that gave rise to the cell through differentiation or reprogramming; or (iv) removal, addition, or alteration of favorable cellular functions / properties at the cellular level through enhanced or newly acquired cellular functions or attributes that are exhibited in the mature cell product originally derived from genome editing or modifications performed on iPSC, progenitor, or dedifferentiated cellular sources.

[0109] "HLA-deficient," including HLA class I-deficient, HLA class II-deficient, or both, refers to any cell that lacks or no longer maintains surface expression of complete MHC complexes comprising HLA class I protein heterodimers and / or HLA class II heterodimers, or has reduced or diminished levels that are lower than those naturally detectable by other cells or synthetic methods.

[0110] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that have been further modified by introducing genes expressing proteins related to improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as, but not limited to, non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3z, 41BBL, CD47, CD113, and PDL1. "Modified HLA-deficient" cells also include cells other than iPSCs.

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

[0112] "Chimeric Fc receptor," abbreviated as CFcR, is a term used to describe engineered Fc receptors in which their native transmembrane and / or intracellular signaling domains have been modified or replaced with non-native transmembrane and / or intracellular signaling domains. In some chimeric Fc receptor embodiments, in addition to one or both of the transmembrane and signaling domains being non-native, one or more stimulatory domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance receptor-induced cell activation, expansion, and function. Unlike chimeric antigen receptors (CARs), which contain an antigen-binding domain for a target antigen, chimeric Fc receptors bind to an Fc fragment, or the Fc region of an antibody, or the Fc region contained in an engager or binding molecule, and activate cells regardless of whether the targeted cell is in close proximity. For example, Fcγ receptors can be engineered to contain selected transmembrane, stimulatory, and / or signaling domains within the intracellular region, which respond to IgG binding at the extracellular domain, thereby generating CFcR. In one example, CFcR is generated by engineering the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domain. To further improve the binding affinity of CD16-based CFcR, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcR with a high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is eliminated or the extracellular domain of the receptor is replaced so that it is not cleavable, i.e., is not susceptible to shedding, thereby resulting in 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 facilitating antibody-dependent cellular cytotoxicity (ADCC). As used herein, "high-affinity CD16," "uncleavable CD16," or "high-affinity uncleavable CD16 (hnCD16)" refers to natural or non-natural variants of CD16. Wild-type CD16 has low affinity and undergoes ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes upon NK cell activation. F176V and F158V are exemplary high-affinity CD16 polymorphic variants. CD16 variants in which the cleavage site (positions 195-198) in the membrane-proximal region (positions 189-212) has been modified or eliminated do not undergo shedding. The cleavage site and membrane-proximal region are described in detail in WO2015 / 148926, the complete disclosure of which is incorporated herein by reference. The CD16 S197P variant is an engineered, non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high-affinity and non-cleavable. Another exemplary high-affinity, non-cleavable CD16 (hnCD16) variant is an engineered CD16 containing an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0114] I. Cells and Compositions Useful for Adoptive Cell Therapy with Enhanced Properties Provided herein is a strategy for systematically manipulating the regulatory circuits of clonal iPSCs without affecting the differentiation potential of iPSCs or the cellular developmental biology of the iPSCs and their derivatives, while enhancing the therapeutic properties of derived cells differentiated from iPSCs. iPSC-derived cells are functionally improved and suitable for adoptive cell therapy after a combination of selective modalities is introduced into the cells at the iPSC level through genomic manipulation. Prior to the present invention, it was unclear whether modified iPSCs containing one or more provided gene edits would still have the ability to enter cellular development and / or mature and generate functional differentiated cells while retaining regulated activity. Unexpected failures during directed cell differentiation from iPSCs have been attributed to a number of factors, including, but not limited to, developmental stage-specific gene expression or lack thereof, the requirement for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure the differentiation protocol to allow for changes in cell phenotype and / or function. The present application has demonstrated that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from the individual or combined genomic modifications retained in the effector cells following iPSC differentiation.

[0115] 1. CD3 Surface Presentation in the Absence of Endogenous TCR The alpha-beta T cell receptor (αβTCR) is an antigen-specific receptor essential for immune responses and is present on the cell surface of αβT lymphocytes. Binding of the TCRαβ to peptide-major histocompatibility complex (pMHC) triggers TCR-CD3 intracellular activation, recruits numerous signaling molecules, and initiates signaling pathway branching and integration, resulting in the recruitment of transcription factors critical for gene expression and T cell development and function. NKT cells, a subset of T cells that also express the αβTCR, differ from conventional αβT cells in that their TCRs are composed of a standard invariant TCRα chain (Vα24-Jα18 in humans) and a TCRβ chain (Vβ11 in humans) that utilizes a restricted Vβ segment, limiting diversity and recognizing a limited number of lipid antigens presented by CD1d.

[0116] Disrupting the constant region of TCR alpha or TCR beta (TRAC or TRBC) either by direct editing of T cells or by editing iPSCs as a source to obtain modified derivative T cells allows for the development of TCR neg One approach to producing T cells is to disrupt (or in this example, truncate) TRAC or TRBC and generate TCR-negative cells (TCR) by inserting a 2A sequence operably linked to either the endogenous or exogenous promoter of TRAC or TRBC at a preselected location in TRAC or TRBC. neg In certain embodiments, TCR neg In another embodiment, the cells are iPSCs. neg The cells are NK lineage cells. As used herein, "TCR negative" or "TCR negThe term " refers to the lack of expression of an endogenous TCR, either due to disruption of TCR gene expression (e.g., T lineage cells, primary or iPSC-derived T lineage cells, etc.) or the natural lack of TCR gene expression despite the presence of a TCR locus in the genome (e.g., iPSCs, or NK lineage cells, primary or iPSC-derived NK lineage cells). Subsequent directed differentiation of clonally selected engineered iPSCs into hematopoietic cells allows for the generation of iPSC-derived immune effector cells and / or homogenous populations thereof without TCR expression.

[0117] In some embodiments, targeted cleavage or disruption using a self-cleaving peptide such as 2A can optionally occur concomitantly with integration of one or more exogenous genes of interest at the cleavage or disruption site, and expression of the integrated genes can be driven by an operably linked exogenous promoter or, upon integration, by the endogenous promoter of TCR alpha or TCR beta, resulting in a TRAC or TRBC knockout and thus TCR negativity, while expressing one or more exogenous genes inserted into the TRAC or TRBC locus.

[0118] iPSC-derived TCR-negative T cells obtained using this approach (with or without exogenous gene incorporation) can be used in allogeneic adoptive cell therapy without the need for HLA matching, reduce alloreactivity, and prevent graft-versus-host disease (GvHD). However, TCR blockage has also been shown to eliminate the CD3 signaling complex from the T cell surface despite intracellular expression of the CD3 subunit gene. The lack of cell-surface CD3 can alter cell expansion and / or viability and reduce the functional potential of the cells due to incompatibility with technologies that require cell-surface CD3 recognition and binding, including, but not limited to, BiTE, BiKE, or TRiKE (or collectively referred to as engager) technologies, CD3 / CD28 T cell activation bead technologies, and anti-CD3 antibody or CD3-CAR stimulation technologies. Furthermore, TCR blockage can alter cell expansion and / or viability and reduce the functional potential of the cells. negWhen iPSCs are used for directed T cell differentiation, it may also affect T cell developmental biology and T cell functional maturation. However, overexpression of CD3 in TCR-negative cells does not appear to restore cell surface presentation of the CD3 complex and / or CD3 signaling. Cells that do not express TCR despite the presence of the TCR gene, e.g., TCR neg With respect to the NK cells or NK precursor cells disclosed herein, the cell surface CD3 complex, or one or more subunits or subdomains thereof (cs-CD3), can function as an acquired CD3-associated cell surface triggering receptor for binding with molecules including, but not limited to, antibodies or functional variants thereof, and / or bi- or multispecific engagers that recognize the CD3 receptor. To address unresolved problems in the art, among other provided advantages described herein, the present invention provides NK cells or NK precursor cells that lack endogenous TCR expression (TCR), whether due to loss of endogenous TCR gene expression by genetic disruption (such as in derived T lineage cells) or natural lack of TCR gene expression (such as in derived NK lineage cells). neg In the case of IgG1A-IgG2A cells, the following designs are detailed in the present application, which are shown in Figures 1A-C, to reconstitute and / or provide surface CD3 presentation.

[0119] Design 1: Non-binding recombinant TCR (nb-rTCR) As shown in Figure 1(a), in this design 1, a targeted genome editing tool is used to knock out endogenous TCRα in cells (TCRα - / - ), TCR negative (TCR neg ), whereas knockout of TCRβ (TCRβ - / - ) is optional, or vice versa, and targeted genome editing tools can be used to knock out endogenous TCRβ in cells to produce (TCRβ - / - ), TCR negative (TCR neg ), whereas knockout of TCRα (TCRα - / -) is optional. In embodiments involving a TCRα knockout, a polynucleotide encoding the full-length or partial-length constant region of TCRα (transgenic TRAC, or tgTRAC) is then introduced into the cell or, upon targeted TRAC knockout, is integrated into TRAC, and expression of the polynucleotide is driven by the endogenous promoter of TCRα, or alternatively, by an exogenous promoter operably linked to the polynucleotide. In some embodiments, the polynucleotide encoding the full-length or partial-length constant region of TCRα further comprises an appropriate N-terminal signal peptide associated with the full-length or partial-length constant region of TCRα. Endogenous TCRβ (TCRβ - / - In embodiments in which the TCRβ full-length or partial-length constant region is knocked out, a polynucleotide encoding the full-length or partial-length constant region of TCRβ (tgTCRβ or tgTRBC) is introduced into the cell, and expression of tgTCRβ or tgTRBC is driven by the endogenous promoter of TCRβ, or alternatively, by an exogenous promoter. In some embodiments, the polynucleotide encoding the full-length or partial-length constant region of TCRβ further comprises an appropriate N-terminal signal peptide linked to the full-length or partial-length constant region of TCRβ. In some embodiments, the exogenous promoter comprises a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In some embodiments, the exogenous promoter comprises one of CMV, EF1α, PGK, CAG, and UBC. In one embodiment, the exogenous promoter comprises at least CAG.

[0120] In some embodiments, a polynucleotide encoding a complete or partial TCR alpha constant region (tgTRAC) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 1. In some embodiments, a polynucleotide encoding a TCR beta comprising at least a complete or partial constant region (tgTRBC) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments of polynucleotides encoding an N-terminal signal peptide and a full-length or partial-length TCR alpha or TCR beta constant region, the polynucleotide further comprises a linker peptide between the signal peptide and the sequence associated with the TCR constant region. In some embodiments of polynucleotides encoding an N-terminal signal peptide and a full-length TCR α or TCR β constant region, the polynucleotide further comprises a poly-A tail at the C-terminus. In some embodiments of polynucleotides encoding an N-terminal signal peptide and a partial-length TCR α or TCR β constant region, integration of the polynucleotide is at a site within the endogenous constant region (e.g., an exon) and is in-frame, i.e., in-frame with the remaining endogenous sequence of the TCR α or TCR β constant region downstream of the integration site, such that a full-length transgenic / chimeric TRAC or TRBC is formed with one portion of that sequence being exogenous / transgenic and another portion being endogenous. In some embodiments of Design 1, at least one of the endogenous TCR α and TCR β is engineered to essentially remove its respective variable region while presenting its respective transgenic constant region on the cell surface upon expression. In some embodiments, only one of the endogenous TCRα and TCRβ is engineered to display a transgenic constant region and a wild-type TCR subunit (TCRα or TCRβ) on the cell surface, while essentially removing the associated variable region.In some embodiments, both endogenous TCRα and TCRβ are engineered to remove their respective variable regions while displaying both transgenic constant regions on the cell surface upon expression, as provided. Exemplary N-terminal signal peptides include MALPVTALLLPLALLLHA (SEQ ID NO: 4; CD8asp) or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 5; IgKsp), or any signal peptide sequence known in the art or functional variants thereof. Exemplary linker peptides include DYKDDDDK (SEQ ID NO: 6; FLAG), or any linker peptide sequence known in the art or functional variants thereof. SEQ ID NO:1: [Sequence Table 1] TIFF0007783746000001.tif32169 [Sequence Table 2] TIFF0007783746000002.tif26167 SEQ ID NO: 3 [Sequence Table 3] TIFF0007783746000003.tif26167

[0121] In the present application, we have discovered that the transgenic constant regions of either the TCR subunits tgTRAC or tgTRBC (which can form a recombinant TCR complex (rTCR) by associating with other TCR subunits (endogenous / wild-type or transgenic; in the transgenic case, with or without their respective variable regions)) and the endogenous CD3 subunit do not allow peptide-MHC binding due to the lack of the TCRα or TCRβ variable regions involved in antigen recognition. The resulting cells regain canonical TCR / CD3 signaling through cell surface-presenting endogenous CD3 (cs-CD3) complexes, but are not alloreactive due to the endogenous TCR knockout and rTCR lacking the TCRα variable region. Thus, in view of Design 1, cells or populations thereof are provided herein, wherein the cells are iPSCs, clonal iPSCs, clonal iPS cell line cells, or derivative cells obtained by differentiation of said iPSCs, and the cells are derived from iPSCs in which the endogenous TCR is knocked out (TCRneg The recombinant TCR complex comprises disruption of at least one of the endogenous TCRα and TCRβ constant regions, such as tgTRAC and / or tgTRBC, and one or both exogenous polynucleotides encoding the disrupted TCRα (tgTRAC) and / or TCRβ (tgTRBC) constant regions, whereby tgTRAC and / or tgTRAB enable cell surface presentation of endogenous CD3 (cs-CD3). Recombinant TCR complexes containing at least one of tgTRAC and tgTRBC do not bind to antigenic peptides presented by MHC because they lack both variable regions (Vα and Vβ) of the TCR subunits, and are therefore referred to as non-binding recombinant TCRs (nb-rTCRs).

[0122] Design 2: Defined recombinant TCR (d-rTCR) As shown in Figure 1A, in this design 2, both endogenous TCRα and TCRβ were knocked out in cells using genome editing tools (TCRα - / - and TCRβ - / - or TCR neg TCRβ neg ), TCR neg Concurrently with or following the TCR knockout, a first polynucleotide encoding a TCR α (tgTCRα) comprising a defined variable region of TCR α and a complete or partial constant region, and a second polynucleotide encoding a TCR β (tgTCRβ) comprising a defined variable region of TCR β, and a complete or partial constant region, are inserted into the TCR cells. negThe defined TCR alpha or TCR beta variable region is introduced into a cell. The defined TCR alpha or TCR beta variable region can be of any given specificity, as long as its sequence is specified or can be specified. In some embodiments, one or both of the first and second polynucleotides are driven by the endogenous promoters of TCR alpha and TCR beta, respectively. In some other embodiments, one or both of the first and second polynucleotides are driven by an exogenous promoter. In some embodiments, the second polynucleotide is driven by the endogenous promoter of TCR beta, while in some other embodiments, the second polynucleotide is driven by an exogenous promoter. In some embodiments, the exogenous promoter comprises a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In some embodiments, the exogenous promoter comprises one of CMV, EF1 alpha, PGK, CAG, and UBC. In one embodiment, the exogenous promoter comprises at least CAG. In some embodiments, polynucleotides encoding full-length or partial-length TCR alpha constant regions and given defined variable regions comprise sequences that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 1. In some embodiments, polynucleotides encoding full-length or partial-length TCR beta constant regions and given defined variable regions comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 2 or SEQ ID NO: 3. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. In some embodiments of the polynucleotide encoding the full-length TCR alpha or TCR beta constant region, the polynucleotide further comprises a poly-A tail at the C'-terminus.In some embodiments of polynucleotides encoding partial-length TCR α or TCR β constant regions, integration of the polynucleotide is at a site within the endogenous constant region and is in-frame with the remaining endogenous sequence of the TCR α or TCR β constant region downstream of the integration site, such that a full-length transgenic / chimeric TRAC or TRBC is formed with part of that sequence being exogenous / transgenic and another part being endogenous. Sequences of TCR α or TCR β variable regions can be found, for example, in the Universal Protein Resource (UniProt) database, and some non-limiting examples of defined TCR α or TCR β variable regions are listed below in Tables A and B, respectively. [Table 1] [Table 2] [Table 3] [Table 4]

[0123] Invariant NKT cells are a unique subset of T cells that express a canonical invariant TCR α chain (Vα24-Jα18 in humans, or iTCRα) and a TCR β chain that uses a restricted Vβ segment (Vβ11 in humans, or iTCRβ), resulting in a highly conserved TCR and CD1d-dependent antigen presentation. To take advantage of this property of invariant NKT TCRs (iTCRs or iTCRαβ), in some embodiments of Design 2, defined TCRs are constructed such that polynucleotides encoding full-length or partial-length TCR α constant regions and given defined variable regions have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to the exemplary sequence SEQ ID NO:44. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. SEQ ID NO: 44 [Sequence Table 4] TIFF0007783746000008.tif26169 SEQ ID NO: 45 [Sequence Table 5] TIFF0007783746000009.tif31160

[0124] In the present application, it has been discovered that transgenic TCRα (tgTCRα) having a constant region and a defined variable region, and optionally transgenic TCRβ (tgTCRβ) having a constant region and a defined variable region, can form a recombinant TCR complex (rTCR) by associating with an endogenous CD3 subunit comprising the CD3ζ chain, with or without peptide-MHC binding, depending on the specificity of the variable regions of the tgTCRα and tgTCRβ. In addition to genetic engineering of transgenic TCR subunits for defined recombinant TCRs, other approaches utilizing the TCRα and TCRβ of invariant NKT cells include reprogramming isolated NKT cells into iPSCs and differentiating the iPSCs into T cells, such that the derived T cells contain the TCRα and TCRβ of invariant NKT cells (iTCRα, iTCRβ, and iTCR complex) using the reprogramming and differentiation compositions and methods disclosed herein. The resulting cells differentiated from genetically engineered iPSCs or iNKT-reprogrammed iPSCs have no or known, defined MHC-binding specificity, but regain canonical TCR / CD3 signaling through cell surface-presenting endogenous CD3 (cs-CD3). Thus, in consideration of Design 2, provided herein are cells or populations thereof, wherein the cells are iPSCs, clonal iPSCs, clonal iPS cell line cells, or derivative cells obtained by differentiation of iPSCs, and the cells comprise an exogenous polynucleotide encoding a tgTCRα with a complete or partial constant region and a defined variable region, and an exogenous polynucleotide encoding a tgTCRβ with a complete or partial constant region and a defined variable region, respectively, disrupting endogenous TCRα and endogenous TCRβ, and wherein the endogenous CD3 molecule, when expressed, is present on the cell surface (cs-CD3).

[0125] Design 3: Recombinant pre-TCRα (p-rTCR) with optional non-binding TCRβ Pre-TCRα is a type I transmembrane receptor protein encoded by a developmental control gene in immature thymocytes, the early stage of T cell development. Pre-TCRα covalently binds to TCRβ and CD3 subunits to form the pre-TCR complex. Among other structural and functional differences, pre-TCRα possesses a relatively long cytoplasmic tail compared to the TCRα chain. As shown in Figure 1A, in this design, TCR-negative cells can be generated by using genome editing tools to generate at least one TCRα knockout (TCRα) gene. neg ), with the knockout of endogenous TCRβ optional. Concurrently with or following the TCR knockout, a first polynucleotide encoding a full-length or partial-length pre-TCRα (tgpTCRα) is inserted into the TCR neg The TCR is introduced into the cell. neg In some embodiments, where the cells further comprise a TCRβ knockout, the second polynucleotide encoding a complete or partial TCRβ constant region, with or without a given defined variable region (tgTCRβ or tgTRBC), is a TCRβ knockout. neg In some embodiments of polynucleotides encoding a full-length TCR α or TCR β constant region, the polynucleotide further comprises a poly-A tail at the C'-terminus. In some embodiments of polynucleotides encoding a partial-length TCR α or TCR β constant region, integration of the polynucleotide is at a site within the endogenous constant region and is in-frame with the remaining endogenous sequence of the TCR α or TCR β constant region downstream of the integration site, such that a full-length transgenic / chimeric TRAC or TRBC is formed with part of that sequence being exogenous / transgenic and another part being endogenous.

[0126] In some embodiments, a first polynucleotide encoding a full-length or partial-length pre-TCR alpha (tgpTCR alpha) is operably linked to an endogenous promoter of TCR alpha upon integration. In some embodiments, the first polynucleotide encoding a full-length or partial-length pre-TCR alpha (tgpTCR alpha) is driven by an exogenous promoter. In some embodiments, a second polynucleotide encoding a full or partial TCR beta constant region, with or without a given defined variable region, is operably linked to an endogenous promoter of TCR beta upon integration. In some embodiments, the second polynucleotide encoding a full or partial TCR beta constant region, with or without a given defined variable region, is driven by an exogenous promoter. In some embodiments, the exogenous promoter comprises a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In some embodiments, the exogenous promoter comprises one of CMV, EF1 alpha, PGK, CAG, and UBC. In one embodiment, the exogenous promoter comprises at least CAG. In some embodiments, a polynucleotide encoding tgpTCRα comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to the exemplary sequence SEQ ID NO:23. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. In some embodiments, a polynucleotide encoding tgpTCRα comprises partial-length SEQ ID NO:23, which is represented herein as SEQ ID NO:24. In some embodiments of polynucleotides encoding tgpTCRα or any functional variant thereof comprising full-length or partial-length SEQ ID NO:23, the encoded tgpTCRα further comprises a signal peptide known in the art. One non-limiting exemplary signal peptide comprises the peptide represented by SEQ ID NO:22. SEQ ID NO: 22 [Sequence Table 6] TIFF0007783746000010.tif555 sequence number 23 [Sequence Table 7] TIFF0007783746000011.tif31162 SEQ ID NO: 24 [Sequence Table 8] TIFF0007783746000012.tif33169

[0127] In some embodiments, a polynucleotide encoding a TCR β comprising a complete or partial constant region and a given defined variable region comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to exemplary sequences SEQ ID NO:2 or SEQ ID NO:3. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. The defined TCR β variable region can be of any given specificity, such that its sequence has been or can be identified. Non-limiting defined TCR β variable regions are exemplified in Table B above and are contained in SEQ ID NO:45 (underlined).

[0128] It was previously unclear whether iPSCs with pre-TCRα expression controlled by a promoter (exogenous or endogenous TCRα promoter) different from the native promoter (i.e., the pre-TCRα promoter) still have the ability to differentiate into functional effector T cells. Here, we sought to show that the cellular developmental biology of iPSCs containing transgenic pre-TCRα (tgpTCRα) controlled by a non-native promoter can be maintained to the extent that directed differentiation into iPSC-derived T cells can be performed to generate functional T cells. This is surprising because normally, the expression of endogenous pre-TCRs is developmentally controlled. Furthermore, the tgpTCRα TCR negAlthough iPSC-derived T cells lack peptide-MHC binding capacity, they contain a surface recombinant pre-TCR complex (rpTCR) expressed by associating with endogenous CD3 subunits containing the CD3ζ chain. Without being limited by theory, the transgenic pre-TCR / CD3 complex may nonetheless drive the maturation of iPSC-derived T cells through standard CD3 signaling via the cell surface-presented endogenous CD3 (cs-CD3) complex. In light of the above, the scope of the present application also includes various methods for upregulating and / or preventing downregulation of endogenous pre-TCRα. Overexpressed pre-TCRα in cells other than early / immature thymocytes associates with expressed endogenous TCRβ and CD3 subunits to enable CD3 cell surface presentation, despite the lack of peptide-MHC binding capacity.

[0129] Thus, in consideration of Design 3, provided herein are cells or populations thereof, wherein the cells are iPSCs, clonal iPSCs, clonal iPS cell line cells, or derivative cells obtained from differentiation of said iPSCs, wherein the cells have an endogenous TCR knocked out (TCR neg ), and an exogenous polynucleotide encoding a peptide comprising at least full-length or partial-length pre-TCRα, wherein expression of pre-TCRα in the absence of TCRα associates with endogenous or transgenic TCRβ in cells, resulting in reconstitution of the cell surface CD3 complex (cs-CD3), but also contributes to the directed differentiation of iPSCs into functional derived effector cells, including T cells.

[0130] Design 4: Nonbinding recombinant TCR-anchored CD3 (nb-rTCR-CD3) As shown in FIG. 1B, in this design 4, one or both of the endogenous TCR α and endogenous TCR β are knocked out in cells using genome editing tools (TCR neg ;TCRα - / - and TCRβ - / - Concurrently with or following TCR knockout, an exogenous polynucleotide may be inserted into the TCR negThe polynucleotides introduced into the cells contain a first polynucleotide encoding a recombinant TCRα comprising a TCRα constant region, a full-length or partial-length ectodomain of CD3ε, and one of CD3δ and CD3γ, and / or a second polynucleotide encoding a recombinant TCRβ comprising a TCRβ constant region, a full-length or partial-length ectodomain of CD3ε, and one of CD3δ and CD3γ that is not contained in the recombinant TCRα, so that one heterodimer between CD3ε and CD3δ encoded by one polynucleotide and / or another heterodimer between CD3ε and CD3γ encoded by another polynucleotide can be formed on the cell surface.

[0131] In some embodiments, the recombinant TCR α comprises a complete or partial TCR α constant region at its C-terminus fused to full-length or partial-length CD3ε and CD3δ ectodomains at its N-terminus (tgCD3(ε-δ)-TRAC). In some embodiments, the recombinant TCR α comprises a complete or partial TCR α constant region at its C-terminus fused to full-length or partial-length CD3ε and CD3γ ectodomains at its N-terminus (tgCD3(ε-γ)-TRAC). In some embodiments, the recombinant TCR β comprises a complete or partial TCR β constant region at its C-terminus fused to full-length or partial-length CD3ε and CD3γ ectodomains at its N-terminus (tgCD3(ε-γ)-TRBC). In some embodiments, the recombinant TCR β comprises a complete or partial TCR β constant region at its C-terminus fused to full-length or partial-length CD3ε and CD3δ ectodomains at its N-terminus (tgCD3(ε-δ)-TRBC). In some embodiments of polynucleotides encoding full-length TCR alpha or TCR beta constant regions, the polynucleotide further comprises a poly-A tail at the C-terminus. In some embodiments of polynucleotides encoding partial-length TCR alpha or TCR beta constant regions, integration of the polynucleotide is at a site within the respective endogenous constant region and is in-frame with the remaining endogenous sequence of the TCR alpha or TCR beta constant region downstream of the integration site, such that a full-length transgenic / chimeric TRAC or TRBC is formed with part of that sequence being exogenous / transgenic and another part being endogenous.

[0132] In some other embodiments in which both the first and second polynucleotides are introduced into a cell, the recombinant TCR α is encoded by a first polynucleotide comprising tgCD3(ε-δ)-TRAC and the recombinant TCR β is encoded by a second polynucleotide comprising tgCD3(ε-γ)-TRBC, or the recombinant TCR α is encoded by a first polynucleotide comprising tgCD3(ε-γ)-TRAC and the recombinant TCR β is encoded by a second polynucleotide comprising tgCD3(ε-δ)-TRBC. Thus, in such embodiments, one heterodimer between CD3ε and CD3δ encoded by one polynucleotide and another heterodimer between CD3ε and CD3γ encoded by another polynucleotide can form on the cell surface.

[0133] In some embodiments in which only one of the first and second polynucleotides is introduced into a cell, the other TCR subunit is either wild-type / endogenous or engineered to contain only the constant region with its endogenous variable region removed, either replaced or not replaced with a defined variable region: for example, tgTRAC or tgTRBC (without variable regions) of Design 1 in Figure 1A, or tgTCRα or tgTCRβ (with defined variable regions) of Design 2 in Figure 1A. Thus, as shown in Design 4 in Figure 1B, in one embodiment in which a first polynucleotide containing tgCD3(ε-δ)-TRAC is introduced into a cell to provide a recombinant TCR α subunit, another polynucleotide containing tgTRBC or tgTCRβ is also introduced into the cell to provide a recombinant TCR β subunit. Thus, in this embodiment, one heterodimer between endogenous CD3ε and endogenous CD3γ, and another heterodimer between CD3ε and CD3δ encoded by a polynucleotide comprising tgCD3(ε-δ)-TRAC, can be formed on the cell surface. In yet another embodiment of Design 4 of Figure 1B, a second polynucleotide comprising tgCD3(ε-γ)-TRBC is introduced into the cell to provide a recombinant TCR β subunit, and another polynucleotide comprising tgTRAC or tgTCRα is also introduced into the cell to provide a recombinant TCR α subunit, such that one heterodimer between endogenous CD3ε and endogenous CD3δ, and another heterodimer between CD3ε and CD3γ encoded by a polynucleotide comprising tgCD3(ε-γ)-TRBC, can be formed on the cell surface.

[0134] In some embodiments, the first polynucleotide is driven by the endogenous promoter of TCR α, while in some other embodiments, the first polynucleotide is driven by an exogenous promoter. In some embodiments, the second polynucleotide is driven by the endogenous promoter of TCR β, while in some other embodiments, the second polynucleotide is driven by an exogenous promoter. In some embodiments, the exogenous promoter of either the recombinant TCR α or the recombinant TCR β comprises a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In some embodiments, the exogenous promoter comprises one of CMV, EF1α, PGK, CAG, and UBC. In one embodiment, the exogenous promoter comprises at least CAG. In some embodiments, the polynucleotide encoding the TCR alpha constant region comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the TCR beta constant region comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, a polynucleotide encoding a full-length or partial-length CD3ε ectodomain comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 25. In some embodiments, a polynucleotide encoding a full-length or partial-length CD3δ ectodomain comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 26.In some embodiments, a polynucleotide encoding a full-length or partial-length CD3γ ectodomain comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to exemplary sequence SEQ ID NO: 27. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. In some embodiments of a polynucleotide encoding a full-length or partial-length CD3ε, CD3δ, or CD3γ ectodomain, the polynucleotide further comprises a nucleic acid encoding a signal peptide. In some embodiments, the signal peptide comprises one of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or any other signal peptide known in the art. In some embodiments of a polynucleotide encoding a full-length or partial-length CD3ε ectodomain, the polynucleotide further comprises a nucleic acid encoding a signal peptide of SEQ ID NO: 28. In some embodiments of a polynucleotide encoding a full-length or partial-length CD3δ ectodomain, the polynucleotide further comprises a nucleic acid encoding a signal peptide of SEQ ID NO: 29. In some embodiments of a polynucleotide encoding a full-length or partial-length CD3γ ectodomain, the polynucleotide further comprises a nucleic acid encoding a signal peptide of SEQ ID NO: 30. SEQ ID NO: 25 [Sequence Table 9] TIFF0007783746000013.tif19169 SEQ ID NO: 26 [Sequence Table 10] TIFF0007783746000014.tif20168 SEQ ID NO: 27 [Sequence Table 11] TIFF0007783746000015.tif19168 SEQ ID NO: 28 [Sequence Table 12] TIFF0007783746000016.tif653 SEQ ID NO: 29 [Sequence Table 13] TIFF0007783746000017.tif450 SEQ ID NO:30 [Sequence Table 14] TIFF0007783746000018.tif452

[0135] In some embodiments of a polynucleotide encoding a tgCD3(ε-δ)-TRAC fusion protein, the polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to exemplary sequence SEQ ID NO: 31, wherein each of the two linker sequences (SEQ ID NO: 33 and SEQ ID NO: 34) contained in SEQ ID NO: 31 can be replaced with any known in the art. In some embodiments of a polynucleotide encoding a tgCD3(ε-γ)-TRBC fusion protein, the polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween identity to the exemplary sequence SEQ ID NO: 32, and each of the two linker sequences (SEQ ID NO: 33 and SEQ ID NO: 34) contained in SEQ ID NO: 32 can be replaced with any known in the art. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. In some embodiments of the recombinant TCR α or TCR β fusion proteins provided herein, the fusion protein further comprises a signal peptide known in the art. One non-limiting exemplary signal peptide comprises the peptide represented by SEQ ID NO: 28. SEQ ID NO: 31 [Sequence Table 15] TIFF0007783746000019.tif42170 SEQ ID NO: 32 [Sequence Table 16] TIFF0007783746000020.tif57168 [Sequence Table 17] TIFF0007783746000021.tif463 SEQ ID NO: 34 [Sequence Table 18] TIFF0007783746000022.tif537

[0136] In the present application, we have discovered that TCRα or TCRβ constant regions fused to CD3ε and one of CD3δ and CD3γ ectodomains can associate with transgenic TCRβ or TCRα constant regions, with or without the fused CD3ε and one of CD3δ and CD3γ ectodomains, to form CD3ε / CD3δ and CD3ε / CD3γ heterodimers. The associated transgenic TCRα and TCRβ subunits can further associate with endogenous CD3ζ to support cell surface expression of CD3 ectodomain (cs-CD3) and signaling through endogenous CD3ζ, although without peptide-MHC binding potential. Thus, in consideration of Design 4, there is provided herein a cell or population thereof, wherein the cell is an iPSC, a clonal iPSC, a clonal iPS cell line cell, or a derived cell obtained by differentiation of said iPSC, wherein the cell is a first exogenous polynucleotide encoding a tgTCRα comprising an interruption with each of an endogenous TCRα constant region and an endogenous TCRβ constant region, and a fused complete or partial TCRα constant region, and a complete or partial ectodomain of one of CD3ε and CD3δ and CD3γ (tgCD3(ε-δ / γ)-TRAC), and a fused complete or partial TCRβ constant region. and at least one of a second exogenous polynucleotide (tgCD3(ε-γ / δ)-TRBC) encoding a tgTCRβ containing CD3ε and a complete or partial ectodomain of one of CD3δ and CD3γ, wherein the ectodomain of the CD3 subunit is present on the cell surface (cs-CD3) when expressed; when the cell contains only the first exogenous polynucleotide, the cell further comprises a tgTRBC or tgTCRβ provided herein; and when the cell contains only the second exogenous polynucleotide, the cell further comprises a tgTRAC or tgTCRα provided herein.

[0137] Design 5: CD3 chimeric chain (ccCD3) As shown in Figure 1B, in this design 5, the cell surface-displayed CD3 (cs-CD3) is in the form of a CD3 chimeric chain (ccCD3), which is constructed to include a full-length or partial-length CD3ε ectodomain, a full-length or partial-length ectodomain of either CD3γ or CD3δ, and a full-length or partial-length endodomain of CD3ζ containing at least one ITAM (immunoreceptor tyrosine-based activation motif). Cells containing a polynucleotide encoding the CD3 chimeric chain may further include disruption of either or both endogenous TCRα and TCRβ. Using genome editing tools, targeted editing of TCRα and / or TCRβ can be performed to enhance the expression of TCRα and TCRβ. neg When generating cells, at least one polynucleotide encoding the CD3 chimeric chain is introduced into the cells simultaneously with or subsequent to TCR knockout. In some embodiments, the polynucleotide is introduced into TRAC or TRBC and driven by the endogenous promoter of TCRα or TCRβ, respectively, while in some other embodiments, the introduced polynucleotide is driven by an exogenous promoter. In some embodiments, the exogenous promoter comprises a constitutive, inducible, time-specific, tissue-specific, or cell type-specific promoter. In some embodiments, the exogenous promoter comprises one of CMV, EF1α, PGK, CAG, and UBC. In one embodiment, the exogenous promoter comprises at least CAG.

[0138] In some embodiments, the CD3 chimeric chain comprises a full-length or partial-length CD3ε ectodomain, a full-length or partial-length CD3γ ectodomain, and a full-length or partial-length CD3ζ endodomain containing at least one ITAM (tgCD3(ε-γ)-ζ), where the CD3 chimeric chain is a fusion protein having either ectodomain at its N-terminus, and the two ectodomains form a heterodimer. In some embodiments, the CD3 chimeric chain comprises a full-length or partial-length CD3ε ectodomain, a full-length or partial-length CD3δ ectodomain, and a full-length or partial-length CD3ζ endodomain containing at least one ITAM (ITAM(tgCD3(ε-δ)-ζ)), where the CD3 chimeric chain is a fusion protein having either ectodomain at its N-terminus, and the two ectodomains form a heterodimer. In some embodiments of the CD3 chimeric chain, the internal domain of CD3 zeta comprises two ITAMs, and in some embodiments of the CD3 chimeric chain, the internal domain of CD3 zeta comprises all three ITAMs.

[0139] In some embodiments, a polynucleotide encoding a full-length or partial-length CD3ε ectodomain comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 25. In some embodiments, a polynucleotide encoding a full-length or partial-length CD3δ ectodomain comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween, when compared to exemplary sequence SEQ ID NO: 26. In some embodiments, polynucleotides encoding full-length or partial-length CD3γ ectodomains comprise a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to exemplary sequence SEQ ID NO: 27. In some embodiments of polynucleotides encoding full-length or partial-length CD3ε, CD3δ, or CD3γ ectodomains, the polynucleotide further comprises a nucleic acid encoding a signal peptide. In some embodiments, the signal peptide comprises one of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, or any other signal peptide known in the art. In some embodiments of polynucleotides encoding full-length or partial-length CD3ε ectodomains, the polynucleotide further comprises a nucleic acid encoding the signal peptide of SEQ ID NO: 28. In some embodiments of polynucleotides encoding full-length or partial-length CD3δ ectodomains, the polynucleotide further comprises a nucleic acid encoding the signal peptide of SEQ ID NO: 29. In some embodiments of the polynucleotide encoding a full-length or partial-length CD3γ ectodomain, the polynucleotide further comprises a nucleic acid encoding the signal peptide of SEQ ID NO:30.In some embodiments, a polynucleotide encoding a full-length or partial-length CD3ζ endodomain comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity to an exemplary sequence, SEQ ID NO: 35, including CD3ζ ITAM1, ITAM2, and ITAM3 (SEQ ID NOs: 36-38). In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. SEQ ID NO: 35 [Sequence Table 19] TIFF0007783746000023.tif26168 SEQ ID NO: 36 [Sequence Table 20] TIFF0007783746000024.tif569 SEQ ID NO: 37 [Sequence Table 21] TIFF0007783746000025.tif567 SEQ ID NO: 38 [Sequence Table 22] TIFF0007783746000026.tif569

[0140] In some embodiments of the CD3 chimeric chain, the internal domain of CD3 zeta containing at least one, two, or three ITAMs further comprises one or more signaling domains for signal transduction and / or costimulation: 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3 zeta, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3 zeta 1XX, CS1, or CD8. In one embodiment of the CD3 chimeric chain, the internal domain of CD3 zeta containing at least one, two, or three ITAMs further comprises at least the signaling domain of CD28 (tgCD3(ε-γ / δ)-28 zeta). In some embodiments of a CD3ζ endodomain comprising the signaling domain of CD28, a polynucleotide encoding a full-length or partial-length 28ζ endodomain comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to the exemplary sequence SEQ ID NO: 39, from which any one or two CD3ζ ITAMs may be removed. In some embodiments of a CD3 chimeric chain, the endodomain of CD3ζ comprising at least one, two, or three ITAMs further comprises the signaling domain of 41BB (tgCD3(ε-γ / δ)-BBζ). In some embodiments of a CD3ζ endodomain comprising the signaling domain of 41BB, a polynucleotide encoding a full-length or partial-length BBζ endodomain comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to SEQ ID NO: 40, an exemplary sequence in which any one or two CD3ζ ITAMs may be removed. In some embodiments of a CD3 chimeric chain, the endodomain of CD3ζ comprising at least one, two, or three ITAMs further comprises the signaling domain of CD28 and the signaling domain of 41BB (tgCD3(ε-γ / δ)-(28-BB)ζ).In some embodiments of a CD3ζ endodomain containing both the 28 and 41BB signaling domains, the polynucleotide encoding the full-length or partial-length 28BBζ endodomain comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to SEQ ID NO: 41, an exemplary sequence from which any one or two CD3ζ ITAMs may be removed. In one embodiment of a polynucleotide encoding tgCD3(ε-γ)-(28 / BB)ζ, the encoded polypeptide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to the exemplary sequence SEQ ID NO:42, which, in yet some other embodiments, can be augmented by removing any one or two CD3ζ ITAMs, replacing the linker sequence with any other linker sequence known in the art, or replacing or adding the CD28 signaling domain with a 41BB signaling domain. In one embodiment of tgCD3(ε-δ)-(28 / BB)ζ, the encoded polypeptide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to the exemplary sequence SEQ ID NO:43, which, in some other embodiments, may be augmented by removing any one or two CD3ζ ITAMs, replacing the linker sequence with any other linker sequence known in the art, or replacing the CD28 signaling domain with or further including a 41BB signaling domain. In some other embodiments of the encoded CD3 chimeric chain tgCD3(ε-γ)-(28 / BB)ζ or tgCD3(ε-δ)-(28 / BB)ζ, the polypeptide further comprises the signal peptide of SEQ ID NO:28 or any other signal peptide known in the art. In embodiments, the sequence identity is at least 80%.In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. SEQ ID NO: 39 [Sequence Table 23] TIFF0007783746000027.tif26167 SEQ ID NO: 40 [Sequence Table 24] TIFF0007783746000028.tif27168 SEQ ID NO: 41 [Sequence Table 25] TIFF0007783746000029.tif27168 SEQ ID NO: 42 [Sequence Table 26] TIFF0007783746000030.tif48169 SEQ ID NO: 43 [Sequence Table 27] TIFF0007783746000031.tif46164

[0141] In the present application, a CD3 chimeric chain provided herein has been discovered, which is a fusion protein comprising a full-length or partial-length CD3ε ectodomain, at least one full-length or partial-length ectodomain of CD3δ and CD3γ, a CD3ζ endodomain containing at least one ITAM, and optionally one or more signaling domains, and which is capable of binding to a TCR. neg When expressed in cells containing the chimeric CD3 ectodomain, the chimeric CD3 ectodomain can be presented on the cell surface. Furthermore, cell surface expression of the CD3 ectodomain does not have peptide-MHC binding potential, but allows CD3 binding-triggered signaling through the fused CD3ζ endodomain.

[0142] Thus, in consideration of Design 5, provided herein is a cell or population thereof, wherein the cell is an iPSC, a clonal iPSC, a clonal iPS cell line cell, or a derivative cell obtained by differentiation of an iPSC, wherein the cell comprises an interruption of at least one of an endogenous TCR alpha constant region and an endogenous TCR beta constant region, and at least an exogenous polynucleotide encoding a CD3 chimeric chain fusion protein (ccCD3), wherein the fusion protein comprises a full-length or partial-length ectodomain of CD3 epsilon, a full-length or partial-length ectodomain of any one of CD3 delta and CD3 gamma, a full-length or partial-length endodomain of CD3 zeta having at least one ITAM, and optionally one or more signaling domains, wherein the ectodomain of the CD3 chimeric chain is present on the cell surface (cs-CD3) when expressed.

[0143] TCRs disclosed herein in various designs in Figures 1A-C neg The cell surface CD3 complex, or one or more of its subunits or subdomains (cs-CD3) in TCR cells neg As further provided, the TCR (cs-CD3) can function as a CD3-associated cell surface triggering receptor for binding to molecules including, but not limited to, a CD3-specific antibody or functional variant thereof, a CAR, and / or an engager, as further described below. neg The cs-CD3 cells or populations thereof may further comprise one or more of hnCD16 knock-in, CAR, partial or full-length peptides of cell surface-expressed exogenous cytokines or their receptors, B2M knock-out or knock-down, CIITA knock-out or knock-down, introduced expression of HLA-G or non-cleavable HLA-G, CD38 knock-out, and additional engineered modalities described herein. neg cs-CD3, CD16, CAR, CD38 negative, IL15 (fusion protein with receptor or truncated variant), B2M - / - CIITA - / - , and B2M - / - CIITA - / -A master cell bank is provided that contains single-cell sorted, expanded, clonally engineered iPSCs having at least one phenotype provided herein, including, but not limited to, HLA-G, and the cell bank provides a platform for additional iPSC manipulations and a renewable source for manufacturing off-the-shelf, engineered, homogenous cell therapy products that are well-defined, uniform in composition, and can be mass-produced at scale in a cost-effective manner.

[0144] 2.hnCD16 knock-in CD16 has been identified as two isoforms of the Fc receptors FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and facilitating antibody-dependent cellular cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high-affinity CD16," "uncleavable CD16," or "high-affinity uncleavable CD16" refer to various CD16 variants. Wild-type CD16 has low affinity and undergoes downregulation, including ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes upon NK cell activation. F176V (also referred to as F158V in some publications) is an exemplary CD16 polymorphic allele / variant with high affinity, while the S197P variant is an example of a genetically engineered, non-cleavable version of CD16. Engineered CD16 variants containing both F176V and S197P have high affinity and are non-cleavable, as described in more detail in WO2015 / 148926, the full disclosure of which is incorporated herein by reference. In addition, chimeric CD16 receptors in which the ectodomain of CD16 is essentially replaced with at least a portion of the CD64 ectodomain can also achieve the desired high affinity and non-cleavable characteristics of CD16 receptors capable of performing ADCC. In some embodiments, the replaced ectodomain of the chimeric CD16 comprises one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoforms or polymorphic variants).

[0145] Thus, in some embodiments, the high-affinity, non-cleavable CD16 receptor (hnCD16) comprises both F176V and S197P, and in some embodiments, F176V and the cleavage region is eliminated. 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 therebetween, identity when compared to any of the exemplary sequences SEQ ID NOS: 7, 8, and 9, each of which comprises at least a portion of the CD64 ectodomain. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. SEQ ID NOS: 7, 8, and 9 are encoded by exemplary SEQ ID NOS: 10-12, respectively. As used herein and throughout this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using art-recognized mathematical algorithms. SEQ ID NO:7: [Sequence Table 28] TIFF0007783746000032.tif31170 SEQ ID NO: 8 [Sequence Table 29] TIFF0007783746000033.tif30169 SEQ ID NO: 9 [Sequence Table 30] TIFF0007783746000034.tif37146 SEQ ID NO: 10 [Sequence Table 31] TIFF0007783746000035.tif81157 SEQ ID NO: 11 [Sequence Table 32] TIFF0007783746000036.tif78159 SEQ ID NO: 12 [Sequence Table 33] TIFF0007783746000037.tif78159

[0146] Thus, among other editing techniques contemplated and described herein, provided herein are clonal iPSCs engineered to contain a high-affinity, uncleavable CD16 receptor (hnCD16), which can differentiate into effector cells containing the hnCD16 introduced into the iPSCs. In some embodiments, the derived effector cells containing hnCD16 are NK cells. In some embodiments, the derived effector cells containing hnCD16 are T cells. The exogenous hnCD16 expressed in iPSCs or derived cells thereof exhibits high affinity binding not only to ADCC antibodies or fragments thereof, but also to bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domain of hnCD16 and its variants, CD16, or CD64. Bispecific, trispecific, or multispecific engagers or binders are further described below in this application (see Section I.7). Accordingly, at least one aspect of the present application provides derived effector cells or cell populations thereof preloaded with one or more preselected ADCC antibodies via high affinity binding to the extracellular domain of hnCD16 expressed on the derived effector cells, in an amount 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 CD16 having F176V and S197P.

[0147] In some other embodiments, the native CD16 transmembrane and / or intracellular domains of hnCD16 are further modified or replaced to produce chimeric Fc receptors (CFcRs) that contain non-native transmembrane domains, non-native stimulatory domains, and / or non-native signaling domains. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor than the receptor that provides the extracellular domain. In the present illustration, a CFcR based on CD16 or a variant thereof does not have a transmembrane domain, stimulatory domain, or signaling domain derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR comprises a non-native transmembrane domain derived from a CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T cell receptor polypeptide. In some embodiments, the exogenous hnCD16-based CFcR comprises a non-native stimulatory / inhibitory domain derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some embodiments, the exogenous hnCD16-based CFcR comprises a non-native signaling domain derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one hnCD16 embodiment, a chimeric receptor is provided comprising a transmembrane domain and a signaling domain, both derived from one of IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides.One particular embodiment of an hnCD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the full-length or partial extracellular domain of CD64 or CD16, wherein the extracellular domain of CD16 comprises F176V and S197P. Another embodiment of an hnCD16-based chimeric Fc receptor comprises the transmembrane domain and signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the full-length or partial extracellular domain of CD64 or CD16, wherein the extracellular domain of CD16 comprises F176V and S197P.

[0148] Various embodiments of the hnCD16-based chimeric Fc receptor described above can bind with high affinity to the Fc region of an antibody or fragment thereof, or to the Fc region of a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory and / or signaling domains of the chimeric receptor enable effector cell activation and cytokine secretion, as well as killing of tumor cells targeted by the antibody or bispecific, trispecific, or multispecific engager or binder having a tumor antigen-binding component and an Fc region. Without being limited by theory, through the non-native transmembrane domain, stimulatory domain, and / or signaling domain of the hnCD16-based chimeric Fc receptor, or through an engager binding to the ectodomain, the CFcR can contribute to the killing capacity of the effector cell while increasing the likelihood of effector cell proliferation and / or expansion. The antibody and engager can bring antigen-expressing tumor cells and CFcR-expressing effector cells into close proximity, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, or multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding effector cells expressing hnCD16-based CFcRs in attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0149] Unlike the endogenous CD16 receptor expressed by primary NK cells, which is cleaved from the cell surface after NK cell activation, the various non-cleavable versions of CD16 in derived NK cells avoid CD16 shedding and maintain constant expression. In derived NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicating improved cell function. Non-cleavable CD16 also enhances antibody-dependent cellular cytotoxicity (ADCC) and the binding of bispecific, trispecific, or multispecific engagers. ADCC is an NK cell-mediated lysis mechanism via CD16 binding to antibody-coated target cells. The additional high-affinity feature of the introduced hnCD16 in derived NK cells also enables in vitro loading of ADCC antibodies via hnCD16 prior to administering the cells to subjects requiring cell therapy. As provided, hnCD16, in some embodiments, may comprise F176V and S197P, or may comprise a complete or partial ectodomain derived from CD64 as exemplified by SEQ ID NO: 7, 8, or 9, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides derived NK cells or cell populations thereof preloaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed in Section V below.

[0150] Unlike primary NK cells, mature T cells from primary sources (i.e., native / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing expressed exogenous uncleavable CD16 could differentiate into functional derived T cells that not only express exogenous CD16 but also perform their functions via an adaptive CDCC mechanism without compromising T cell developmental biology. This adaptive ADCC in derived T cells can further be used as an approach for dual targeting and / or to rescue antigen escape, which often occurs with CAR-T cell therapy, where tumors recur with reduced or lost expression of the antigen targeted by the CAR-T or with mutated antigens that evade recognition by the CAR (chimeric antigen receptor). When the derived T cells have adaptive ADCC via exogenous CD16 expression and the antibody targets a tumor antigen different from that targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent the recurrence or recurrence of the targeted tumor, which is common with CAR-T therapy. Such a strategy of achieving dual targeting while reducing and / or preventing antigen escape applies equally to NK cells expressing one or more CARs. Various CARs that can be used in this strategy of reducing and preventing antigen escape are described in more detail below.

[0151] Thus, the present invention provides derived T cells comprising exogenous CD16. In some embodiments, the hnCD16 contained in the derived T cells comprises F176V and S197P. In some other embodiments, the hnCD16 contained in the derived T cells comprises a complete or partial ectodomain 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, a stimulatory domain, and a signaling domain. As described, such derived T cells possess an acquisition mechanism for targeting tumors with monoclonal antibodies medicated by ADCC to enhance the therapeutic effect of the antibody. As disclosed, the present application also provides derived T cells, or cell populations thereof, preloaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed in Section V below.

[0152] Further provided in the present application is a master cell bank comprising single-cell sorted, expanded, clonally engineered iPSCs having at least one phenotype provided herein, including but not limited to CD16, which cell bank provides a platform for additional iPSC manipulations and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products, including but not limited to derived NK cells and T cells, that are well-defined, uniform in composition, and can be mass-produced at scale in a cost-effective manner.

[0153] 3. CAR expression Any CAR design known in the art can be applied to genetically engineered iPSCs and their derived effector cells. A chimeric antigen receptor (CAR) is a fusion protein that generally includes an ectodomain, which includes an antigen recognition region, a transmembrane domain, and an endodomain. In some embodiments, the ectodomain may further include a signal peptide or 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 is capable of specifically binding to an antigen. In some embodiments, the antigen recognition domain is capable of specifically binding to an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, and the tumor can be a liquid or solid tumor. In some embodiments, the CAR is suitable for activating either T cells or NK cells expressing the CAR. In some embodiments, a CAR containing an NK-specific signaling component is specific for NK cells. In certain embodiments, the T cells are derived from CAR-expressing iPSCs, and the derived T cells can include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof. In certain embodiments, the NK cells are derived from CAR-expressing iPSCs.

[0154] In certain embodiments, the antigen recognition region comprises a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding 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 a whole antibody.Non-limiting examples of antigens that can be targeted by a CAR include ADGRE2, carbonic anhydrase IX (CAIX), CCRI, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD269 (BCMA), CDS, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGF, and the like. RvIII, 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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0155] In some embodiments, the transmembrane domain of the CAR comprises the full length or at least a portion of a native or modified transmembrane region of a CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T-cell receptor polypeptide.

[0156] In some embodiments, the signaling peptide of the endodomain (or intracellular domain) comprises the full length or at least a portion of a polypeptide of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the signaling peptide of the CAR comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to at least one ITAM (immunoreceptor tyrosine-based activation motif) of CD3ζ.

[0157] In certain embodiments, the endodomain further comprises at least one costimulatory signaling region, which may comprise the full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.

[0158] In one embodiment, the CAR applied to the cells provided herein comprises a costimulatory domain derived from CD28 and a signaling domain comprising native or modified ITAM1 of CD3ζ, and is represented by an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 13. In a further embodiment, the CAR comprising a costimulatory 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 can be connected to the transmembrane domain via the hinge, and the CAR comprises an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 14. In an embodiment, the sequence identity is at least 80%. In an embodiment, the sequence identity is at least 90%. In an embodiment, the sequence identity is at least 95%. In an embodiment, the sequence identity is 100%. SEQ ID NO: 13 [Sequence Table 34] TIFF0007783746000038.tif25149 SEQ ID NO: 14 [Sequence Table 35] TIFF0007783746000039.tif26142

[0159] In another embodiment, the CAR applied to the cells provided herein comprises a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3ζ, and is represented by an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 15. The CAR comprising a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3ζ may further comprise a CD8 hinge, and the amino acid sequence of such a structure is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. SEQ ID NO: 15 [Sequence Table 36] TIFF0007783746000040.tif32152 SEQ ID NO: 16 [Sequence Table 37] TIFF0007783746000041.tif39153

[0160] Non-limiting CAR strategies include heterodimeric, conditionally activated CARs via dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9,587,020); split CARs, which are homologous recombination of antigen-binding, hinge, and endodomains to generate a CAR (see, e.g., U.S. Publication No. 2017 / 0183407); multi-chain CARs, which allow for non-covalent binding between two transmembrane domains connected to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Publication No. 2014 / 0134142); CARs with bispecific antigen-binding domains (see, e.g., U.S. Patent No. 9,447,194) or with a pair of antigen-binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Patent No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Invest. 2016;126(8):3036-3052); inducible CARs (see, e.g., U.S. Publication Nos. 2016 / 0046700, 2016 / 0058857, 2017 / 0166877); switchable CARs (see, e.g., U.S. Publication No. 2014 / 0219975); and any other designs known in the art.

[0161] Thus, provided herein are derived cells obtained from the differentiation of genomically engineered iPSCs, wherein both the iPSCs and the derived cells are TCR neg It includes one or more CARs along with additional modified modalities, including, but not limited to, cs-CD3 and / or hnCD16. negFurther provided is a master cell bank comprising single-cell sorted, expanded, clonally engineered iPSCs having at least one phenotype provided herein, including, but not limited to, one or both of cs-CD3 and hnCD16, wherein the cell bank provides a platform for additional iPSC manipulation and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products that are well-defined, compositionally uniform, and can be mass-produced at scale in a cost-effective manner. In one particular embodiment, the iPSCs and their derivatives are TCR neg The derivative cells comprise a CAR that targets cs-CD3, hnCD16, and a selected tumor or viral antigen, and the derivative cells are NK cells or T cells, which can be used with one or more ADCC antibodies or bispecific, trispecific, or multispecific engagers that target tumor antigens different from those targeted by the CAR to avoid or reduce tumor antigen escape while achieving dual targeting of the same tumor via hnCD16 binding.

[0162] In further embodiments, CAR-containing iPSCs and their derived T cells have the CAR inserted into the TCR constant region, resulting in TCR knockout and placing CAR expression under the control of the endogenous TCR promoter. Additional insertion sites include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In some embodiments, the derived TCR-negative CAR-T cells derived from the engineered iPSCs further comprise an hnCD16 with an ectodomain native to CD16 (F176V and / or S197P) or derived from CD64, and native or non-native transmembrane, stimulatory, and signaling domains. In another embodiment, the CAR-containing iPSCs and their derived NK cells have the CAR inserted 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. Master cell banks containing single-cell sorted and expanded clonally engineered iPSCs with at least one phenotype provided herein, including but not limited to a CAR, are further provided. The cell banks provide a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogenous cell therapy products that are well-defined, uniform in composition, and can be mass-produced at scale in a cost-effective manner.

[0163] 4. Exogenously Introduced Cytokines and / or Cytokine Signaling Avoiding systemic administration of high doses of clinically relevant cytokines reduces the risk of dose-limiting toxicity associated with such practice while establishing cytokine-mediated cell autonomy. To achieve lymphocyte autonomy without the need for additional soluble cytokine administration, partial or full-length peptides of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors are introduced into cells to allow cytokine signaling, regardless of whether the cytokine itself is expressed, thereby maintaining or improving cell growth, proliferation, expansion, and / or effector function while reducing the risk of cytokine toxicity. In some embodiments, the introduced cytokine and / or its respective native or modified receptor for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or temporary.

[0164] Figure 2 presents several construct designs using IL15 as an illustrative example. The transmembrane (TM) domain of any of the designs in Figure 2 may be native to the IL15 receptor or may be modified with or replaced by the transmembrane domain of any other membrane-bound protein.

[0165] Figure 2 Design 1: IL15 and IL15Rα are co-expressed by using a self-cleaving peptide to mimic the trans-presentation of IL15 without eliminating the cis-presentation of IL15.

[0166] Design 2 in Figure 2: IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without eliminating cis-presentation of IL15 and ensuring membrane binding of IL15.

[0167] Design 3 in Figure 2: IL15Rα with a truncated intracellular domain is fused to IL15 at its C-terminus via a linker, mimicking IL15 trans-presentation, maintaining IL15 membrane binding, and eliminating cis-presentation and / or any other potential signaling pathways mediated by normal IL15R via its intracellular domain. The intracellular domain of IL15Rα is believed to be important for the receptor to be expressed in IL15-responsive cells and for responder cell expansion and function. Such truncated constructs comprise an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, which can be encoded by the exemplary nucleic acid sequence represented by SEQ ID NO: 18. In one embodiment of truncated IL15 / IL15Rα, the construct does not include the last four amino acids "KSRQ" of SEQ ID NO: 17 and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 21. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. SEQ ID NO: 17 [Sequence Table 38] TIFF0007783746000042.tif44157 SEQ ID NO: 18 [Sequence Table 39] TIFF0007783746000043.tif102170 SEQ ID NO: 21 [Sequence Table 40] TIFF0007783746000044.tif44157

[0168] Those skilled in the art will understand that the above signal peptide and linker sequences are exemplary and in no way limit the variations thereof suitable for use as signal peptides or linkers. Many suitable signal peptide or linker sequences are known and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence may be substituted with another sequence without altering the activity of the functional peptide provided by the signal peptide or linked by the linker.

[0169] Design 4 in Figure 2: Because the constructs in Design 3 were shown to function in promoting effector cell survival and expansion and because the cytoplasmic domain of IL15Rα can be omitted without adversely affecting the autonomous characteristics of IL15-equipped effector cells in such designs, Design 4 offers another practical alternative to Design 3. Essentially, the entire IL15Rα is removed, except for the sushi domain, which is fused to IL15 on one end and the transmembrane domain on the other (mb-Sushi), with an optional linker between the sushi and transmembrane domains. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of any membrane-bound protein. In constructs such as Design 4, unwanted signaling through IL15Rα, including cis-presentation, is eliminated while only the desired trans-presentation of IL15 is retained. In some embodiments, a component comprising IL15 fused to a Sushi domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 19, which may be encoded by the exemplary nucleic acid sequence represented by SEQ ID NO: 20. In embodiments, the sequence identity is at least 80%. In embodiments, the sequence identity is at least 90%. In embodiments, the sequence identity is at least 95%. In embodiments, the sequence identity is 100%. SEQ ID NO: 19 [Sequence Table 41] TIFF0007783746000045.tif32156 SEQ ID NO: 20 [Sequence Table 42] TIFF0007783746000046.tif71155

[0170] Those skilled in the art will understand that the above signal peptide and linker sequences are exemplary and in no way limit the variations thereof suitable for use as signal peptides or linkers. Many suitable signal peptide or linker sequences are known and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence may be substituted with another sequence without altering the activity of the functional peptide provided by the signal peptide or linked by the linker.

[0171] Design 5 in Figure 2: Native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker, allowing constitutive signaling and maintaining membrane-bound and trans-expression of IL15.

[0172] Design 6 in Figure 2: Native or modified common receptor γC is fused at the C-terminus to IL15 via a linker for constitutive signaling and membrane-bound trans-presentation of the cytokine. Common receptor γC is also known as common gamma chain or CD132 and IL2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukin receptors, including but not limited to the IL2, IL4, IL7, IL9, IL15, and IL21 receptors.

[0173] Design 7 of Figure 2: Engineered IL15Rβ that forms homodimers in the absence of IL15 is useful for producing constitutive signaling of cytokines.

[0174] In some embodiments, one or more of the cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or their receptors, can be introduced into iPSCs and their derivative cells upon iPSC differentiation using one or more of the designs in Figure 2. In some embodiments, IL2 or IL15 cell surface expression and signaling is via the constructs illustrated in any one of Designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling is via the constructs illustrated in Designs 5, 6, or 7, 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 2 may be native to the respective cytokine receptor or may be modified or replaced with the transmembrane domain of any other membrane-bound protein.

[0175] In addition to induced pluripotent cells (iPSCs), clonal iPSCs, clonal iPS cell line cells, or iPSC-derived cells containing at least one engineered modality disclosed herein, a master cell bank containing single-cell sorted, expanded, clonally engineered iPSCs with at least exogenously introduced cytokine and / or cytokine receptor signaling as described in this section is provided, which cell bank provides a platform for additional iPSC manipulation and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products that are well-defined, compositionally uniform, and can be mass-produced at scale in a cost-effective manner. In iPSCs and derived cells containing both CAR and exogenous cytokine and / or cytokine receptor signaling, the CAR and IL can be expressed in separate constructs or co-expressed in a bicistronic construct containing both the CAR and IL. In some further embodiments, IL15 in a form represented by any of the construct designs in Figure 2 can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence, for example, depicted as CAR-2A-IL15 or IL15-2A-CAR. Thus, IL15 and CAR are in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises IL15 of design 3 in Figure 2. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises IL15 of design 3 in Figure 2. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises IL15 of design 7 in Figure 2. Upon expression of CAR-2A-IL15 or IL15-2A-CAR, the self-cleaving 2A peptide dissociates the expressed CAR and IL15, and the dissociated IL15 is displayed on the cell surface. The CAR-2A-IL15 or IL15-2A-CAR bicistronic design allows for coordinated expression of CAR and IL15 in both timing and amount, and under the same regulatory mechanism, which can be selected, for example, to incorporate an inducible promoter for expression of a single ORF.Self-cleaving peptides are found in foot-and-mouth disease viruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine teschovirus-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 members of cardioviruses such as Theiloviruses (e.g., Theiler's murine encephalomyelitis virus) and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are also referred to as "F2A," "E2A," "P2A," and "T2A," respectively.

[0176] Bicistronic CAR-2A-IL15 or IL15-2A-CAR embodiments disclosed herein for IL15 also contemplate expression of any other cytokine provided herein, e.g., IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, IL2 cell surface expression and signaling is via a construct depicted in any of Designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling is via a construct depicted in Designs 5, 6, or 7, using either a common receptor and / or cytokine-specific receptors.

[0177] In iPSCs and derived cells thereof, which contain both CAR and cytokine receptor signaling, including but not limited to exogenous cytokines and / or IL15, the iPSCs and derived cells may be TCR neg It may further comprise cs-CD3 and / or CD16.

[0178] 5. HLA-I and HLA-II deficiency To avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins must be matched for histocompatibility with the allogeneic recipient. Provided herein are iPSC cell lines and their differentiated derivatives in which expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21) or by deletion or reduction of expression levels of HLA class I-associated 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 cell surface expression of all HLA class I heterodimers. B2M-negative cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II-associated genes, including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator and functions through the activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-negative cells are HLA-II deficient. For example, iPSC lines and their derivatives that lack both B2M and CIITA expression and therefore have both HLA-I and HLA-II deficiency are provided herein, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoid recognition and killing by host (allogeneic) T cells.

[0179] In some cell types, lack of class I expression leads to lysis by NK cells. To overcome this "loss of self" response, HLA-G can optionally be knocked in to prevent NK cell recognition and killing of HLA-I-deficient effector cells derived from engineered iPSCs. In one embodiment, HLA-I-deficient iPSCs and their derived cells further comprise an HLA-G knockin. In some embodiments, the provided HLA-I-deficient iPSCs and their derived cells further comprise one or both of a CD58 knockout and a CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and facilitate the migration of cells, including immune cells. While CD58 knockout is more efficient at reducing allogeneic NK cell activation than CD54 knockout, double knockout of both CD58 and CD54 has been shown to most effectively reduce NK cell activation. Some observations suggest that CD58 and CD54 double knockout is even more effective than HLA-G overexpression in HLA-I-deficient cells in overcoming the "loss of self" effect, without adversely affecting the differentiation potential of PSCs and the function of derived effector cells, including derived T cells and NK cells. negProvided herein is a master cell bank containing single-cell sorted, expanded clonally engineered iPSCs that are cs-CD3, HLA-I and HLA-II deficient, and one or more of hnCD16, CAR, and IL. As provided above, in some embodiments, the HLA-I and HLA-II deficient iPSCs and their derived cells have an exogenous polynucleotide encoding HLA-G. In some embodiments, the HLA-I and HLA-II deficient iPSCs and their derived cells are CD58 null. In some other embodiments, the HLA-I and HLA-II deficient iPSCs and their derived cells are CD54 null. In yet some other embodiments, the HLA-I and HLA-II deficient iPSCs and their derived cells are CD58 null and CD54 null. The cell bank provides a platform for further iPSC manipulation and a renewable source for producing off-the-shelf, engineered, homogenous cell therapy products that are well-defined, uniform in composition, and can be mass-produced at large scale in a cost-effective manner.

[0180] 6.CD38 knockout The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated cancer cell depletion typically results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). In addition to ADCC, immune effector mechanisms that cooperate with therapeutic antibodies may also include phagocytosis (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0181] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells and NK cells, as well as activated T and B cells. During hematopoiesis, CD38 is expressed in association with CD34 +It is expressed on stem cells and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages during the final stages of maturation, which continue through the plasma cell stage. As a type II transmembrane glycoprotein, CD38 performs cellular functions as both a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 mediates the synthesis of NAD + CD38 catalyzes the synthesis and hydrolysis of ATP to ADP-ribose, thereby producing the second messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which are important in the process of cell adhesion (this process is calcium-dependent). As a receptor, CD38 recognizes CD31 and regulates cytokine release and cytotoxicity in activated NK cells. CD38 associates with cell surface proteins in lipid rafts and mediates the release of cytosolic Ca. 2+ It has also been reported to regulate flux and mediate signaling in lymphoid and myeloid cells.

[0182] In malignant tumor treatment, the systemic use of T cells transduced with the CD38 antigen-binding receptor has been shown to lyse the CD38+ fraction of CD34+ hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells, resulting in incomplete therapeutic responses and reduced or eliminated efficacy due to impaired recipient immune effector cell function. Additionally, in multiple myeloma patients treated with the CD38-specific antibody daratumumab, a reduction in NK cells was observed in both bone marrow and peripheral blood, whereas other immune cell types, such as T cells and B cells, were unaffected despite CD38 expression (Casneuf et al., Blood Advances. 2017;1(23):2105-2114). Without being limited by theory, the present application provides a strategy to maximize the potential of CD38-targeted cancer therapy by overcoming the depletion or reduction of effector cells induced by CD38-specific antibodies and / or CD38 antigen-binding domains due to fratricide. Additionally, because CD38 is upregulated on activated lymphocytes, such as T or B cells, in recipients of allogeneic effector cells, inhibiting the activation of these recipient lymphocytes using a CD38-specific antibody, such as daratumumab, reduces and / or prevents allorejection of these effector cells, thereby increasing effector cell survival and persistence. Accordingly, the present application also provides strategies to enhance effector cell persistence and / or survival through the use of CD38-specific antibodies, secreted CD38-specific engagers, or CD38CARs (chimeric antigen receptors) for recipient T and B cell activation, i.e., reducing or preventing allorejection by lymphodepletion of activated T and B cells, often prior to adoptive cell transfer. Specifically, the provided strategies involve generating a master cell bank containing CD38 knockout iPSC lines, single-sorted and expanded clonal CD38-negative iPSCs, and directing the differentiation of engineered iPSC lines to CD38-negative (CD38 neg) to obtain derived effector cells, which, when a CD38-targeted therapeutic moiety is used in conjunction with the effector cells, are protected from fratricide and allogeneic rejection, among other advantages. In addition, anti-CD38 monoclonal antibody therapy significantly depletes a patient's activated immune system without adversely affecting the patient's hematopoietic stem cell compartment. CD38-negative derivative cells have the ability to resist CD38 antibody-mediated depletion and can be effectively administered in combination with anti-CD38 or CD38-CAR without the use of toxic conditioning agents, thereby reducing and / or replacing chemotherapy-based lymphodepletion. Single-cell sorted and expanded clonal CD38 neg The master cell bank containing iPSCs was designed to detect TCR neg It provides a platform for additional iPSC manipulation, including but not limited to one or more of cs-CD3, hnCD16, CAR, IL, and HLA I and II deficiency, and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products that are well-defined, uniform in composition, and can be mass-produced at scale in a cost-effective manner.

[0183] In one embodiment provided herein, the CD38 knockout in the iPSC line is a biallelic knockout. As disclosed herein, the provided CD38-negative iPSC line comprises at least a TCR gene that, when expressed, provides a cell surface CD3 complex, or one or more subunits or subdomains thereof (CS-CD3). negand one or more polynucleotides encoding one or more exogenous proteins, wherein the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, including, but not limited to, mesodermal cells with secondary hemogenic endothelial (HE) potential, secondary HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, when an anti-CD38 antibody is used to induce ADCC or an anti-CD38 CAR is used for targeted cell killing, CD38 neg The iPSCs and / or their derived effector cells are not eliminated by anti-CD38 antibodies, anti-CD38CARs, or recipient activated T cells or B cells, thereby increasing the persistence and / or survival of the iPSCs and their effector cells in the presence of and / or after exposure to such therapeutic moieties. In some embodiments, the effector cells have increased persistence and / or survival in vivo in the presence of and / or after exposure to such therapeutic moieties.

[0184] 7. Additional Modifications In some embodiments, the TCR neg cs-CD3, and CD16, CAR, IL, HLA I and II deficient, and CD38 - / - iPSCs and their derived effector cells containing one or more of the following may additionally contain deletions or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and at least one of any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention also includes the introduction or increased expression of at least one of a surface triggering receptor for binding to a TCR, an Fc receptor, an engager, and a bispecific, multispecific, or universal engager.

[0185] Bispecific or multispecific engagers are fusion proteins consisting of two or more single-chain variable fragments (scFvs) or other functional variants of different antibodies, where at least one scFv binds to an effector cell surface molecule or "surface triggering receptor" and at least another scFv binds to tumor cells via a tumor-specific surface molecule. As used herein, the term "surface triggering receptor" refers to a receptor that can trigger or initiate an immune response, e.g., a cytotoxic response, of an effector cell. Surface triggering receptors can be engineered or can be endogenous surface proteins expressed on effector cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, the surface triggering receptor facilitates binding of the bispecific or multispecific antibody between the effector cell and a specific target cell, e.g., a tumor cell, regardless of the effector cell's native receptor and cell type. In some other embodiments, one or more exogenous surface triggering receptors can be introduced into effector cells using the methods and compositions provided herein, i.e., via manipulation of iPSCs, to generate a master cell bank containing clonally engineered iPSCs that are optionally single-cell sorted and expanded, and then direct the differentiation of the iPSCs into T cells, NK cells, or any other effector cells that contain the same genotype as the source iPSCs.

[0186] This approach can also be used to generate iPSCs containing universal surface triggering receptors, which can then be differentiated into populations of various effector cell types that express the universal surface triggering receptor. "Universal" means that the surface triggering receptor can be expressed and activated on any effector cell, regardless of cell type, and all effector cells that express the universal receptor can bind or link to an engager with the same epitope recognizable by the surface triggering receptor, regardless of the engager's tumor-binding specificity. In some embodiments, engagers with the same tumor-targeting specificity are used to bind to different universal surface triggering receptors. In some embodiments, engagers with different tumor-targeting specificities are used to bind to the same universal surface triggering receptor. Thus, one or more effector cell types can be used to kill a specific type of tumor cell or to kill more than one type of tumor. Surface triggering receptors generally contain a costimulatory domain for effector cell activation and an anti-epitope specific for the epitope of the engager, or vice versa; the surface triggering receptor contains an epitope that can recognize or is specific for the anti-epitope of the engager. For example, a bispecific engager is specific for the anti-epitope / epitope of the surface triggering receptor at one end and a tumor antigen at the other end.

[0187] As provided herein, the various forms of cell surface-displayed CD3 molecules disclosed herein serve, among other functions, as CD3-associated cell surface triggering receptors for engager recognition, which are particularly useful in TCR-negative cells where expressed CD3 molecules are not present on the cell surface despite their expression. In some embodiments, the CD3-associated surface triggering receptor for engager recognition is contained in a complete or partial CD3 molecule (i.e., a subunit or subdomain of the CD3 complex) that is expressed and presented on the cell surface of an effector cell. In some embodiments, the complete or partial CD3 molecule presented on the effector cell surface comprises (1) at least one full-length or partial-length ectodomain from one or more CD3 subunits, including at least CD3ε, CD3δ, and / or CD3ε, and optionally (2) a full-length or partial-length endodomain of CD3ζ. In some embodiments, all subunits or subdomains of the complete or partial CD3 molecule that comprises the CD3-associated surface triggering receptor are endogenous. In some embodiments, at least one subunit or subdomain of a complete or partial CD3 molecule comprising a CD3-associated surface triggering receptor is exogenous. In some embodiments, a bispecific antibody that binds a CD3-associated cell surface triggering receptor disclosed herein is CD3-CD19. In yet another embodiment, the bispecific antibody is CD3-CD33. In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and tumor cell antigen-binding domains, e.g., modified IL15 (referred to in some publications as TriKE or trispecific killer engager) as a linker for effector NK cells to facilitate effector cell expansion.

[0188] In addition to CD3, additional effector cell surface molecules, or surface triggering receptors, that can be used for bispecific or multispecific engager recognition or binding include, but are not limited to, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, and chimeric Fc receptors disclosed herein. In some embodiments, the CD16 expressed on the surface of effector cells for engager recognition is an hnCD16 comprising the CD16 (including F176V and optionally S197P) or CD64 extracellular domain, and a native or non-native transmembrane domain, stimulatory domain, and / or signaling domain, as described in Section I.2. 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 the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the full-length or partial sequence extracellular domain of CD64 or CD16, and wherein the extracellular domain of CD16 comprises F176V and optionally S197P.

[0189] 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.

[0190] In view of the above, in one embodiment, for binding CD3 on effector cells, the bispecific antibody is CD3-CD19. In another embodiment, the bispecific antibody is CD3-CD33. For binding CD16 on effector cells, the bispecific antibody is CD16-CD30 or CD64-CD30. In another embodiment, the bispecific antibody is CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and tumor cell antigen-binding domains, e.g., modified IL15 (referred to in some publications as TriKE or trispecific killer engager) as a linker for effector NK cells to facilitate effector cell expansion. 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. Linkers can also be derived from other cytokines, including, but not limited to, IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21.

[0191] 8. Genetically engineered iPSC lines and iPSC-derived cells provided herein In light of the above, the present application provides a TCR comprising, when expressed, a cell surface CD3 complex, or one or more subunits or subdomains thereof (cs-CD3). neg cells (TCR neg cs-CD3) or a population thereof, the cells being induced pluripotent cells (iPSCs), clonal iPSCs, clonal iPS cell line cells, or TCR negiPSC-derived cells obtained from directed differentiation of cs-CD3 iPSCs. A master cell bank containing single-cell sorted, expanded, clonally engineered iPSCs with the phenotypes described herein is also provided, which provides a renewable source for the production of off-the-shelf, engineered, homogenous cell therapy products that are well-defined, uniform in composition, and can be mass-produced at scale in a cost-effective manner.

[0192] In some embodiments, iPSC-derived cells are hematopoietic cells, including, but not limited to, mesodermal cells with secondary hemogenic endothelial (HE) potential, secondary HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, iPSC-derived hematopoietic cells include effector cells, such as T cells, NK cells, regulatory cells, which express TCR neg and when expressed, comprises a cell surface CD3 complex, or one or more subunits or subdomains thereof (cs-CD3). In a further embodiment, the present application provides an iPSC-derived TCR comprising a cell surface CD3 complex, or one or more subunits or subdomains thereof (cs-CD3). neg T cells (TCR neg cs-CD3) or a population thereof, and T cells are provided with TCR neg Obtained from directed differentiation of cs-CD3 iPSCs.

[0193] As used herein, TCR neg Also, TCR negative, TCR - / -, "TCR null," or TCR knockout, which includes cells that lack endogenous TCR expression, either originally (e.g., NK cells or iPSC-derived NK cells), or by gene expression control, or by genome editing iPSC cells (e.g., iPSCs, iPSCs reprogrammed from T cells (TiPSCs)) or T cells to knock out the endogenous TCR or one or more subunits thereof, or by obtaining TCR-negative derivative cells differentiated from iPSCs with TCR knockout. Thus, as disclosed, the TCR knocked out in a cell is the endogenous TCR complex. Interfering with the expression of either the TCRα or TCRβ constant region of the TCR in a cell is one of many ways to knock out the endogenous TCR complex of a cell. TCR neg Cells negatively affect cell functions that require cell surface CD3 recognition, binding, and / or signaling, including TCR neg It was discovered that despite the expression of all CD3 subunits in TCR cells, they were unable to present the CD3 complex on the cell surface. neg In a cell, the cell surface CD3 complex disclosed herein, or one or more subunits or subdomains thereof (cs-CD3), can function as a CD3-associated cell surface triggering receptor for binding with molecules, including, but not limited to, antibodies or functional variants thereof and / or engagers, as described herein.

[0194] Also provided herein are iPSCs or iPSC-derived cells comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), wherein the cells are optionally TCR negative. When expressed, cs-CD3 functions as a CD3-related cell surface triggering receptor. TCR negIn some embodiments of the CD3-associated surface triggering receptor provided on the cell, the receptor is contained in a complete or partial endogenous CD3 molecule that, when expressed, is presented on the effector cell surface, and even when expressed, is not otherwise present on the TCR neg Presentation of endogenous CD3 molecules not present in cells is enabled by association with a recombinant TCR comprising one or more of full-length or partial-length exogenous TCR alpha, exogenous TCR beta, and any variant thereof provided herein. In some embodiments, the TCR neg Cell surface presentation of complete or partial endogenous CD3 molecules in cells is made possible by additionally expressing at least a combination of TCRs in the cells, including a nonbinding recombinant TCR (nb-rTCR), a defined recombinant TCR (d-rTCR), and / or a recombinant pre-TCR.

[0195] In some embodiments, a TCR comprising a CD3-associated surface triggering receptor neg The cells contain a non-binding recombinant TCR (nb-rTCR), which contains one or both of tgTRAC (transgenic TCR alpha constant region) and tgTRBC (transgenic TCR beta constant region), and thus, TCR neg The iPSCs or iPSC-derived cells contain one or more polynucleotides encoding tgTRAC and / or tgTRBC. neg In some embodiments of the cell, the polynucleotide is inserted into the TRAC locus, and the inserted polynucleotide disrupts expression of endogenous TRAC, thereby resulting in endogenous TCR knockout, and optionally, the inserted polynucleotide is driven by the endogenous promoter or a heterologous promoter of TRAC. negIn some embodiments of the cells, the polynucleotide is inserted into the TRBC locus, and the inserted polynucleotide disrupts expression of endogenous TRBC, thereby resulting in endogenous TCR knockout, and optionally, the inserted polynucleotide is driven by the TRBC's endogenous promoter or a heterologous promoter.

[0196] In some embodiments, a TCR comprising a CD3-associated surface triggering receptor neg The cells contain defined recombinant TCRs (d-rTCRs), which include tgTCRα (transgenic TCRα) and tgTCRβ (transgenic TCRβ), each of which contains a respective defined variable region in addition to a respective constant region (i.e., TRAC and TRBC), and thus are TCRs. neg The iPSC or iPSC-derived cells contain one or more polynucleotides encoding tgTCRα and / or tgTCRβ. In some embodiments, the defined variable regions are derived from TCR alpha and beta of T cells with known TCR specificity. In some embodiments, the defined variable regions are derived from TCR alpha and beta of invariant NKT cells. TCRs comprising a polynucleotide encoding tgTCRα neg In some embodiments of the cell, the polynucleotide is inserted into the TRAC locus, and the inserted polynucleotide disrupts expression of endogenous TRAC, thereby resulting in an endogenous TCR knockout, and optionally the inserted polynucleotide is driven by the endogenous promoter or a heterologous promoter of TRAC. neg In some embodiments of the cells, the polynucleotide is inserted into the TRBC locus, and the inserted polynucleotide disrupts expression of endogenous TRBC, thereby resulting in endogenous TCR knockout, and optionally, the inserted polynucleotide is driven by the TRBC's endogenous promoter or a heterologous promoter.

[0197] In some embodiments, a TCR comprising a CD3-associated surface triggering receptor neg The cells comprise a recombinant pre-TCR (p-rTCR), which comprises a tgpTCRα (transgenic pre-TCRα), and optionally a tgTCRβ or tgTCRβ, which comprises a defined variable region, and thus a TCR neg The iPSC or iPSC-derived cell contains at least a polynucleotide encoding tgpTCRα. neg In some embodiments of the cell, the polynucleotide is inserted into the TRAC locus, and the inserted polynucleotide disrupts expression of endogenous TRAC, thereby resulting in endogenous TCR knockout, and optionally the inserted polynucleotide is driven by the endogenous promoter of TRAC or a heterologous promoter. neg In some embodiments of the cells, the tgTRBC or tgTCRβ-encoding polynucleotide is inserted into the TRBC locus, the inserted polynucleotide disrupts expression of endogenous TRBC, thereby resulting in endogenous TCR knockout, and optionally, the inserted tgTRBC or tgTCRβ-encoding polynucleotide is driven by the TRBC's endogenous promoter or a heterologous promoter, each of which has a defined variable region.

[0198] TCR neg In some embodiments of the CD3-associated surface triggering receptor in a cell, the receptor is contained in a complete or partial CD3 molecule that includes at least one exogenous subunit or subdomain from one or more of CD3ε, CD3δ, and CD3γ. In one embodiment, a TCR neg The CD3-related surface triggering receptor for engager recognition in cells is contained in a partial CD3 molecule that includes at least the full-length or partial-length ectodomain of CD3ε. In one embodiment, the TCR negThe CD3-related surface triggering receptor for cell engager recognition is contained in a partial CD3 molecule comprising at least a full-length or partial-length ectodomain of CD3ε and, additionally, a full-length or partial-length ectodomain of CD3γ or CD3δ. In one embodiment, the CD3 molecule comprises at least a full-length or partial-length ectodomain of CD3ε, CD3γ, and / or CD3δ, wherein the full-length or partial-length ectodomain is fused to the constant region of TCRα or TCRβ, and the partial fusion protein comprising TRAC or TRBC, respectively, can form a heterodimer with endogenous CD3ζ. Thus, a TCR having a CD3-related surface triggering receptor neg In one embodiment of the iPSC or iPSC-derived cell, the cell comprises at least one of: (i) a transgenic fusion protein comprising full-length or partial-length ectodomains of CD3ε and CD3δ and a TCR α constant region (tgCD3(ε-δ)-TRAC), (ii) a transgenic fusion protein comprising full-length or partial-length ectodomains of CD3ε and CD3γ and a TCR β constant region (tgCD3(ε-γ)-TRBC), (iii) a transgenic fusion protein comprising full-length or partial-length ectodomains of CD3ε and CD3γ and a TCR α constant region (tgCD3(ε-γ)-TRAC), and / or (iv) a transgenic fusion protein comprising full-length or partial-length ectodomains of CD3ε and CD3δ and a TCR β constant region (tgCD3(ε-δ)-TRBC). TCR with CD3-associated surface triggering receptor neg In some embodiments of the cell, the cell comprises a heterodimer comprising a transgenic fusion protein comprising a TCR alpha constant region fused to at least a full-length or partial-length ectodomain of CD3 epsilon, and a transgenic fusion protein comprising a TCR beta constant region fused to at least a full-length or partial-length ectodomain of CD3 epsilon.

[0199] TCR negIn some embodiments of the CD3-associated surface triggering receptor in a cell, the receptor is comprised in a complete or partial CD3 molecule that includes at least one exogenous subunit or subdomain from one or more of CD3ε, CD3δ, CD3γ, and / or CD3ζ, and optionally one or more signaling domains from 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8 for signaling and / or costimulation, and all subunits or subdomains including the signaling domains are fused to form a chimeric chain. In one embodiment, a TCR neg The CD3-related surface triggering receptor for engager recognition in cells is comprised in a CD3 chimeric chain comprising at least a full-length or partial-length ectodomain of CD3ε, a full-length or partial-length ectodomain of one or both of CD3δ and CD3γ, and a full-length or partial-length endodomain of CDζ. In one embodiment, a TCR neg The CD3-associated surface triggering receptor for engager recognition in cells is contained in a CD3 chimeric chain comprising a full-length or partial-length ectodomain of CD3ε, a full-length or partial-length ectodomain of one or both of CD3δ and CD3γ, and a full-length or partial-length endodomain of CDζ, and further comprising the cytoplasmic signaling domains of one or both of CD28 and 41BBL. Thus, a TCR having a CD3-associated surface triggering receptor negIn one embodiment of the iPSC or iPSC-derived cells, the cells are expressed as a transgenic fusion protein comprising (i) full-length or partial-length ectodomains of CD3ε and CD3γ and a full-length or partial-length endodomain of CD3ζ [tgCD3(ε-γ)-ζ], (ii) full-length or partial-length ectodomains of CD3ε and CD3δ and a full-length or partial-length endodomain of CD3ζ [tgCD3(ε-δ)-ζ], or (iii) a transgenic fusion protein comprising a full-length or partial-length ectodomain of CD3ε, a full-length or partial-length ectodomain of CD3γ or CD3δ, a full-length or partial-length endodomain of CD3ζ, and a signaling domain of CD28. (iv) a transgenic fusion protein comprising a full-length or partial-length ectodomain of CD3ε, a transgenic fusion protein comprising a full-length or partial-length ectodomain of CD3γ or CD3δ, a full-length or partial-length endodomain of CD3ζ, and the signaling domain of 41BB [tgCD3(ε-γ / δ)-BBζ], and / or (v) a transgenic fusion protein comprising a full-length or partial-length ectodomain of CD3γ or CD3δ, a full-length or partial-length endodomain of CD3ζ, a signaling domain of CD28, and the signaling domain of 41BB [tgCD3(ε-γ / δ)-(28-BB)ζ].

[0200] Further provided herein are iPSCs comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3) and a polynucleotide encoding high-affinity, non-cleavable CD16 (hnCD16), wherein the iPSCs are capable of directed differentiation to produce functional derivative hematopoietic cells. In some embodiments, the effector cells comprise T cells. In some embodiments, the effector cells comprise NK cells.

[0201] Provided herein are iPSCs comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and a polynucleotide encoding a target-specific chimeric antigen receptor (CAR), wherein the iPSCs are capable of directed differentiation to produce functional derivative effector cells.

[0202] Further provided are iPSCs comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and a polynucleotide encoding at least one exogenous cytokine and / or its receptor (IL) that enables cytokine signaling that contributes to cell survival, persistence, and / or expansion, wherein the iPSC line is capable of directed differentiation to produce functional derivative hematopoietic cells with improved survival, persistence, expansion, and effector cell function. In some embodiments, the cs-CD3-containing iPSCs are TCR-negative (TCR neg In some embodiments, the exogenously introduced cytokine signaling includes signaling of any one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or complete peptide of the cytokine and / or its respective receptor for cytokine signaling is expressed on the cell surface. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary. In some embodiments, the transient / transient expression of the cell surface cytokine / cytokine receptor is mediated by retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA, including mRNA. In some embodiments, the TCR negThe exogenous cell surface cytokines and / or receptors contained in the cs-CD3 iPSCs or derived cells thereof enable IL7 signaling. In some embodiments, TCR neg The exogenous cell surface cytokines and / or receptors contained in the cs-CD3 iPSCs or derived cells thereof enable IL10 signaling. In some embodiments, TCR neg The exogenous cell surface cytokines and / or receptors contained in the cs-CD3 iPSCs or derived cells enable IL15 signaling. neg In some embodiments of cs-CD3 IL iPSCs, IL15 expression is mediated by construct 3 of Figure 2. neg In some embodiments of the cs-CD3 IL iPSCs, IL15 expression is via construct 4 of Figure 2. The TCR of the above embodiment neg cs-CD3 IL iPSCs and their derivatives can autonomously maintain or improve cell growth, proliferation, expansion, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo. In some embodiments, TCR neg cs-CD3 IL iPSCs and their derived effector cells can be used with anti-CD38 antibodies to induce ADCC without eliminating the effector cells, thereby synergistically increasing the persistence and / or survival of iPSCs and their effector cells.

[0203] Further provided are iPSCs comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and a CD38 knockout, wherein the iPSCs are capable of directed differentiation to produce functional derivative hematopoietic cells with improved survival, persistence, expansion, and effector cell function. In some embodiments, the cs-CD3-containing iPSCs are TCR-negative (TCR negThe cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells and NK cells, as well as activated T cells and B cells. In some embodiments, when CD3 engagers, including anti-CD38 antibodies, CD38-binding CARs, or anti-CD38 scFvs, are used to induce ADCC and / or tumor cell targeting, TCR neg cs-CD3 CD38 - / - iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without effector cell elimination, i.e., without causing a reduction or depletion of CD38-expressing effector cells, thereby increasing the persistence and / or survival of iPSCs and their effector cells.

[0204] Also provided are iPSCs comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), a B2M knockout, a CIITA knockout, and optionally a polynucleotide encoding HLA-G, wherein the iPSCs are capable of directed differentiation to produce functional derivative hematopoietic cells. In some embodiments, the cs-CD3-containing iPSCs are TCR-negative (TCR neg In some embodiments, the TCR neg cs-CD3 B2M - / - CIITA - / - iPSCs and their derived effector cells are deficient in both HLA-I and HLA-II.

[0205] In view of the above, provided herein are iPSCs comprising one or more polynucleotides encoding one or more exogenous proteins that, when expressed, provide a cell surface CD3 complex or one or more subunits or subdomains thereof (cs-CD3), and optionally one, two, three, or all four of hnCD16, CAR, exogenous cytokine / receptor, and B2M / CIITA knockout; if B2M is knocked out, a polynucleotide encoding HLA-G, or alternatively, one or both of CD58 and CD54 knockout, are optionally introduced, and the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells. In some embodiments, the cs-CD3-containing iPSCs are TCR-negative (TCR neg In this application, TCR neg Also included are functional iPSC-derived hematopoietic cells comprising one, two, three, or all four of hnCD16, B2M / CIITA knockout, CAR, and exogenous cytokine / receptor, where if B2M is knocked out, a polynucleotide encoding HLA-G, or alternatively, one or both of CD58 and CD54 knockout are optionally introduced, and the derived hematopoietic cells include, but are not limited to, mesodermal cells with secondary hemogenic endothelial (HE) potential, secondary HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0206] Another aspect provided herein is a TCR neg, cs-CD3, and iPSCs or iPSC-derived cells comprising a truncated fusion protein of IL15 and IL15Rα, wherein the fusion protein does not comprise an intracellular domain. These embodiments, shown in FIG. 2 as "IL15Rα(ΔICD) fusion" and "IL5 / mb-Sushi," are further collectively abbreviated as IL15Δ throughout this application. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 21. neg iPSCs or iPSC-derived cells comprising cs-CD3, a truncated IL15 / IL15Rα fusion protein lacking the intracellular domain (IL15Δ), further comprising one or more of CD38 knockout, hnCD16, CAR, exogenous cytokine / receptor, and B2M / CIITA knockout, where if B2M is knocked out, a polynucleotide encoding HLA-G is optionally introduced, and the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, including, but not limited to, primary hemogenic endothelial (HE) potential, secondary HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0207] Thus, the present application provides iPSCs and their functional derived hematopoietic cells comprising any one of the following genotypes in Table 1. The "IL" provided in Table 1 represents one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on the specific cytokine / receptor expression selected. When IL represents IL15, it also includes IL15Δ, which is detailed above as a truncated fusion protein of IL15 and IL15Rα, but does not include the intracellular domain. Furthermore, when iPSCs and their functional derived hematopoietic cells have a genotype that includes both a CAR and an IL, the CAR and IL are optionally included in a bicistronic expression cassette containing a 2A sequence. By comparison, in some other embodiments, the CAR and IL are in separate expression cassettes included in the iPSCs and their functional derived hematopoietic cells. In one particular embodiment, both a CAR and an IL are included in the iPSC and its functional derived effector cells, where the IL is IL15 from construct 3 or 4 in Figure 2, and the IL15 construct is included in the expression cassette together with or separate from the CAR. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

[0208] 9. Antibodies for immunotherapy In some embodiments, in addition to the genomically engineered effector cells provided herein, additional therapeutic agents, including antibodies or antibody fragments that target antigens associated with a condition, disease, or indication, can be used with these effector cells in combination therapy. 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, tumor- or virus-specific antigens activate the administered iPSC-derived effector cells, enhancing their killing capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include anti-CD20 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, ibritumomab, ocrelizumab), anti-CD22 (inotuzumab, moxetumomab, epratuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 ( Antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include, but are not limited to, anti-EGFR (alemtuzumab), anti-EGFR (certuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells further include bispecific or multispecific antibodies that target target cells while targeting two or more antigens or epitopes on the target cells or recruiting effector cells (T cells, NK cells, or macrophage cells) to the target cells.Such bispecific or multispecific antibodies function as engagers that can direct effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils, to tumor cells and activate immune effector cells, demonstrating great potential for maximizing the benefits of antibody therapy. The engager is specific for at least one tumor antigen and at least one surface triggering receptor on immune effector cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs), or multispecific killer cell engagers, or universal engagers compatible with multiple immune cell types.

[0209] In some embodiments, the iPSC-derived effector cells comprise hematopoietic lineage cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise NK cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise T cells comprising a genotype listed in Table 1. In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises at least a TCR negThe CD3 engager comprises a bispecific or multispecific antibody that binds iPSC-derived NK cells or T cells containing cs-CD3 and cells bearing cell surface CD3. The CD3 engager comprises at least a first variable segment that binds to cs-CD3 and a second variable segment that binds to one of the following: ADGRE2, carbonic anhydrase IX (CAIX), CCRI, 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, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, or a combination thereof. 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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A and a second variable segment that binds to an antigen comprising at least one of: MAGE-A1, MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and / or a pathogen antigen.

[0210] In some embodiments of the CD3 engager, the engager comprises at least a first variable segment that binds to cs-CD3 and a second variable segment that binds to an antigen including at least one of BCMA, CD19, CD20, CD33, CD38, CD52, CD123, CEA, EGFR, EpCAM, GD2, GPA33, HER2, MICA / B, PDL1, and / or PSMA. In yet some other embodiments of the CD3 engager, the engager comprises a second variable segment that binds to an antigen including at least one of CD19, CD33, CD123, CEA, EpCAM, GPA33, HER2, and / or PSMA. In some other embodiments of the CD3 engager, the engager is at least one of blinatumomab, catumaxomab, ertumaxomab, RO6958688, AFM11, MT110 / AMG110, MT111 / AMG211 / MEDI-565, AMG330, MT112 / BAY2010112, MOR209 / ES414, MGD006 / S80880, MGD007, and / or FBTA05. In one embodiment, the combination comprises a CD3 engager and a TCR neg In one embodiment, the combination comprises a CD3 engager and an iPSC-derived NK cell comprising cs-CD3 and hnCD16. neg In some further embodiments, the iPSC-derived NK cells in combination with the CD3 engager comprise TCR neg The IL15 comprises cs-CD3, hnCD16, IL15, and a CAR targeting one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, and PDL1, wherein the IL15 is expressed simultaneously with or separately from the CAR, and the IL15 is in any one of the forms presented in constructs 1-7 in Figure 2. In some specific embodiments, the IL15, when expressed simultaneously with or separately from the CAR, is in the form of construct 3, 4, or 7.

[0211] 10. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that, if uninhibited, can suppress or downregulate immune responses. It is now clear that tumors exploit certain immune checkpoint pathways as a primary mechanism of immune resistance, particularly against tumor antigen-specific T cells. Checkpoint inhibitors (CIs) are antagonists that reduce checkpoint gene expression or gene products or decrease the activity of checkpoint molecules, thereby blocking the inhibitory checkpoint and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, and these agents have led to long-term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a significant concern. One aspect of the present application provides a therapeutic approach to overcoming CI resistance by including genomically engineered functional iPSC-derived cells in combination with CIs. In one embodiment of the combination therapy, the iPSC-derived cells are NK cells. In another embodiment of the combination therapy, the iPSC-derived cells are T cells. In addition to exhibiting direct antitumor capabilities, the derived NK cells provided herein have been shown to have the ability to resist PDL1-PD1-mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at tumor sites. Thus, tumor infiltration of T cells facilitated by functionally potent, genome-engineered derived NK cells indicates that the NK cells can synergize with T cell-targeted immunotherapies, including checkpoint inhibitors, to alleviate local immune suppression and reduce tumor burden.

[0212] In one embodiment, a TCR-derived TCR for checkpoint inhibitor combination therapy negThe NK cells comprise cs-CD3, and optionally one, two, three, or all four of hnCD16 expression, B2M / CIITA knockout, CAR expression, CD38 knockout, and expression of an exogenous cell surface cytokine and / or receptor, and if B2M is knocked out, optionally a polynucleotide encoding HLA-G or a knockout of one or both of CD58 and CD54. In some embodiments, the derived NK cells comprise any one of the genotypes listed in Table 1. In some embodiments, the derived NK cells additionally comprise deletions or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D, E, F, F, G, H, H, I, I, M, N, O, P, P, R, S, T ... 2A and induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, an Fc receptor, an engager, and a bispecific, multispecific, or universal engager.

[0213] In another embodiment, a TCR-derived TCR for checkpoint inhibitor combination therapy negThe T cells comprise cs-CD3, and optionally one, two, three, or all four of hnCD16 expression, B2M / CIITA knockout, CAR expression, CD38 knockout, and expression of an exogenous cell surface cytokine and / or receptor, and if B2M is knocked out, optionally a polynucleotide encoding HLA-G or a knockout of one or both of CD58 and CD54. In some embodiments, the derived T cells comprise any one of the genotypes listed in Table 1. In some embodiments, the derivative T cells additionally comprise deletions or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A and induced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, an Fc receptor, an engager, and a bispecific, multispecific, or universal engager.

[0214] The derived NK cells or T cells described above may be TCR neg In some embodiments, the iPSC clonal line is derived from differentiation of an iPSC clonal line comprising cs-CD3, and optionally one, two, three, or all four of hnCD16 expression, B2M / CIITA knockout, CAR expression, CD38 knockout, and exogenous cell surface cytokine expression, wherein if B2M is knocked out, a polynucleotide encoding HLA-G or knockout of one or both of CD58 and CD54 is optionally introduced. In some embodiments, the iPSC clonal line is derived from differentiation of an iPSC clonal line comprising cs-CD3, and optionally one, two, three, or all four of hnCD16 expression, B2M / CIITA knockout, CAR expression, CD38 knockout, and exogenous cell surface cytokine expression, wherein ... hnCD16 expression, B2M / CIITA knockout, CAR2A Further comprising introduced or increased expression of at least one of a surface triggering receptor for binding to an R, a CAR, an Fc receptor, an engager, and a bispecific, multispecific, or universal engager.

[0215] Suitable checkpoint inhibitors for combination therapy with derived 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, CD73 (NT5 E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0216] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of whole antibodies and may be more cost-effective to produce, easier to use, or more sensitive than whole antibodies. In some embodiments, the one, two, or three or more checkpoint inhibitors comprise at least one of atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR), IPH43 (anti-MICA), IPH33 (anti-TLR3), lilimumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0217] In some embodiments, antagonists that inhibit any of the above checkpoint molecules are microRNA-based, as many miRNAs are found to be regulators of immune checkpoint expression (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.

[0218] Some embodiments of the combination therapies with provided iPSC-derived NK cells or T cells include at least one checkpoint inhibitor that targets at least one checkpoint molecule, and the iPSC-derived cells have a genotype listed in Table 1. Some other embodiments of the combination therapies with provided derived NK cells or T cells include two, three, or more checkpoint inhibitors, such that two, three, or more checkpoint molecules are targeted. In some embodiments of the combination therapies including at least one checkpoint inhibitor and iPSC-derived cells having a genotype listed in Table 1, the checkpoint inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the checkpoint inhibitor is produced by the iPSC-derived cells by expressing an exogenous polynucleotide sequence encoding the antibody, or fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the checkpoint-inhibiting antibody, or fragment or variant thereof, is co-expressed with the CAR, either in a separate construct or in a bicistronic construct including both the CAR and sequences encoding the antibody or fragment thereof. In some further embodiments, the sequence encoding the antibody or fragment thereof can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence, depicted as, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences for the checkpoint inhibitor and CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered, expressed, and secreted as a payload by derived effector cells capable of infiltrating the tumor microenvironment (TME), binding to the TME counteracts inhibitory checkpoint molecules, allowing effector cell activation by activating modalities such as CARs or activating receptors.In some embodiments, the checkpoint inhibitor co-expressed with the CAR inhibits 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, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR. In some embodiments, the checkpoint inhibitor co-expressed with the CAR in the derivative cells having a genotype listed in Table 1 is selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is atezolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is nivolumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0219] In some other embodiments of the combination therapies comprising iPSC-derived cells and at least one antibody that inhibits a checkpoint molecule provided herein, the antibody is not produced by or in the iPSC-derived cells and is additionally administered before, together with, or after administration of iPSC-derived cells having a genotype listed in Table 1. In some embodiments, the administration of one, two, three or more checkpoint inhibitors in the combination therapy with the provided derived NK cells or T cells is simultaneous or sequential. In one embodiment of the combination therapy comprising derived NK cells or T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments of combination therapies comprising derived NK cells or T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is atezolizumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising derived NK cells or T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is nivolumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising derived NK cells or T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is pembrolizumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof.

[0220] II. Methods for targeted genome editing at selected loci in iPSCs Genome editing, or genomic 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 targeted cell. Targeted genome editing (interchangeably referred to as "targeted genomic editing" or "targeted gene editing") allows for insertion, deletion, and / or replacement at preselected sites within the genome. When endogenous sequences are deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or down due to the deletion of the sequence. Therefore, targeted editing can also be used to precisely disrupt the expression of endogenous genes. The term "targeted integration" is similarly used herein to refer to a process involving the insertion of one or more exogenous sequences, with or without deletion of endogenous sequences at the insertion site. In comparison, randomly integrated genes are subject to position effects and silencing, and their expression is unreliable and unpredictable. For example, centromeric and subtelomeric regions are particularly prone to transgene silencing. Reciprocally, newly integrated genes can affect surrounding endogenous genes and chromatin, potentially altering cellular behavior or supporting cellular transformation. Therefore, inserting exogenous DNA into preselected loci, such as safe harbor loci or genomic safe harbors (GSH), is crucial for safety, efficiency, copy number control, and reliable gene response regulation.

[0221] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is induced by the host cell's enzymatic machinery via homologous sequences flanking the exogenous polynucleotide to be inserted.

[0222] Alternatively, targeted editing can be achieved at higher frequencies through the specific introduction of double-strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in the random insertion or deletion (in / del) of a small number of endogenous nucleotides. In contrast, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homologous arms, the exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) via homologous recombination, resulting in "targeted integration." In some cases, the targeted integration site is intended to be within the coding region of a selected gene, allowing targeted integration to disrupt gene expression and simultaneously result in knock-in and knock-out (KI / KO) in a single editing step.

[0223] One or more transgenes can be inserted into a selected location of a gene locus of interest (GOI) to simultaneously knock out the genes. Suitable loci for simultaneous knock-in and knock-out (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. Each site-specific targeting homology arm for site-selective insertion allows the transgene to be expressed either under the endogenous promoter of that site or under the exogenous promoter included in the construct. When inserting two or more transgenes into a selected location (e.g., into the CD38 locus), a linker sequence, such as a 2A linker or IRES, is placed between any two transgenes. The 2A linker encodes a self-cleaving peptide derived from FMDV, ERAV, PTV-I, or TaV (referred to as "F2A," "E2A," "P2A," and "T2A," respectively), allowing separate proteins to be produced from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter can be CAG or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, including, but not limited to, CMV, EF1α, PGK, and UBC.

[0224] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR (clustered regularly interspaced palindromic repeats) systems. Additionally, the DICE (dual integrase cassette exchange) system, which utilizes phiC31 and Bxb1 integrases, is also a promising tool for targeted integration.

[0225] ZFN is a targeted nuclease 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 the zinc finger binding domain, and its structure is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A "designed" zinc finger domain is a domain that does not exist in nature, whose design / composition is primarily based on rational criteria, such as the application of substitution rules and computerized algorithms to process information from databases storing information on existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261, and also WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496. A "selected" zinc finger domain is a domain not found in nature, and its production primarily results from empirical processes such as phage display, interaction trapping, or hybrid selection. ZFNs are described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of FokI nuclease with a zinc finger DNA binding domain.

[0226] TALENs are targeted nucleases containing a nuclease fused to a TAL effector DNA-binding domain. "Transcription activator-like effector DNA-binding domain," "TAL effector DNA-binding domain," or "TALE DNA-binding domain" refers to the polypeptide domain of a TAL effector protein involved in binding the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA-binding domain, and activate gene transcription at these sequences via their transactivation domain. The specificity of the TAL effector DNA-binding domain depends on the effector's variable number of imperfect 34-amino acid repeats, which contain polymorphisms at select repeat positions called repeat variable dinucleotides (RVDs). TALENs are described in more detail in U.S. Patent Application Publication No. 2011 / 0145940, incorporated herein by reference. The most recognized example of a TALEN in the art is a polypeptide fusion of FokI nuclease to a TAL effector DNA binding domain.

[0227] Another example of a targeted nuclease that finds use in the subject methods is a targeted Spo11 nuclease, which is a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain having specificity for a DNA sequence of interest, e.g., a zinc finger DNA-binding domain, a TAL effector DNA-binding domain, etc. See, e.g., U.S. Patent Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0228] Additional 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.

[0229] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases; and the like.

[0230] As an illustrative example, CRISPR / Cas9 requires two key components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When coexpressed, the two components form a complex and are recruited to a target DNA sequence containing a PAM and a seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target a selected sequence. These two components can be delivered to mammalian cells via transfection or transduction. When using the CRISPR / Cpf system, a Cpf endonuclease (Cpf1, MAD7, and many more known in the art) and (2) a gRNA, which often does not require tracrRNA, are required to guide the Cpf endonuclease to target a selected sequence.

[0231] DICE-mediated insertion uses a pair of recombinases, e.g., phiC31 and Bxb1, to provide unidirectional integration of exogenous DNA, strictly limited by each enzyme's own small attB and attP recognition sites. These target att sites do not naturally occur in mammalian genomes and must therefore first be introduced into the genome at the desired integration site. See, e.g., U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0232] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method for targeted integration comprises introducing the construct into a cell to allow site-specific homologous recombination by the cellular host enzyme machinery. In another embodiment, the method for targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a ZFN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow ZFN-mediated insertion. In yet another embodiment, the method for targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a TALEN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow TALEN-mediated insertion. In another embodiment, the method for targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, introducing a Cas9 expression cassette, and introducing a gRNA comprising a guide sequence specific to the desired integration site into the cell to allow Cas9-mediated insertion. In yet another embodiment, a method for targeted integration in a cell comprises introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for the DICE recombinase to enable targeted integration via DICE.

[0233] Promising sites for targeted integration include, but are not limited to, safe harbor loci or genomic safe harbors (GSHs), which are intragenic or extragenic regions of the human genome that, theoretically, can accommodate predictable expression of the newly integrated DNA without adversely affecting the host cell or organism. A useful safe harbor must allow sufficient transgene expression to obtain the desired levels of vector-encoded protein or non-coding RNA. The safe harbor must also not predispose cells to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it ideally must meet several criteria, including but not limited to: no disruption of regulatory elements or genes as determined by sequence annotation; an intergenic region within a gene-dense region or a convergent position between two genes transcribed in opposite directions; distance between vector-encoded transcriptional activators and promoters of neighboring genes, particularly cancer-related and microRNA genes, to minimize the possibility of long-range interactions; and apparent ubiquitous transcriptional activity, as reflected by widespread spatial and temporal expressed sequence tag (EST) expression patterns indicative of ubiquitous transcriptional activity. This latter feature is particularly important in stem cells, where chromatin remodeling during differentiation typically results in the silencing of some loci and the potential activation of others. Within a region suitable for exogenous insertion, the precise locus selected for insertion should lack repetitive elements and conserved sequences, allowing for easy design of primers for homologous arm amplification.

[0234] Suitable sites for human genome editing, or specifically targeted integration, include, but are not limited to, the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the human ortholog of the mouse ROSA26 locus. Additionally, the human ortholog of the mouse H11 locus may also be suitable for insertion using the compositions and targeted integration methods disclosed herein. Furthermore, the collagen and HTRP loci may also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be necessary, particularly in stem cells for specific integration events, and optimization of the insertion strategy, including promoter selection, exogenous gene sequence and placement, and construct design, is often required.

[0235] In the case of targeted in / dels, the editing site is often contained in the endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted in / del is associated with immune response control and regulation. In some other embodiments, the endogenous gene containing the targeted in / del is associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response control and regulation, or proteins that inhibit the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem and / or progenitor cells and their derived cells.

[0236] Thus, one aspect of the present invention provides a method for targeted integration at a selected locus that includes a genomic safe harbor, or a preselected locus that is known or proven to be safe and well-regulated for continuous or transient gene expression, such as the B2M, TAP1, TAP2, tapasin, TRAC, or CD38 loci provided herein. In one embodiment, the genomic safe harbor for the targeted integration method includes one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genomic safe harbor. In some embodiments, the targeted integration is at one or more loci where gene knockdown or knockout as a result of integration is desired, including, but not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0237] In one embodiment, a method for targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing a construct comprising a pair of homologous arms specific for a desired integration site and one or more exogenous sequences to enable site-specific homologous recombination by the cellular host enzyme machinery, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0238] In another embodiment, a method for targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, to allow for ZFN-mediated insertion. In yet another embodiment, a method for targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site to enable TALEN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.In another embodiment, a method for targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a gRNA comprising a Cas9 expression cassette and a guide sequence specific for a desired integration site to allow Cas9-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable targeted integration via DICE, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0239] Furthermore, as provided herein, the above-described methods for targeted integration under safe harbor can be used to insert any polynucleotide of interest, such as polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and proteins that promote stem and / or progenitor cell engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival. In some other embodiments, the 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. Suitable suicide gene systems 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). In addition, some suicide gene systems are cell type specific; for example, genetic modification of T lymphocytes with the B cell molecule CD20 allows them to be eliminated by administration of the mAb rituximab. Furthermore, modified EGFR-containing epitopes recognized by cetuximab can be used to deplete genetically engineered cells when exposed to cetuximab. Thus, one aspect of the present invention provides a method for targeted integration of one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0240] In some embodiments, one or more exogenous polynucleotides integrated by the methods herein are driven by an operably linked exogenous promoter included in a construct for targeted integration. The promoter may be inducible or constitutive, and may be time-specific, tissue-specific, or cell-type specific. Constitutive promoters suitable for the methods of the present invention include, but are not limited to, the cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0241] The exogenous polynucleotides integrated by the methods herein can be driven at the integration site by an endogenous promoter in the host genome. In one embodiment, the methods of the present invention are used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the methods of the present invention are used for targeted integration at the ROSA26 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the methods of the present invention are used for targeted integration at the H11 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the methods of the present invention are used for targeted integration at the collagen locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the methods of the present invention are used for targeted integration at the HTRP locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous HTRP promoter. In theory, only correct insertion at the desired location will allow gene expression of the exogenous gene driven by the endogenous promoter.

[0242] In some embodiments, one or more exogenous polynucleotides included in a construct for targeted integration methods 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 increase the physical separation between the portions and maximize access to enzymatic machinery. The linker peptide of the linker sequence can be composed of amino acids selected to make the physical separation between the portions (exogenous polynucleotides and / or the proteins or peptides encoded therefrom) more flexible or more rigid depending on the function involved. The linker sequence can be cleavable by a protease or chemically cleavable to generate separate portions. Examples of enzymatic cleavage sites in a linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is naturally produced by the host or is exogenously introduced. Alternatively, the cleavage site in the linker can be cleavable upon exposure to a selected chemical, such as cyanogen bromide, hydroxylamine, or low pH. The optional linker sequence may serve a purpose other than providing a cleavage site. The linker sequence should allow for effective positioning of a moiety relative to another adjacent moiety for proper function. The linker may also be a simple amino acid sequence of sufficient length to prevent steric hindrance between the moieties. In addition, the linker sequence may provide for post-translational modifications, including, but not limited to, phosphorylation sites, biotinylation sites, sulfation sites, gamma-carboxylation sites, etc. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single, undesired conformation. To provide flexibility, the linker may be composed primarily 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 contains glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, a G4S linker peptide separates the terminal processing domain and the endonuclease domain of the fusion protein. In other embodiments, a 2A linker sequence allows two separate proteins to be produced from a single translation. Suitable linker sequences can be easily identified empirically. In addition, the suitable size and sequence of the linker sequence can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some embodiments, any two consecutive linker sequences are different.

[0243] Introduction of a construct containing an exogenous polynucleotide into cells for targeted integration can be achieved using known methods for gene transfer into cells. In one embodiment, the construct comprises a viral vector backbone, such as an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a Sendai viral vector. In some embodiments, a plasmid vector is used to deliver and / or express the exogenous polynucleotide into target cells (e.g., pAl-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.). In other embodiments, an episomal vector is used to deliver the exogenous polynucleotide into target cells. In some embodiments, recombinant adeno-associated viruses (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV does not integrate into the host genome. In addition, episomal rAAV vectors mediate homology-directed gene targeting at a much higher rate than traditional targeting plasmid transfection. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions into target sites in the genome of iPSCs. In some embodiments, the genomically modified iPSCs and their derivatives obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0244] III. Methods for obtaining and maintaining genome-engineered iPSCs The present invention provides methods for obtaining and maintaining genomically engineered iPSCs containing one or more targeted edits at one or more desired sites, wherein the targeted edits remain intact and functional at each selected edit site in expanded genomically engineered iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions, i.e., targeted integrations and / or indels, at selected sites into the genome of iPSCs and derived cells. The many advantages of obtaining genomically engineered iPSCs via iPSC editing and differentiation as provided herein compared to directly manipulating patient-derived peripheral blood-derived primary effector cells 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 resulting effector cells have elongated telomeres and are resilient due to reduced attrition; and the effector cell population is homogenous in terms of editing site, copy number, and absence of allelic variation, random mutation, and expression diversity, primarily due to the ability to perform clonal selection on the engineered iPSCs provided herein.

[0245] In certain embodiments, genomically engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and expanded long-term as single cells in a cell culture medium shown in Table 2 as fate-maintaining medium (FMM), where the iPSCs retain the targeted edits and functional modifications at the selected sites. The composition of the medium may be present in the medium in amounts within the optimal ranges shown in Table 2. iPSCs cultured in FMM have been shown to remain undifferentiated, maintaining a basal or naive profile, maintaining genomic stability without the need to wash or select the culture, and readily give rise to all three somatic lineages, in vitro differentiation via embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation via teratoma formation. See, e.g., U.S. Patent Application No. 61 / 947,979, the disclosure of which is incorporated herein by reference. [Table 11]

[0246] In some embodiments, genomically engineered iPSCs containing one or more targeted integrations and / or in / dels are maintained, passaged, and expanded in medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and free of or essentially free of a TGFβ receptor / ALK5 inhibitor, wherein the iPSCs retain intact and functional targeted edits at the selected sites.

[0247] Another aspect of the present invention provides methods for generating genomically engineered iPSCs through targeted editing of iPSCs, or by first generating genomically engineered non-pluripotent cells through targeted editing and then reprogramming the selected / isolated genomically engineered non-pluripotent cells to obtain iPSCs containing the same targeted edits as the non-pluripotent cells. A further aspect of the present invention provides genomically engineered non-pluripotent cells undergoing simultaneous reprogramming by introducing targeted integrations and / or targeted in / dels into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and the reprogramming conditions include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genomic engineering and reprogramming, the targeted integrations and / or targeted in / dels can be introduced into the non-pluripotent cells prior to or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and, optionally, small molecules.

[0248] In some embodiments, for simultaneous genome manipulation and reprogramming of non-pluripotent cells, targeted integrations and / or in / dels may also be introduced into non-pluripotent cells after the multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and the vector carrying the construct is introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181, and CD30.

[0249] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor, and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 2). In some embodiments, genomically engineered iPSCs by any of the methods described above are further maintained and expanded using a mixture comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 2).

[0250] In some embodiments of the method of generating genomically engineered iPSCs, the method comprises genomically engineering iPSCs by introducing one or more targeted integrations and / or in / dels into the iPSCs to obtain genomically engineered iPSCs having at least one genotype listed in Table 1. Alternatively, the method of generating genomically engineered iPSCs comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genomically engineered non-pluripotent cell comprising a targeted integration and / or in / del at a selected site, and (b) contacting the genomically engineered non-pluripotent cell with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, to obtain a genomically engineered iPSC comprising a targeted integration and / or in / del at the selected site. Alternatively, a method for generating genomically engineered iPSCs includes: (a) contacting non-pluripotent cells with a small molecule composition containing one or more reprogramming factors and, optionally, a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells; (b) introducing one or more targeted integrations and / or in / dels into the reprogrammed non-pluripotent cells for genome manipulation; and (c) obtaining genomically engineered iPSCs containing targeted integrations and / or in / dels at selected sites. Any of the above methods may further include single-cell sorting of the genomically engineered iPSCs to obtain clonal iPSCs. Clonal expansion of the genomically engineered iPSCs generates a master cell bank containing single-cell sorted and expanded clonally engineered iPSCs having at least one phenotype as provided in Table 1 herein. The master cell bank is then cryopreserved, providing a platform for additional iPSC manipulations and a renewable source for manufacturing off-the-shelf, engineered, homogenous cell therapy products that are well-defined, uniform in composition, and can be mass-produced at scale in a cost-effective manner.

[0251] The reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in PCT / US2015 / 018801 and PCT / US16 / 57136 (the disclosures of which are incorporated herein by reference). One or more reprogramming factors may be in the form of a polypeptide. Reprogramming factors may also be in the form of polynucleotides, and are introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced via a plasmid combination. See, e.g., U.S. Patent Application No. 62 / 571,105, the disclosure of which is incorporated herein by reference.

[0252] In some embodiments, non-pluripotent cells are transfected with multiple constructs containing different exogenous polynucleotides and / or different promoters via multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides may include genes encoding suicide genes, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, potential drug targets, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNAs, including, but not limited to, siRNAs, shRNAs, miRNAs, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue- or cell-type-specific promoters. Thus, the polynucleotides can be expressed under conditions that activate the promoter, such as in the presence of an inducer, or in specific differentiated cell types. 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, e.g., capase-9 driven by CAG. These constructs, containing different exogenous polynucleotides and / or different promoters, can be introduced into non-pluripotent cells either simultaneously or sequentially. Non-pluripotent cells subjected to targeted integration of multiple constructs can be simultaneously contacted with one or more reprogramming factors to initiate reprogramming simultaneously with genome manipulation, thereby obtaining genomically engineered iPSCs containing multiple targeted integrations in the same pool of cells. This robust method thus enables simultaneous reprogramming and manipulation strategies to lead to clonal genomically engineered hiPSCs with multiple modalities integrated into one or more selected target sites.In some embodiments, the genomically modified iPSCs and derived cells th...

Claims

1. A cell or population thereof, (i) the cell is an induced pluripotent stem cell (iPSC), a clonal iPSC, or a clonal iPS cell line cell, or a derivative cell obtained from differentiation of the iPSC; (ii) the cells lack expression of an endogenous TCR (TCR ｎｅｇ ), (iii) the cells comprise one or more polynucleotides encoding one or more exogenous proteins that provide a cell surface CD3 complex (cs-CD3), which, when expressed, lacks antigen-recognition activity; (iv) the cells or T cells derived therefrom induce the formation of cs-CD3 comprising (a) a CD3ζ endodomain, and (b) one or more of a CD3ε ectodomain, a CD3δ ectodomain, and a CD3γ ectodomain; (v) the one or more exogenous proteins are (a) nb-rTCR, a nonbinding recombinant TCR characterized by (1) a transgenic TCRα constant region lacking the TCRα variable region (tgTARC) or (2) a transgenic TCRβ constant region lacking the TCRβ variable region (tgTABC); (b) p-rTCR, a recombinant pre-TCR containing the extracellular and intracellular domains of pre-TCRα; (c) nb-rTCR-CD3, a non-binding recombinant TCR comprising a fusion protein containing (1) a transgenic TCR alpha constant region, tgTRAC, or a transgenic TCR beta constant region, tgTRBC, (2) a CD3 epsilon ectodomain, and (3) a CD3 delta ectodomain or a CD3 gamma ectodomain; or (d) ccCD3, a CD3 chimeric chain comprising a fusion protein between (1) the CD3ε ectodomain, (2) the CD3δ ectodomain or the CD3γ ectodomain, and (3) the CD3ζ ectodomain; Including, (vi) the derivative cells comprise derivative T cells or precursor cells thereof; A cell or a group thereof.

2. the one or more polynucleotides (i) a transgenic fusion protein (tgCD3(ε-δ)-TRAC) comprising the ectodomains of CD3ε and CD3δ and the TCRα constant region; (ii) a transgenic fusion protein (tgCD3(ε-γ)-TRBC) comprising the ectodomains of CD3ε and CD3γ and the TCRβ constant region; (iii) a transgenic fusion protein (tgCD3(ε-γ)-TRAC) comprising the ectodomains of CD3ε and CD3γ and the TCRα constant region; (iv) a transgenic fusion protein (tgCD3(ε-δ)-TRBC) comprising the ectodomains of CD3ε and CD3δ and the TCRβ constant region; (v) a transgenic fusion protein (tgCD3(ε-γ)-ζ) comprising the ectodomains of CD3ε and CD3γ and the endodomain of CD3ζ; (vi) a transgenic fusion protein (tgCD3(ε-δ)-ζ) comprising the ectodomains of CD3ε and CD3δ and the endodomain of CD3ζ; (vii) a transgenic fusion protein comprising the ectodomain of CD3ε, the ectodomain of CD3γ or CD3δ, the endodomain of CD3ζ, and the signaling domain of CD28 (tgCD3(ε-γ / δ)-28ζ); (viii) a transgenic fusion protein comprising the ectodomain of CD3ε, the ectodomain of CD3γ or CD3δ, the endodomain of CD3ζ, and the signaling domain of 41BB (tgCD3(ε-γ / δ)-BBζ); and / or (ix) a transgenic fusion protein (tgCD3(ε-γ / δ)-(28-BB)ζ) comprising the ectodomain of CD3γ or CD3δ, the endodomain of CD3ζ, the signaling domain of CD28, and the signaling domain of 41BB; The cell or population thereof according to claim 1, wherein the cell or population thereof encodes an exogenous protein comprising one or more of the following: wherein the one or more polynucleotides encoding tgTRAC, tgTCRα, p-rTCR, tgCD3(ε-δ)-TRAC, tgCD3(ε-γ)-TRAC, tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, or tgCD3(ε-γ / δ)-(28-BB)ζ are inserted into the TRAC locus and, optionally, are operably linked to the endogenous promoter of TRAC upon insertion; or 3. The cell or population thereof according to claim 2, wherein the one or more polynucleotides encoding tgTRBC, tgTCRβ, tgCD3(ε-γ)-TRBC, tgCD3(ε-δ)-TRBC, tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, and / or tgCD3(ε-γ / δ)-(28-BB)ζ are inserted into the TRBC locus and, optionally, are operably linked to an endogenous promoter of TRBC upon insertion.

4. the transgenic fusion protein comprises the constant region of TCRα and, optionally, an N-terminal signal peptide; or the transgenic fusion protein comprises the constant region of TCRα and a C-terminal poly-A tail, and optionally an N-terminal signal peptide; or the transgenic fusion protein comprises the constant region of TCRα, and optionally an N-terminal signal peptide, and the insertion of the polynucleotide is in an exon of the constant region and in frame; or the transgenic fusion protein comprises the constant region of TCRβ and optionally an N-terminal signal peptide; or the transgenic fusion protein comprises the constant region of TCRβ and a C-terminal polyA tail and, optionally, an N-terminal signal peptide; or the transgenic fusion protein comprises a constant region of TCRβ, and optionally an N-terminal signal peptide, and the insertion of the polynucleotide is in an exon of the constant region and in frame. A cell or population thereof according to claim 3.

5. The one or more polynucleotides inserted into the TRAC and / or TRBC locus disrupt expression of TRAC and TRBC, respectively, and inhibit TCR. ｎｅｇ The cell or population thereof according to claim 3, which results in a cell.

6. 6. The cell or population thereof of claim 5, wherein the one or more polynucleotides inserted into the TRAC and / or TRBC loci are driven by (i) the endogenous promoters of TRAC and TRBC, respectively, or (ii) a heterologous promoter.

7. (i) the nb-rTCR comprises one or both of a tgTRAC and a tgTRBC; (ii) the p-rTCR comprises a tgpTCRα, and optionally a tgTRBC or a tgTCRβ; or 2. The cell or population thereof of claim 1, wherein (iii) the ccCD3 comprises at least one of the fusion proteins: tgCD3(ε-γ)-ζ, tgCD3(ε-δ)-ζ, tgCD3(ε-γ / δ)-28ζ, tgCD3(ε-γ / δ)-BBζ, and tgCD3(ε-γ / δ)-(28-BB)ζ, wherein the fusion protein further comprises a cytoplasmic domain comprising the ectodomain of a CD3ε, CD3δ, and / or CD3γ protein and the endodomain of a CDζ protein, and optionally one or both of a CD28 signaling domain and a 41BB signaling domain.

8. The cell or population thereof according to claim 2, wherein the nb-rTCR-CD3 comprising the fusion protein tgCD3(ε-δ)-TRAC further comprises tgTRBC or tgTCRβ, or the nb-rTCR-CD3 comprising the fusion protein tgCD3(ε-γ)-TRBC further comprises tgTRAC or tgTCRα.

9. (i) the cell comprises one of the recombinant TCRs: nb-rTCR or p-rTCR, and the recombinant TCR forms a complex with an endogenous CD3 polypeptide, thereby enabling cell surface presentation of the endogenous CD3 polypeptide and its signal transduction; or (ii) The cell or population thereof according to claim 7, wherein the cell contains nb-rTCR-CD3 or ccCD3, thereby enabling cell surface presentation of exogenous CD3 polypeptide and its signal transduction.

10. The cells (i) HLA-I deficiency, (ii) HLA-II deficiency, (iii) introduced expression of HLA-G or non-cleavable HLA-G; (iv) exogenous CD16, (v) chimeric antigen receptor (CAR), (vi) a cell surface-expressed exogenous cytokine or its receptor peptide; (vii) at least one of the genotypes listed in Table 1; (viii) deletion or reduced expression of at least one of B2M, CD38, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region; and (ix) HLA-E, 41BBL, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ 2. The cell or population thereof of claim 1, further comprising one or more of introduced or increased expression of at least one of an R, a CAR, an Fc receptor, an engager, and a surface triggering receptor for binding with a bi- or multispecific or universal engager.

11. The cell or population thereof of claim 10, wherein the cell further comprises exogenous CD16.

12. The CD16 (a) F176V and S197P in the ectodomain of CD16; (b) an ectodomain derived from CD64; (c) a non-native (or non-CD16) transmembrane domain; (d) a non-native (or non-CD16) intracellular domain; (e) a non-native (or non-CD16) signaling domain; (f) a non-native stimulatory domain, and (g) The cell or population thereof described in claim 11, which comprises at least one of a transmembrane, signaling, and stimulatory domain not derived from CD16 but derived from the same or a different polypeptide.

13. The cells further comprise a chimeric antigen receptor (CAR), the CAR comprising: (i) T cell-specific or NK cell-specific; (ii) is a bispecific antigen-binding CAR; (iii) is a switchable CAR; (iv) is a dimerized CAR; (v) is a split CAR; (vi) is a multi-chain CAR; (vii) is an inducible CAR; (viii) is co-expressed with another CAR; (ix) co-expressed with a cell surface-expressed exogenous cytokine or its receptor peptide, optionally in a separate or bicistronic construct; (xi) co-expressed with a checkpoint inhibitor, optionally in a separate or bicistronic construct; (xii) specific for CD19 or BCMA; and / or (xiii) ADGRE2, carbonic anhydrase IX (CAIX), 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, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase Zerb-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 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A specific for any one of: MAGE-A1, MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms' tumor protein (WT-1), and pathogen antigens; 11. The cell or population thereof of claim 10, wherein the CAR of any one of (i) to (xiii) is optionally inserted into a TRAC locus and / or is driven by the endogenous promoter of a TCR, and / or the TCR is knocked out by the CAR insertion.

14. The cells containing cell surface-expressed exogenous cytokine or its receptor, or exogenous cytokine or its receptor peptide, (a) comprises at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors; or (b) (i) Co-expression of IL15 and IL15Rα by using a self-cleaving peptide; (ii) a fusion protein of IL15 and IL15Rα; (iii) an IL15 / IL15Rα fusion protein having a truncated intracellular domain of IL15Rα; (iv) a fusion protein of IL15 and the membrane-binding Sushi domain of IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and common receptor γC, wherein the common receptor γC is native or modified; and (vii) at least one homodimer of IL15Rβ; (Any one of (i) to (vii) may be co-expressed with the CAR in a separate or bicistronic construct); Optionally, (c) a polynucleotide encoding IL15Δ comprising an amino acid sequence having at least 90%, 95%, or 99% identity to SEQ ID NO: 17, 19, or 21, and optionally (d) transiently expressed; A cell or population thereof according to claim 10.

15. The cell or population thereof of claim 1 , wherein the derivative cells comprise derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell precursor cells, or derived T cells.

16. A clonal master cell bank comprising cells of a clonal iPSC cell line according to any one of claims 1 to 15.

17. A composition comprising a cell or a population thereof according to any one of claims 1 to 15.

18. A composition for therapeutic use comprising a derivative cell according to any one of claims 1 to 15 and one or more therapeutic agents.

Images