Stealth strategies engaging immune recognition pathways for use in allogeneic cell therapy

Abstract

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

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 328,730, filed April 7, 2022, U.S. Provisional Application No. 63 / 341,970, filed May 13, 2022, U.S. Provisional Application No. 63 / 382,620, filed November 7, 2022, and U.S. Provisional Application No. 63 / 386,880, filed December 9, 2022, the disclosures of which are incorporated by reference herein in their entireties.

[0002] Incorporation by reference of sequence listing The sequence listing entitled 184143-642601_SL.xml, created on April 5, 2023 and having a size of 85,569 bytes, is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates generally to the field of off-the-shelf immune cell products. More specifically, the present disclosure relates to a strategy for developing multifunctional effector cells that can provide therapeutically relevant properties in vivo. The cell products developed according to the present disclosure address critical limitations of patient-derived cell therapy. [Background technology]

[0004] The field of adoptive cell therapy is currently focused on using patient- and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapy and to provide the therapy to all patients who may benefit from it. There is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote better patient outcomes. Lymphocytes, such as T cells and natural killer (NK) cells, are potent antitumor effectors that play a key role in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains challenging and there is an unmet need for improvement. Thus, there remains a significant opportunity 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 rate, cell attrition, loss of transfused cells (viability and / or persistence), tumor escape due to target loss or lineage switching, precision of tumor targeting, off-target toxicity, off-tumor effects, to efficacy against solid tumors, e.g., the tumor microenvironment and associated immune suppression, recruitment, trafficking, and invasion.

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

[0007] In some embodiments, the iPSC-derived non-pluripotent cells of the present application include, but are not limited to, CD34 cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells (HSCs), hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, and B cells. In some embodiments, 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. In some embodiments, the engineered clonal iPSC differentiation strategy to obtain genetically engineered derivative cells provides that the generation potential of the iPSC in directed differentiation is not adversely affected by the engineered modalities of the iPSC, and that the engineered modalities function as intended in the derivative cells. Moreover, such a strategy overcomes the current barrier in engineering primary lymphocytes, such as T cells or NK cells, obtained from peripheral blood, which are difficult to engineer because such cells often lack reproducibility and homogeneity, resulting in cells that exhibit poor cell persistence with high cell death and low cell proliferation. Moreover, the strategies disclosed herein can avoid the generation of heterogeneous effector cell populations that are otherwise obtained using initially heterogeneous primary cell sources.

[0008] Some aspects of the present invention provide genomically engineered iPSCs obtained after, simultaneously with, or prior to the reprogramming process using a method that includes the following (I), (II), or (III), respectively, reflecting a strategy of genomic engineering.

[0009] (I): Genetically engineering iPSCs by performing one or both of the following (i) and (ii) in any order: (i) introducing one or more constructs into iPSCs to allow targeted integration at selected sites, (ii)(a) introducing one or more double strand breaks at the selected sites into the 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 generate targeted indels at the selected sites, thereby obtaining genomically engineered iPSCs capable of differentiating into partially or fully differentiated cells.

[0010] (II): Genetically reprogramming 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 the following in any order: (a) one or more constructs that allow targeted incorporation at the selected site, (b) one or more double-strand breaks at the selected site using at least one endonuclease that can recognize the selected site. The cells of step (II)(ii)(b) are then cultured to allow endogenous DNA repair to generate targeted in / dels at the selected site. The resulting genomically engineered iPSCs thus 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 to reprogram and obtain genomically engineered iPSCs, which includes (i) and (ii): (i) introducing into the non-pluripotent cells one or both of the following (a) and (b) in any order: (a) one or more constructs enabling targeted incorporation at the selected sites, (b) one or more double strand breaks at the selected sites using at least one endonuclease capable of recognizing the selected sites, where the breaks allow endogenous DNA repair to generate targeted in / dels at the selected sites, 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 targeted editing at the selected sites. Thereby, genomically engineered iPSCs are obtained that contain at least one functional targeted genome edit, and the genomically engineered iPSCs can be differentiated into partially or fully differentiated cells.

[0012] In one embodiment of the above-described 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, pharma- ceutically 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 derived cells. In some embodiments, the exogenous polynucleotides for insertion are operably linked to one or more exogenous promoters, including (1) CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, or (2) one or more endogenous promoters contained at the selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of the genomic safe harbor. In some embodiments, the genomically engineered iPSCs generated using the methods described above comprise one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, where if the genomically engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated into different safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotides encode partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors. In some embodiments, the partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors encoded by the exogenous polynucleotide are in the form of a fusion protein.

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

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

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

[0016] The present invention also provides, in various aspects and embodiments, the following:

[0017] One aspect of the present application provides a cell or population thereof, (i) the cell is (a) an immune cell, (b) an induced pluripotent cell (iPSC), or (c) a derived cell (e.g., a derived effector cell) obtained from differentiating an iPSC, and (ii) the cell comprises (a) an exogenous polynucleotide encoding an alloimmune defense receptor (ADR), and optionally (b) one or both of a CD38 knockout and an endogenous TCR knockout, or one or more of a CD38 knockout, an endogenous TCR knockout, and a knockout of one or both of CD58 and CD54. In some embodiments, the cell has improved resistance to host immune alloreactivity compared to a cell that does not comprise the exogenous polynucleotide. In various embodiments of the cell or population thereof, (i) the iPSCs are clonal iPSCs, single cell dissociated iPSCs, iPSC cell line cells, or iPSC master cell bank (MCB) cells; or (ii) the derived cells are derived CD34 + cells, derived hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, or derived B lineage cells; or (iii) the derivative cells include derived effector cells that have one or more functional attributes not present in the corresponding primary T, NK, NKT, and / or B cells.

[0018] In some embodiments of the cells of the population, the ADR is specific for 41BB or CD38. In some embodiments, the ADR comprises (i) a 41BB-specific ligand operably linked to a signaling domain that promotes effector cell activation, or (ii) a CD38-binding domain operably linked to a signaling domain that promotes effector cell activation. In one embodiment of the cells of the population, the 41BB-specific ligand is 4-1BBL, an antibody or fragment thereof that targets 4-1BB, or a 4-1BBL-Fc fusion. In some embodiments of the cells of the population, the signaling domain comprises CD3zeta from DAP12 or a functional fragment thereof, an Fc receptor, or a combination thereof. In various embodiments of the cells of the population, the ADR further comprises one, two, three, or more costimulatory domains. In some aspects of the cells of the population, one, two, three or more costimulatory domains are derived from the intracellular signaling domains of CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, or CD40. In some embodiments, the ADR comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:8.

[0019] In various embodiments of the cells of the population, the cells are characterized by: (i) a chimeric antigen receptor (CAR); (ii) a knockout of one or both of CD58 and CD54; (iii) exogenous CD16 or a variant thereof; (iv) a cytokine signaling complex comprising a cell surface expressed exogenous cytokine and / or a partial or complete peptide of its receptor; (v) at least one of the genotypes listed in Table 2; (vi) a disruption of least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (vii) CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AR, an antigen-specific TCR, an Fc receptor, an antibody or functional variant or fragment thereof, introduction of at least one of a surface triggering receptor for coupling with a checkpoint inhibitor, an engager, and an agonist. In some embodiments of the cells of the population, the cells have therapeutic properties including one or more of: (i) increased cytotoxicity, (ii) improved persistence and / or survival, (iii) enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site, (iv) improved tumor penetration, (v) enhanced ability to reduce tumor immune suppression, (vi) improved ability to rescue tumor antigen escape, (vii) controlled apoptosis, (viii) enhanced or acquired ADCC, and (ix) ability to avoid fratricide, compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue that do not have the same gene edit.

[0020] In some embodiments of the cells of the population, the exogenous CD16 or variant thereof comprises at least one of: (a) high affinity non-cleavable CD16 (hnCD16); (b) F176V and S197P in the ectodomain of CD16; (c) a complete or partial ectodomain derived from CD64; (d) a non-native (or non-CD16) transmembrane domain; (e) a non-native (or non-CD16) intracellular domain; (f) a non-native (or non-CD16) signaling domain; (g) a non-native stimulatory domain; and (h) a transmembrane domain, signaling domain, and stimulatory domain not derived from CD16 and derived from the same or a different polypeptide. In some embodiments of the cells of the population, the CAR is: (i) T cell-specific or NK cell-specific; (ii) bispecific antigen-binding CAR; (iii) switchable CAR; (iv) dimerized CAR; (v) split CAR; (vi) multi-chain CAR; (vii) inducible CAR; (viii) co-expressed with another CAR; (ix) co-expressed with a cytokine signaling complex comprising a cell surface-expressed exogenous cytokine and / or partial or complete peptide of its receptor, optionally in a separate construct or in a bicistronic construct. (x) optionally co-expressed with a checkpoint inhibitor in a separate construct or in a bicistronic construct; (xi) specific for at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1; and / or (xii) specific for ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), IL-1, IL-2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-35, IL-36, IL-47, IL-48, IL-59, IL-59, IL-59, IL-59, IL-59, IL-60, IL-71, IL-82, IL-83, IL-84, IL-85, IL-86, IL-87, IL-88, IL-89, IL-99, IL-190, IL-191, IL-192, IL-193, IL-194, IL-195, IL-196, IL-197, IL-198, IL-199,antigen, CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor 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 LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PRAME)and specific for any one of: p53 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 a pathogen antigen, and optionally, the CAR of any one of (i)-(xii) is inserted into the TCR locus and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by the CAR insertion.

[0021] In some embodiments of the cells of the population, the cytokine signaling complex comprises (a) a partial or complete peptide of at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (b) a fusion protein comprising (i) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is truncated, (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, or (vi) a fusion protein of IL15 and common receptor γC, where common receptor γC is native or modified. (b) at least one of (i) to (vii) a fusion protein of IL7 and IL7Rα, (ii) a fusion protein of IL7 and common receptor γC, wherein common receptor γC is native or modified, and (iii) a homodimer of IL7Rβ, wherein (c) at least one of (i) to (iii) is optionally co-expressed with the CAR, either in a separate construct or in a bicistronic expression cassette, and optionally (d) is transiently expressed. In some embodiments of the cells of the population, the cells are NK lineage cells or T lineage cells, and (i) the NK lineage cells or T lineage cells have improved infiltration and / or retention at tumor sites, (ii) the NK lineage cells are capable of recruiting and / or migrating T cells to tumor sites, or (iii) the NK lineage cells or T lineage cells are capable of reducing tumor immune suppression in the presence of one or more checkpoint inhibitors. In some embodiments of the cells of the population, the checkpoint inhibitors are PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2A and antagonists against one or more checkpoint molecules, including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some embodiments, the checkpoint inhibitor comprises (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.

[0022] In various embodiments of the cells of the population, the cells comprise (i) one or more exogenous polynucleotides integrated at a safe harbor locus or a selected locus, or (ii) more than two exogenous polynucleotides integrated at different safe harbor loci or two or more selected loci. In some embodiments, the safe harbor locus includes at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, or the selected locus is one of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and / or integration of the exogenous polynucleotide knocks out expression of a gene at the locus. In some embodiments, the TCR locus is the constant region of TCR alpha and / or TCR beta.

[0023] In one embodiment of the cells of the population, the cells comprise (i) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR), (ii) a CD38 knockout and an exogenous CD16, and (iii) a TCR knockout, and the cells or population thereof have improved resistance to host immune alloreactivity compared to cells that do not have all of (i), (ii), and (iii). In one embodiment of the cells or population thereof, the cells comprise (i) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR), (ii) a CD38 knockout, (iii) an exogenous CD16 or variant thereof, (iv) a partial or complete peptide of IL15, and a partial or complete peptide of the IL15 receptor, and (v) a CAR, and the cells or population thereof have improved resistance to host immune alloreactivity compared to cells that do not have all of (i), (ii), and (iii). In one embodiment, the CAR is specific for at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1. In one embodiment, the CAR is specific for CD19.

[0024] In another aspect, the application provides a method of improving resistance of effector cells to host immune alloreactivity comprising: (i) obtaining engineered iPSCs comprising an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR), and optionally one or both of a CD38 knockout and an endogenous TCR knockout, or one or more of a CD38 knockout, an endogenous TCR knockout, and one or both of a CD58 and CD54 knockout; and (ii) differentiating the iPSCs into effector cells, thereby producing effector cells with improved resistance to host immune alloreactivity compared to corresponding cells that do not comprise the exogenous polynucleotide.

[0025] In some embodiments of the method of improving effector cell resistance to host immune alloreactivity, (i) induced pluripotent cells (iPSCs) are engineered to generate genome-edited iPSCs that include one or more exogenous polynucleotides encoding allogeneic immune defense receptors (ADRs), and optionally knock out one or both of CD38 and an endogenous TCR, or knock out CD38, knock out an endogenous TCR, and knock out one or both of CD58 and CD54; or (ii) engineer an induced pluripotent cell (iPSC) to generate genome-edited iPSCs that include one or more exogenous polynucleotides encoding allogeneic immune defense receptors (ADRs), and optionally knock out one or both of CD38 and an endogenous TCR, or knock out one or both of CD58 and CD54. The immune cells are engineered by introducing a leution and optionally knocking out one or both of CD38 and endogenous TCR, or knocking out CD38, knocking out endogenous TCR, and knocking out one or both of CD58 and CD54 to produce genome-edited effector cells comprising an ADR and, optionally, one or both of CD38 knockout and endogenous TCR knockout, or one or more of CD38 knockout, endogenous TCR knockout, and one or both of CD58 and CD54 knockout. In some embodiments, the ADR is specific for 41BB or CD38. In some embodiments of the method of improving the resistance of effector cells, the ADR comprises (i) an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8, or (ii) a CD38 binding domain.In some embodiments of the methods of improving effector cell resistance, the engineered iPSCs have the following features compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue that do not have the same genome edits: (i) a chimeric antigen receptor (CAR); (ii) knockout of one or both of CD58 and CD54; (iii) introduction of exogenous CD16 or a variant thereof; (iv) cell surface expression of exogenous cytokines and / or partial or complete peptides of their receptors. (v) at least one of the genotypes listed in Table 2; (vi) disruption of at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (vii) disruption of CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A. 2A R, introduction of at least one of an antigen-specific TCR, an Fc receptor, an antibody or functional variant or fragment thereof, a checkpoint inhibitor, an engager, and a surface triggering receptor for coupling with an agonist. In some embodiments of the method of improving effector cell resistance, the improved effector cell resistance to host immune alloreactivity is in vivo.

[0026] In another aspect, the present application provides methods of improving in vivo resistance of CAR-T cells to host immune alloreactivity according to the methods provided herein.

[0027] In another aspect, the application provides a composition comprising the cells or populations thereof provided herein. In some embodiments of the composition, the composition further comprises one or more therapeutic agents. In some embodiments of the composition, the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, an effector antibody or a functional variant or fragment thereof, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a supplement thereof, a vector comprising one or more polynucleic acids of interest, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In some embodiments of the composition in which the therapeutic agent comprises a checkpoint inhibitor, the checkpoint inhibitor is selected from the group consisting of (i) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2ACheck for one or more antagonists including R, 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 point molecule, (ii) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (iii) at least one of atezolizumab, nivolumab, and pembrolizumab, or (b) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide. In some embodiments of the composition in which the therapeutic agent is an antibody, the antibody is selected from the group consisting of (a) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, and / or an anti-CD38 antibody; (b) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, and / or an anti-CD38 antibody; (c) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, and / or an anti-CD38 antibody; (d) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, and / or an anti-CD38 antibody; (e) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, and / or an anti-CD38 antibody; or (c) one or more of lutuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-engineered variants or fragments thereof, and functional equivalents and biosimilars thereof, or (d) daratumumab, and the derived effector cells may comprise a CD38 knockout, and optionally expression of CD16 or a variant thereof.

[0028] In some embodiments of the composition, the composition further comprises a sensitizing agent. In some embodiments, the sensitizing agent comprises at least one of a chemotherapeutic agent, external beam radiation, brachytherapy, and a radiopharmaceutical. In some embodiments, the sensitizing agent increases secretion and / or surface expression of a chemokine by tumor cells upon contact with the tumor cells. In some embodiments, the sensitizing agent comprises (i) at least one of calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, iron(iorn)-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90, or (ii) paclitaxel.

[0029] In another aspect, the application provides for the therapeutic use of a composition provided herein by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection.

[0030] In another aspect, the present application provides a master cell bank (MCB) comprising the clonal iPSCs provided herein.

[0031] In another aspect, the application provides a method of producing derived effector cells comprising an allogeneic immune defense receptor (ADR) and optionally one or both of a CD38 knockout and an endogenous TCR knockout, or one or more of a CD38 knockout, an endogenous TCR knockout, and one or both of a CD58 and CD54 knockout, comprising differentiating engineered iPSCs into derived effector cells, the engineered iPSCs comprising an exogenous polynucleotide encoding an ADR and optionally one or both of a CD38 knockout and an endogenous TCR knockout. In some embodiments, the engineered iPSCs express (i) a CAR, (ii) exogenous CD16 or a variant thereof, (iii) a cytokine signaling complex comprising a cell surface expressed exogenous cytokine and / or a partial or complete peptide of its receptor, (iv) at least one of the genotypes listed in Table 2, (v) a disruption of least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (vi) CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, an antigen-specific TCR, an Fc receptor, an antibody or functional variant or fragment thereof, introduction of at least one of a surface triggering receptor for coupling with a checkpoint inhibitor, an engager, and an agonist. In some embodiments of the method of manufacturing, (i) the iPSCs are clonal iPSCs, single cell dissociated iPSCs, iPSC cell line cells, or iPSC Master Cell Bank (MCB) cells, or (ii) the derived cells comprise derived CD34+ cells, derived hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, or derived B lineage cells, or (iii) the derived cells comprise derived effector cells having one or more functional attributes not present in the corresponding primary T, NK, NKT, and / or B cells.

[0032] In some embodiments of the method of production, the exogenous CD16 or variant thereof comprises at least one of: (a) high affinity non-cleavable CD16 (hnCD16); (b) F176V and S197P in the ectodomain of CD16; (c) a complete or partial ectodomain derived from CD64; (d) a non-native (or non-CD16) transmembrane domain; (e) a non-native (or non-CD16) intracellular domain; (f) a non-native (or non-CD16) signaling domain; (g) a non-native stimulatory domain; and (h) a transmembrane domain, signaling domain, and stimulatory domain not derived from CD16 and derived from the same or a different polypeptide. In some embodiments of the manufacture, the CAR is: (i) T cell specific or NK cell specific; (ii) bispecific antigen binding CAR; (iii) switchable CAR; (iv) dimerized CAR; (v) split CAR; (vi) multi-chain CAR; (vii) inducible CAR; (viii) co-expressed with another CAR; (ix) co-expressed, optionally in a separate construct or in a bicistronic construct, with a cytokine signaling complex comprising a cell surface expressed exogenous cytokine and / or partial or complete peptide of its receptor; (x) co-expressed, optionally in a separate construct or in a bicistronic construct, with a checkpoint inhibitor; (xi) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct, ... and / or (xii) specific for at least one of: D79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1; and / or (xiii) specific for at least one of: ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD2 0, 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 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM),EGFRvIII, receptor tyrosine protein kinase erb B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (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 A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-1 6), 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, and optionally, the CAR of any one of (i) to (xii) is inserted into the TCR locus and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by the CAR insertion.

[0033] In some embodiments of the method of manufacturing, the method further comprises genomic engineering of the clonal iPSC to knock in a polynucleotide encoding an ADR, and optionally to (i) knock out one or both of CD38 and exogenous TCR, (ii) knock out one or both of CD58 and CD54, and / or (iii) introduce one or more of exogenous CD16 or variants thereof, CAR, and / or a cytokine signaling complex comprising a partial or complete peptide of a cell surface-expressed exogenous cytokine and / or its receptor. In some embodiments of the method of manufacturing, the genomic engineering comprises targeted editing. In some embodiments, the targeted editing comprises a deletion, an insertion, or an indel, and the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0034] In another aspect, the present application provides a method of producing a clonal master engineered iPSC line using CRISPR, ZFN, or TALEN-mediated editing of a clonal iPSC, the editing comprising knocking in a polynucleotide encoding an allogeneic immune defense receptor (ADR), and optionally one or both of a CD38 knockout and an endogenous TCR knockout, thereby producing an engineered iPSC. In some embodiments of the method of production, the ADR is inserted into one of the following loci: B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, the insertion knocking out expression of a gene at the locus.

[0035] In some embodiments of the method of producing, the method further comprises sorting the engineered iPSCs to obtain single cell dissociated iPSCs comprising a polynucleotide encoding an ADR and optionally one or more of a CD38 knockout and an endogenous TCR knockout, or a CD38 knockout, an endogenous TCR knockout, and a knockout of one or both of CD58 and CD54. In some embodiments of the method of producing, the method further comprises amplifying the single cell dissociated iPSCs to produce a clonal master engineered iPSC population. In some embodiments of the method of producing, the method further comprises cryopreserving the produced clonal master engineered iPSC line. In some embodiments of the method of producing, the method further comprises analyzing the off-target editing and / or karyotype of the engineered iPSCs.

[0036] In another aspect, the application provides clonal master engineered iPSC lines produced using the methods provided herein.

[0037] In another aspect, the present invention provides a method of treating a disease or condition comprising administering to a subject in need thereof a composition provided herein.

[0038] In another aspect, the present application provides a method of treating a disease or condition, comprising administering to a subject in need thereof a cell or population thereof provided herein. In various embodiments of the method of treating, the method further comprises administering to the subject a sensitizing agent, thereby preconditioning tumor cells in the subject. In various embodiments of the method of treating, the method further comprises administering to the subject one or more therapeutic agents.

[0039] In another aspect, the present application provides a method of treating a subject, the method comprising: (a) administering a sensitizing agent to the subject to precondition tumor cells in the subject; and (b) administering a cell or population thereof disclosed herein to the subject after administration of the sensitizing agent, the subject having an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection. In various embodiments of the method of treating, the sensitizing agent comprises at least one of a chemotherapeutic agent, external beam radiation, brachytherapy, and a radiopharmaceutical. In some embodiments, the sensitizing agent increases secretion and / or surface expression of 4-1BB and / or CD38 by the tumor cells upon contact with the tumor cells. In some embodiments, the sensitizing agent is selected from the group consisting of (i) at least one of x-ray irradiation, gamma irradiation, photon irradiation, proton irradiation, and neutron irradiation, or (ii) calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131. , iron(I)-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90; or (iii) paclitaxel.

[0040] In some embodiments of the method of treating, the method further comprises administering one or more therapeutic agents. In some embodiments, the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, an effector, an antibody or functional variant or fragment thereof, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a supplement thereof, a vector comprising one or more polynucleic acids of interest, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In some embodiments of the method of treating, where the therapeutic agent comprises a checkpoint inhibitor, the checkpoint inhibitor is selected from the group consisting of: (i) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2ACheck for one or more antagonists including R, 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 point molecule, (ii) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (iii) at least one of atezolizumab, nivolumab, and pembrolizumab, or (b) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide. In some embodiments of the methods of treating in which the therapeutic agent is an antibody, the antibody is selected from the group consisting of (a) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, and / or an anti-CD38 antibody; (b) rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, ibritumomab, ocrelizumab, inotuzumab, moxetumomab, epratuzumab, trastuzumab, pembrolizumab, and the like. or (c) one or more of lutuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-engineered variants or fragments thereof, and functional equivalents and biosimilars thereof, or (d) daratumumab, and the derived effector cells may comprise a CD38 knockout, and optionally expression of CD16 or a variant thereof.

[0041] In another aspect, the application provides a method of reducing or preventing alloreactivity of host cells to allogeneic effector cells in an adoptive cell therapy provided to a subject in need thereof, the allogeneic effector cells comprising cells or populations thereof provided herein, the method optionally comprising CD38 conditioning. In various embodiments, the host cells comprise alloreactive immune cells comprising primary T cells, primary B cells, and / or primary NK cells. In some embodiments, the CD38 conditioning comprises (i) administering a CD38 antagonist to the subject before, during, or after infusion of allogeneic effector cells into the subject, or (ii) preloading the allogeneic effector cells in vitro with a CD38 antagonist followed by infusion of the allogeneic effector cells into the subject. In some embodiments, the CD38 antagonist comprises (i) an anti-CD38 antibody or CD38-CAR, (ii) daratumumab, isatuximab, or MOR202, and / or (iii) daratumumab. In some embodiments, the method (i) reduces or prevents host cell alloreactivity to allogeneic effector cells, (ii) eliminates or reduces the number of alloreactive host cells, (iii) prolongs the survival and persistence of allogeneic effector cells, (iv) delays reconstitution of host immunity, and / or (v) prevents leakage of allogeneic effector cell protection against host cell alloreactivity via overexpression of HLA-G or HLA-E. In another aspect, the invention provides a method of treating a subject in need of adoptive cell therapy according to the methods provided herein.

[0042] In another aspect, the present invention provides a cell or population thereof, (i) the cell is (a) an immune cell, (b) an induced pluripotent cell (iPSC), or (c) a derived effector cell obtained by differentiating an iPSC, (ii) the cell comprises (a) an exogenous polynucleotide encoding an alloimmune defense receptor (ADR), an exogenous polynucleotide encoding an exogenous CD16 or a variant thereof, an exogenous polynucleotide encoding a cytokine signaling complex comprising a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor, and a CD38 knockout, wherein the cell has improved resistance to host immune alloreactivity compared to a cell not comprising the exogenous polynucleotide. In various embodiments, the cell further comprises a CAR. In various embodiments, the cell further comprises a double knockout of CD58 and CD54.

[0043] In some embodiments of the cells or populations thereof, the ADR comprises a 41BB-specific ligand operably linked to a signaling domain that promotes effector cell activation. In some embodiments, the 41BB-specific ligand is 4-1BBL, an antibody or fragment thereof that targets 4-1BB, or a 4-1BBL-Fc fusion. In some embodiments, the signaling domain comprises CD3ζ or a functional fragment thereof from DAP12, an Fc receptor, or a combination thereof. In some embodiments, the ADR further comprises one, two, three, or more costimulatory domains. In some embodiments, the one, two, three, or more costimulatory domains are derived from the intracellular signaling domains of CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, or CD40. In some embodiments, the ADR comprises (i) an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:8, or (ii) a CD38 binding domain.

[0044] In some embodiments of the cells or populations thereof, the cytokine signaling complex comprises a fusion protein of IL7 and IL7Rα, or an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is truncated. In some embodiments, the exogenous CD16 or variant thereof is high affinity non-cleavable CD16 (hnCD16). In some embodiments, the high affinity non-cleavable CD16 (hnCD16) comprises the ectodomain domain of CD16 with F176V and S197P. In some embodiments, at least one of (i) the exogenous polynucleotide encoding the exogenous CD16 or variant thereof, and (ii) the exogenous polynucleotide encoding the cytokine signaling complex is inserted into the CD38 locus, resulting in knockout of CD38.

[0045] In another aspect, the invention provides a method of treating a subject comprising administering to a subject in need thereof a cell or population thereof as described herein, the subject having an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection, hi various embodiments, the method does not comprise administering a Cy / Flu-based lymphodepletion therapy.

[0046] In another aspect, the invention provides a method of reducing or preventing alloreactivity of host cells to allogeneic effector cells in an adoptive cell therapy provided to a subject in need thereof, the allogeneic effector cells comprising a cell or population thereof described herein, the method optionally comprising CD38 conditioning. In some embodiments, the method does not comprise administering a Cy / Flu-based lymphodepletion therapy.

[0047] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description, taken in conjunction with the accompanying drawings, in which are set forth by way of illustration and example certain embodiments of the invention. [Brief description of the drawings]

[0048] [Figure 1A] FIG. 1 shows exemplary stealth editing of iPSC-derived effector cells to passively avoid or selectively deplete alloreactive immune cells. [Figure 1B] FIG. 1 shows exemplary stealth editing of iPSC-derived effector cells to passively avoid or selectively deplete alloreactive immune cells. [Figure 1C] FIG. 1 shows exemplary stealth editing of iPSC-derived effector cells to passively avoid or selectively deplete alloreactive immune cells. [Figure 1D] FIG. 1 shows exemplary stealth editing of iPSC-derived effector cells to passively avoid or selectively deplete alloreactive immune cells. [Figure 1E] FIG. 1 shows exemplary stealth editing of iPSC-derived effector cells to passively avoid or selectively deplete alloreactive immune cells. [Figure 2A] FIG. 1 shows that ADRs enhance iPSC-derived effector cells by selectively targeting 4-1BB-positive alloreactive immune cells, thereby prolonging their functional persistence and proliferation. [Figure 2B] FIG. 1 shows that ADRs enhance iPSC-derived effector cells by selectively targeting 4-1BB-positive alloreactive immune cells, thereby prolonging their functional persistence and proliferation. [Figure 3A] FIG. 1 shows that a distinct population of alloreactive T cells present before and after Cy / Flu treatment is eliminated by ADR-armed effector cells. [Figure 3B] FIG. 1 shows that a distinct population of alloreactive T cells present before and after Cy / Flu treatment is eliminated by ADR-armed effector cells. [Figure 4A] FIG. 1 shows that ADR-armed effector cells exhibit enhanced functional persistence in mixed lymphocyte reactions. [Figure 4B] FIG. 1 shows that ADR-armed effector cells exhibit enhanced functional persistence in mixed lymphocyte reactions. [Figure 5A] FIG. 1 shows that ADR+ effector cells suppress the expansion of alloreactive T and NK cells. [Figure 5B] FIG. 1 shows that ADR+ effector cells suppress the expansion of alloreactive T and NK cells. [Figure 6A] FIG. 1 shows that ADR+ effector cells selectively deplete alloreactive immune cells expressing 4-1BB. [Figure 6B] FIG. 1 shows that ADR+ effector cells selectively deplete alloreactive immune cells expressing 4-1BB. [Figure 7A] FIG. 1 shows that ADR+ effector cells selectively target both CD4+ and CD8+ alloreactive T cell subsets. [Figure 7B] FIG. 1 shows that ADR+ effector cells selectively target both CD4+ and CD8+ alloreactive T cell subsets. [Figure 8A] FIG. 1 shows that ADR+ effector cells exhibit enhanced functional persistence in the presence of daily tumor challenge. [Figure 8B] FIG. 1 shows that ADR+ effector cells exhibit enhanced functional persistence in the presence of daily tumor challenge. [Figure 8C] FIG. 1 shows that ADR+ effector cells exhibit enhanced functional persistence in the presence of daily tumor challenge. [Figure 9] FIG. 1 shows that iPSCs engineered to harbor TRAC-driven CAR and ADR transgenes can be successfully differentiated into T cells that express CD3 intracellularly and robustly co-express CAR and ADR. [Figure 10] FIG. 1 shows that ADR expression in iPSC-derived iT cells provides protection from primed allogeneic T cell rejection and depletes allogeneic T cells. [Figure 11]FIG. 1 shows that effector cells±ADR exhibit similar cytotoxicity against CD19-expressing tumor targets in vitro. [Figure 12A] FIG. 1 shows that ADR+ effector cells are not impaired in vivo in tumor control in the presence of host alloreactive T cells. [Figure 12B] FIG. 1 shows that ADR+ effector cells are not impaired in vivo in tumor control in the presence of host alloreactive T cells. [Figure 13A] FIG. 1 shows that dual costimulatory ligand-deficient iNKs avert peripheral blood NK self-loss responses. [Figure 13B] FIG. 1 shows that dual costimulatory ligand-deficient iNKs avert peripheral blood NK self-loss responses. [Figure 13C] FIG. 1 shows that dual costimulatory ligand-deficient iNKs avert peripheral blood NK self-loss responses. [Figure 13D] FIG. 1 shows that dual costimulatory ligand-deficient iNKs avert peripheral blood NK self-loss responses. [Figure 14A] FIG. 1 shows that CD38 knockout in iT cells eliminates anti-CD38 ADCC when combined with peripheral blood NK cells. [Figure 14B] FIG. 1 shows that CD38 knockout in iT cells eliminates anti-CD38 ADCC when combined with peripheral blood NK cells. [Figure 15] FIG. 1 shows that preconditioning with anti-CD38 monoclonal antibody protects CAR-iT cells from exhaustion. [Figure 16] FIG. 1 shows that allogeneic T and NK cells are depleted in a manner dependent on both daratumumab and CAR-iT. [Figure 17A] FIG. 1 shows that CD38+ T and pbNK cells are depleted in the presence of daratumumab. [Figure 17B] FIG. 1 shows that CD38+ T and pbNK cells are depleted in the presence of daratumumab. [Figure 17C] FIG. 1 shows that CD38+ T cells and pbNK cells are depleted in a daratumumab-dependent manner. [Figure 17D] FIG. 1 shows that CD38+ T cells and pbNK cells are depleted in a daratumumab-dependent manner. [Figure 18] FIG. 1 shows representative FACS plots obtained from 10-day co-cultures (iNK to PBMC ratio of 8:1) and analyzed for CD38 and 4-1BB expression in CD3+ T cells. [Figure 19A] FIG. 1 shows that CD38-negative iNK cells are resistant to alloreactive pbNK challenge in the presence of daratumumab. [Figure 19B] FIG. 1 shows that CD38-negative iNK cells are resistant to alloreactive pbNK challenge in the presence of daratumumab. [Figure 20A] FIG. 1 shows that ADR-armed CD19-CAR effector cells have intact anti-tumor efficacy in an in vivo disseminated Nalm6 leukemia model in an alloreactive setting. [Figure 20B] FIG. 1 shows that ADR-armed CD19-CAR effector cells have intact anti-tumor efficacy in an in vivo disseminated Nalm6 leukemia model in an alloreactive setting. [Figure 20C] FIG. 1 shows that ADR-armed CD19-CAR effector cells have intact anti-tumor efficacy in an in vivo disseminated Nalm6 leukemia model in an alloreactive setting. [Figure 21] FIG. 1 shows that the addition of anti-CD38 antibody to lympho-depletion conditioning (LDC) delays host immune reconstitution and extends the therapeutic window for adoptive cell therapy in patients treated with engineered CD38KO hnCD16 iNK cells in combination with daratumumab compared to patients treated with engineered iNK cells without daratumumab. [Figure 22]FIG. 1 shows Uniform Manifold Approximation and Projection (UMAP) visualization of lymphocyte profiles in lymphoma and multiple myeloma patients treated with engineered iNK cells in combination with daratumumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Genome modification of iPSCs (induced pluripotent stem cells) can include one or more of polynucleotide insertion, deletion, substitution, and combinations thereof. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types from cells derived from genome-engineered iPSCs. Meanwhile, it is difficult to directly manipulate primary immune cells such as T cells or NK cells, which poses obstacles to the preparation and delivery of engineered immune cells for adoptive cell therapy. In some embodiments, the present invention provides an efficient and reliable targeted approach for stably integrating one or more exogenous genes, including suicide genes and other functional modalities, to confer improved therapeutic properties with respect to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, longevity, self-renewal, persistence, and / or survival to iPSC-derived cells, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell precursors, NK cell precursors, T lineage cells, NKT lineage cells, NK lineage, and immune effector cells with one or more functional characteristics not present in primary NK cells, T cells, and / or NKT cells.

[0050] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0051] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0052] 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.

[0053] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0054] The term "and / or" should be understood to mean either one or both of the alternatives.

[0055] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference 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.

[0056] 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 "substantially the same" or "essentially the same" refers to a reference 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.

[0057] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that is free of a particular substance or source thereof, such as, for example, 95% free, 96% free, 97% free, 98% free, 99% free, etc., of a particular substance or source thereof, or is undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance is (1) not included in the composition at any concentration, or (2) included in the composition at a low concentration that is functionally inactive. A similar meaning may be applied to the term "absent," which refers to the absence of a particular substance or source thereof in a composition.

[0058] Throughout this specification, unless the context requires otherwise, "comprise", "comprises", and "comprising" will be understood to mean the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include", "having", "contain", and "comprise" are used interchangeably.

[0059] "Consisting of" means inclusive of and limited to everything that 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.

[0060] "Consisting essentially of" means the inclusion of 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 that 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.

[0061] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0062] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of the body, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures involve live cells or tissues taken from a living 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 hours or 72 hours or more depending on the circumstances). In certain embodiments, such tissues or cells may be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using live cells or tissues are typically considered to be "in vitro", although in certain embodiments, the term may be used interchangeably with ex vivo.

[0063] The term "in vivo" generally refers to activities that take place inside a living organism.

[0064] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0065] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as, for example, a blood cell or a muscle cell. A differentiated or differentiation-induced cell is a cell that assumes a more specialized ("committed") position within the lineage of a cell. The term "committed" as applied to the process of differentiation refers to a cell that has progressed down a differentiation pathway to a point where it continues to differentiate into a particular cell type or subset of cell types under normal circumstances and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell (i.e., the embryo itself) to form all lineages of an organism or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs), which are unable to give rise to an entire organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells), which are able to give rise to an entire organism.

[0066] As used herein, the term "induced pluripotent stem cells" or iPSCs refers to stem cells produced in vitro from differentiated adult, neonatal or fetal cells that have been induced or modified, i.e., reprogrammed, into cells that can differentiate into tissues of all three germ layers or layers: mesoderm, endoderm, and ectoderm. In some embodiments, the reprogramming process uses reprogramming factors and / or small molecule chemical drivers. The produced iPSCs do not refer to naturally occurring cells.

[0067] As used herein, the term "embryonic stem cell" refers to the naturally occurring pluripotent stem cells of the inner cell mass of blastocyst. Embryonic stem cells are pluripotent and generate all derivatives of the three primary germ layers, i.e., ectoderm, endoderm, and mesoderm, during development. They do not contribute to extraembryonic membranes or placenta, i.e., they are not totipotent.

[0068] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (i.e., ectoderm, mesoderm, and endoderm), but not all three. Thus, pluripotent cells can also be referred to as "partially differentiated cells." Pluripotent cells are known in the art, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell can form many types of cells in a given lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to a state of a cell that has a degree of developmental potential less than totipotency and pluripotency.

[0069] Pluripotency can be determined, in part, by assessing the pluripotency characteristics of the cells, including, but not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) 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 lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation composed of the three somatic lineages, and (vi) formation of embryoid bodies composed of cells from the three somatic lineages.

[0070] Two types of pluripotency have been described so far: a "primed" or "metastable" state pluripotency similar to the epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "ground" state similar to the inner cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the above-mentioned characteristics, the naive or ground state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single cell culture, (iii) global reduction in DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) reduced expression of differentiation markers compared to primed state pluripotent cells. Standard methodologies of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, generally appear to have characteristics of a primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the primed state and characteristics of the ground state are observed unless exogenous transgene expression is maintained.

[0071] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape with a high nuclear to cytoplasmic ratio, prominent presence of nucleoli, and typical intercellular spacing.

[0072] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animals.

[0073] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0074] "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 a nutritional composition in which a cell culture is cultivated.

[0075] "Culturing" or "maintaining" refers to sustaining, propagating (growing), and / or differentiating cells outside of a tissue or body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" or "maintaining" can utilize culture medium as a source of nutrients, hormones, and / or other factors that aid in the growth and / or maintenance of the cells.

[0076] 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.

[0077] As used herein, the term "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refers to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial-hematopoietic conversion. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm through hemangioblasts to definitive hemogenic endothelial cells and hematopoietic precursors.

[0078] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic precursor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including multipotent hematopoietic stem cells (blood cells), myeloid precursors, megakaryocyte precursors, erythroid precursors, and lymphoid precursors. 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) lineages. As used herein, the term "secondary hematopoietic stem cells" refers to cells that are CD34+ / CD35+ / CD36+ / CD37+ / CD38+ / CD39 ... + Refers to hematopoietic cells, which also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0079] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that has completed maturation in the thymus and has a variety of roles in the immune system, including identifying specific foreign antigens in the body and activating and inactivating other immune cells in an MHC class I-restricted manner. A T cell can be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. T cells are CD3 + T cells can be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 +The T cells can be of any type and at any stage of development, including, but not limited to, T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), and the like. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). The term "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 or T cell-like effector cells may also be differentiated from stem or progenitor cells ("derived T cells" or "derived T cell-like effector cells", or collectively "derived T lineage cells"). Derived T cell-like effector cells may have T cell lineage in some respects, but at the same time have one or more functional attributes not present in primary T cells. In this application, T cells, T cell-like effector cells, derived T cells, derived T cell-like effector cells, or derived T lineage cells are collectively referred to as "T lineage cells". In some embodiments, derived T lineage cells are iPSC-derived T cells obtained by differentiating iPSCs, which are also referred to herein as "iT" cells.

[0080] "CD4 +"CD4 cells" refers to a subset of T cells that express CD4 on their surface and are associated with cellular immune responses. They are characterized by their secretion profile after stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. The "CD4" molecule is a 55 kD glycoprotein originally defined as a differentiation antigen for T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as the relevant recognition element in the MHC (major histocompatibility complex) class II-restricted immune response. In T lymphocytes, it defines the helper / inducer subsets.

[0081] "CD8 + "CD8 cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the associated recognition element of major histocompatibility complex class I restricted interactions.

[0082] 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 the T cell receptor (CD3). The NK cells can be any NK cell, e.g., cultured NK cells (e.g., primary NK cells), or NK cells derived from cultured or expanded NK cells, or cell line NK cells (e.g., NK-92), or NK cells obtained from a mammal that is healthy or has a disease state. As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and are phenotypically CD3+ and CD4+ expression. - and CD56 +and refers to a subset of NK cells that 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, CD56 + The isolated subpopulation of NK cells includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. + may be dim or bright expression. NK cells or NK cell-like effector cells may be differentiated from stem or progenitor cells ("derived NK cells" or "derived NK cell-like effector cells", or collectively "derived NK lineage cells"). Derived NK cell-like effector cells may have NK cell lineage in some respects, but at the same time have one or more functional attributes not present in primary NK cells. In this application, NK cells, NK cell-like effector cells, derived NK cells, derived NK cell-like effector cells, or derived NK lineage cells are collectively referred to as "NK lineage cells". In some embodiments, derived NK lineage cells are iPSC-derived NK cells obtained by differentiating iPSCs, which are also referred to herein as "iNK" cells.

[0083] As used herein, the term "NKT cells" or "natural killer T cells" or "NKT lineage cells" refers to CD1d-restricted T cells expressing 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 are recognized. Invariant or type I NKT cells express a very limited TCR repertoire, i.e., a canonical α chain (Vα24-Jα18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, presents a more heterogeneous use of TCRαβ. Type I NKT cells are considered to be suitable for immunotherapy. Adaptive or invariant (Type I) NKT cells can be identified by expression of one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0084] The term "effector cell" generally refers to a specific cell in the immune system that performs a specific activity in response to stimulation and / or activation, or that provides a specific function upon activation. As used herein, the term "effector cell" includes, and in some contexts is interchangeable with, immune cells, "differentiated immune cells," and primary or differentiated cells that have been edited and / or regulated to perform a specific activity in response to stimulation and / or activation. Non-limiting examples of effector cells include primary or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0085] As used herein, terms such as "isolated" refer to a cell or population of cells that has been separated from its original environment, i.e., the environment of the isolated cell is substantially free of at least one component found in the environment in which the "non-isolated" reference cell resides. The term includes cells that are removed from some or all components found in their naturally occurring environment, e.g., isolated from a tissue or biopsy sample. The term also includes cells that are 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 as grown, stored, or persisted in a non-natural 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 isolating a desired cell or population thereof from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0086] As used herein, terms such as "purify" refer to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0087] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, may be referred to as encoding the protein or other product of that gene or cDNA.

[0088] A "construct" refers to a complex of polymers or molecules, including polynucleotides, that are delivered to a host cell, either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct that can direct the delivery or transfer of foreign genetic material to a target cell and can replicate and / or express in the target cell. Thus, the term "vector" includes the construct that is delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0089] "Integration" means that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence in the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted into the chromosomal or mitochondrial DNA of the cell at a preselected site or "integration site". As used herein, the term "integration" further refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of the construct, with or without the deletion of the endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" can further include the replacement of the deleted nucleotide with the endogenous sequence or one or more inserted nucleotides.

[0090] As used herein, the term "exogenous" is intended to mean that the referenced molecule or activity is introduced into the host cell or is non-native to the host cell. The molecule can be introduced, for example, by integrating into a host chromosome or by introducing the encoding nucleic acid into the host genetic material as non-chromosomal genetic material such as a plasmid. Thus, the term used in reference to expression of an encoding nucleic acid 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 a 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.

[0091] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo. A gene or polynucleotide of interest may include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may code for an miRNA, an shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, a variant polypeptide (i.e., a variant of a natural polypeptide having less than 100% sequence identity with the natural polypeptide) or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selection marker, etc.

[0092] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases adenine (A), cytosine (C), guanine (G), and thymine (T); if the polynucleotide is RNA, thymine is uracil (U). 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.

[0093] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, generally referred to in the art as polypeptides or proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0094] As used herein, the term "subunit" refers to each separate polypeptide chain of a protein complex, each separate polypeptide chain being capable of forming a stable folded structure by itself. Many protein molecules are composed of two or more subunits, where the amino acid sequence can be either identical, similar, or completely different for each subunit. 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, contiguous portions of the polypeptide chains are often folded into compact, localized, semi-independent units called "domains". Many protein domains may further comprise independent "structural subunits", also called subdomains, that contribute to a common function of the domain. Thus, as used herein, the term "subdomain" refers to a protein domain inside a larger domain, for example, a binding domain in the ectodomain of a cell surface receptor, or a stimulatory or signaling domain in the endodomain of a cell surface receptor.

[0095] "Operably-linked" or "operatively linked" are interchangeable with "operably connected" or "operatively connected" and refer to the association of nucleic acid sequences (or amino acids in a polypeptide having multiple domains) 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 is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. As a further example, a receptor binding domain can be operably connected to an intracellular signaling domain such that binding of the receptor to a ligand transduces a signal in response to said binding.

[0096] A "fusion protein" or "chimeric protein," as used herein, is a protein created through genetic engineering to join two or more partial or complete polynucleotides encoding sequences coding for separate proteins, such that expression of these joined polynucleotides results in a single peptide or multiple polypeptides having functional properties derived from each of the original proteins or fragments thereof. A linker (or spacer) peptide can be added between the two adjacent polypeptides of different sources in a fusion protein.

[0097] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic properties of source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, which can be further differentiated into a specific cell type, including any hematopoietic lineage cell. Source cell-derived iPSCs, and cells differentiated therefrom, can be collectively referred to as "derived" or "derived" cells, depending on the context. For example, derived effector cells, or derived NK cells or derived T cells, as used throughout this application, are cells differentiated from iPSCs, when compared to their primary counterparts obtained from natural / native sources, such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, genetic imprints conferring preferential therapeutic attributes are incorporated into iPSCs by reprogramming selected source cells that are donor, disease, or therapeutic response specific, or by introducing genetic modification modalities into iPSCs using genome editing. In aspects of source cells obtained from specifically selected donors, diseases, or therapeutic contexts, genetic imprints that contribute preferential therapeutic attributes may include context-specific genetic or epigenetic modifications that represent a retainable phenotype, i.e., preferential therapeutic attributes, that are passed on to descendants of the selected source cells, regardless of whether the underlying molecular events have been identified. Source cells that are donor, disease, or therapeutic response specific may contain genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including, but not limited to, pre-positioned monospecific TCRs, e.g., from virus-specific T cells or invariant natural killer T (iNKT) cells, traceable and desirable genetic polymorphisms, e.g., homozygosity for a point mutation encoding the high affinity CD16 receptor in the selected donor, and selected HLA-matched donor cells that exhibit predetermined HLA requirements, i.e., haplotypes in an expanded population.As used herein, preferential therapeutic properties include improved engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of immune responses, survival rate, and cytotoxicity of derived cells. Preferential therapeutic properties are also associated with antigen-targeting receptor expression, HLA presentation or lack thereof, resistance to tumor microenvironment, induction of bystander immune cells and immune modulation, improved on-target specificity with reduced extratumoral effects, and / or resistance to treatments such as chemotherapy. When derived cells with one or more therapeutic properties are obtained from differentiating iPSCs that incorporate genetic imprints that confer preferential therapeutic properties, such derived cells are also referred to as "synthetic cells." For example, synthetic effector cells, or synthetic NK cells or synthetic T cells as used throughout this application are cells differentiated from genomically modified iPSCs compared to their primary counterparts obtained from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissues. In some embodiments, a synthetic cell has one or more non-native cell functions when compared to its closest corresponding primary cell.

[0098] As used herein, the term "enhanced therapeutic properties" refers to therapeutic properties of a cell that are enhanced compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties compared to typical unmodified and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of immune responses, viability, and cytotoxicity. Therapeutic properties of immune cells are also associated with antigen targeting receptor expression, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modifications, improved on-target specificity with reduced extratumoral effects, and / or resistance to treatments such as chemotherapy.

[0099] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between an immune cell (e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil) and a tumor cell and activate the immune cell. Examples of engagers include, but are not limited to, bi-specific T cell engagers (BiTEs), bi-specific killer cell engagers (BiKEs), tri-specific killer cell engagers (TriKEs), or multispecific killer cell engagers, or universal engagers that are compatible with multiple immune cell types.

[0100] 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, or neutrophils. In some embodiments, the surface triggering receptor promotes bispecific or multispecific antibody binding between an effector cell and a specific target cell (e.g., tumor cell) regardless of the effector cell's native receptor and cell type. Using this approach, it is possible to generate iPSCs that contain a universal surface triggering receptor and differentiate such iPSCs into a population of various effector cell types that express the universal surface triggering receptor. "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 be bound or linked to an engager that can be recognized 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 the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to the universal surface triggering receptor. Thus, one or more effector cell types may engage to kill one specific type of tumor cell or two or more types of tumors. Surface triggering receptors generally contain a costimulatory domain for effector cell activation and an anti-epitope specific to the epitope of the engager. Bispecific engagers are specific to the anti-epitope of the surface triggering receptor on one end and specific to the tumor antigen on the other end.

[0101] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of a cell therapy. In some examples, expression of the safety switch protein is conditionally controlled to address safety concerns of transplanted and engineered cells that have a gene encoding the safety switch protein permanently integrated into their genome. This conditional regulation can be variable and can include post-translational activation via small molecules and control by tissue-specific and / or transient transcriptional regulation. The safety switch protein can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation and / or antibody-mediated depletion. In some examples, the safety switch protein is activated by an exogenous molecule, e.g., a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cell. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product and kills the transduced cells.

[0102] As used herein, the term "pharmaceutical active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. A pharmaceutical active protein has curative, therapeutic or palliative properties against a disease and can be administered to improve, relieve, alleviate, reverse or lessen the severity of the disease. A pharmaceutical active protein also has prophylactic properties and is used to prevent the onset of a disease or to lessen the severity of such a disease or pathological condition once it appears. A "pharmaceutical active protein" includes a whole protein or peptide or a pharmaceutical active fragment thereof. The term also includes a pharmaceutical active analog of a protein or peptide or an analog of a fragment of a protein or peptide. The term pharmaceutical active protein also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharmaceutical active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0103] As used herein, the term "signaling molecule" refers to any molecule that modifies, participates in, inhibits, activates, reduces, or enhances cell signaling. "Signaling" refers to the transmission of a molecular signal in the form of a chemical modification by the recruitment of protein complexes along a pathway that ultimately leads to a biochemical event in the cell. Examples of signaling pathways are 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.

[0104] 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 specific antigen or surface molecule.

[0105] As used herein, the terms "specific" or "specificity" can be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0106] As used herein, the term "adoptive cell therapy" refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes (e.g., T cells, B cells, and / or NK cells), regardless of whether the immune cells are isolated from a human donor or are effector cells obtained from in vitro differentiation of pluripotent cells, whether they are genetically modified, or whether they are primary donor cells that have been passaged, expanded, or immortalized ex vivo after isolation from the donor.

[0107] As used herein, "lymphodepletion" and "lymphatic conditioning" are used interchangeably to refer to the destruction of lymphocytes and T cells, typically prior to immunotherapy. The purpose of lymphatic conditioning prior to administration of adoptive cell therapy is to promote homeostatic proliferation of effector cells, as well as to eliminate regulatory immune cells and other competing elements of the immune system that compete for homeostatic cytokines. Thus, lymphatic conditioning is typically accomplished by administering one or more chemotherapeutic agents to the subject prior to the first dose of adoptive cell therapy. In various embodiments, lymphatic conditioning is performed hours to days prior to the first dose of adoptive cell therapy. Exemplary chemotherapeutic agents useful for lymphatic conditioning include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, sufficient lymphodepletion with anti-CD38 mAb may provide an alternative conditioning process for the present iNK cell therapy without or at a minimum the need for a CY / FLU-based lymphoid conditioning procedure, as further described herein.

[0108] As used herein, "radiation" refers to the emission or transmission of energy in the form of waves or particles. Exemplary forms of radiation include, but are not limited to, electromagnetic radiation (e.g., radio waves, microwaves, infrared, visible light, ultraviolet, X-rays, and gamma radiation), particle radiation (e.g., alpha, beta, proton, and neutron radiation), and acoustic radiation (e.g., ultrasound, sonic, and seismic waves). In various embodiments, the amount of radiation is measured in Gray (Gy), which is defined as the absorption of one Joule of radiation energy per kilogram of material. In radiation therapy, the amount of radiation applied varies depending on the type and stage of the cancer being treated. In curative cases, typical doses for solid epithelial tumors range from 60 to 80 Gy, while lymphomas are typically treated with 20 to 40 Gy. Prophylactic (adjuvant) doses are typically about 45 to 60 Gy in fractions of 1.8 to 2 Gy (e.g., for breast, head, and neck cancers). In various embodiments, radiation can be used as a sensitizing agent as disclosed herein.

[0109] As used herein, "radiation therapy" or "radiotherapy" are used interchangeably to refer to a type of cancer treatment that involves the use of radiation to damage cells by destroying genetic material that controls how cells grow and divide. Both healthy and cancer cells are damaged by radiation therapy, but the goal of radiation therapy is to destroy as few normal, healthy cells as possible. The term "radiotherapy" often refers to external beam radiation therapy, in which high-energy beams (e.g., X-rays, gamma rays, photons, protons, neutrons, ions, and any other form of energy applicable to such treatments) are generated by a machine outside the subject being treated and directed to precise points on the subject's body. However, the term "radiotherapy" also includes brachytherapy, in which seeds, ribbons, or capsules containing or otherwise connected to a source of radiation are placed in or near tumors or cancer cells inside the subject's body. Brachytherapy includes low-dose-rate implants, high-dose-rate implants, and permanent implants. The term "radiotherapy" also includes total body radiation therapy, in which a radioactive drug (e.g., a radiopharmaceutical or radionuclide containing a radioactive peptide) is administered orally or intravenously to a subject and concentrates in the subject's body in areas where tumor or cancer cells are located. Similar to antibody-drug candidates, in which an antibody that binds to a tumor antigen is linked to a toxic drug, radiopharmaceuticals incorporate a radioactive compound linked to a targeting molecule (such as an antibody) that specifically binds to a tumor antigen. Examples of radioactive compounds useful in radiopharmaceuticals include, but are not limited to, calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, iron (iorn)-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90.In various embodiments, radiation therapy may be used as a sensitizing agent as disclosed herein.

[0110] As used herein, "homing" or "trafficking" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate the interaction of a cell to a target site. In some embodiments, a homing molecule is a chemokine receptor. As used herein, a "chemokine receptor" refers to a cell surface molecule that binds to a chemokine. A chemokine receptor can include a naturally occurring or recombinant chemokine receptor or a variant thereof. Exemplary chemokine receptors include, but are not limited to, CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), CC chemokine receptors (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), CX3C chemokine receptors (e.g., CX3CR1), XC chemokine receptors (e.g., XCR1), or variants thereof.

[0111] As used herein, a "therapeutically sufficient amount" includes within its meaning a non-toxic, but sufficient and / or effective amount of the particular therapeutic agent and / or pharmaceutical composition being referred to, to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the stage and severity of the condition being treated. 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.

[0112] Differentiation of pluripotent stem cells requires changes in the culture system, including changes in stimuli in the medium and the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a common and important intermediate to initiate lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development as 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 grow into cystic EBs, at which point they are further processed to continue differentiation, typically days to weeks. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multi-layered cluster of cells. Typically, this is accomplished by one of several methods, including settling the pluripotent cells in droplets, settling the cells in "U" bottom well plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require further differentiation cues to promote EB growth. Therefore, pluripotent stem cell aggregates need to be transferred to differentiation medium that provides cue induction to the selected lineage. EB-based culture of pluripotent stem cells typically generates differentiated cell populations (i.e., ectodermal, mesodermal, and endodermal germ layers) with moderate proliferation within the EB cell clusters. Although EBs have been proven to promote cell differentiation, they give rise to heterogeneous cells with various differentiation states due to inconsistent exposure of the cells in the three-dimensional structure to differentiation cues in the environment. In addition, EBs are laborious to create and maintain. Furthermore, cell differentiation by EB formation is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0113] In contrast, "aggregate formation", unlike "EB formation", can be used to expand a population of pluripotent stem cell-derived cells. For example, during aggregate-based pluripotent stem cell expansion, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates to form larger aggregates that can be mechanically or enzymatically dissociated into smaller aggregates to maintain cell proliferation and increase cell number in culture. Unlike EB culture, cells cultured within aggregates in maintenance culture medium maintain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0114] As used herein, "monolayer differentiation" refers to a differentiation method that is different from the differentiation of three-dimensional multi-layered clusters of cells, i.e. "EB formation".Monolayer differentiation, among other advantages disclosed herein, avoids the need for EB formation to initiate differentiation.Since monolayer culture does not mimic embryonic development as in the case of EB formation, differentiation into specific lineages is considered minimal compared to the differentiation of all three germ layers in EB formation.

[0115] As used herein, "dissociated cells" or "single dissociated cells" refers to cells that have been substantially separated or purified away 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, for example, by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells can be dissociated from a culture plate or other surface. Thus, dissociation includes disrupting the extracellular matrix (ECM) and cellular interactions with the substrate (such as the culture surface) or disrupting the ECM between cells.

[0116] As used herein, "master cell bank" or "MCB" refers to a clonal master engineered iPSC line that is a clonal population of iPSCs that have been engineered to contain one or more therapeutic properties, characterized, tested, qualified, expanded, and shown to reliably serve as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing environment. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic mutations and / or potential contamination by reducing and / or eliminating the total number of times the iPS cell line is passaged, thawed, or handled during the manufacturing process.

[0117] As used herein, "feeder cells" or "feeders" is a term that refers to one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, proliferate, or differentiate, since the feeder cells provide stimuli, growth factors, nutrients, and support the second cell type. Feeder cells may optionally be derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the proliferation and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the above, one particular feeder cell type can be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be a mouse embryonic fibroblast (MEF). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation to specific lineages, enhance proliferation capacity, and promote maturation into specialized cell types, such as effector cells.

[0118] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, that is essentially free of feeder or stromal cells and / or has not been 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. Preconditioned medium contains many mediator substances, including growth factors and cytokines, secreted from feeder cells cultured in the medium. In some embodiments, the feeder-free environment does not contain either feeder cells or stromal cells, nor has it been preconditioned by culturing feeder cells.

[0119] "Functional" as used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, refers to (1) successful transgenic or controlled gene expression, such as inducible or transient expression, at a desired stage of cellular development, achieved by knock-in, knock-out, knock-down gene expression, direct genome editing or modification, or by "passaging" through differentiation from an originally genomically engineered starting cell or reprogramming of that starting cell, at the genetic level, or (2) the genetic modification or alteration of a gene that is obtained in the cell through (i) direct genome editing. (ii) gene expression modifications that are maintained in the cell through "passage" via differentiation or reprogramming of the original genomically engineered starting cell; (iii) downstream gene regulation in the cell as a result of gene expression modifications that are only present in an earlier developmental stage of the cell or only present in the starting cell that gave rise to the cell via differentiation or reprogramming; or (iv) successful removal, addition or modification of a cellular function / characteristic by enhanced or newly achieved cellular function or attribute exhibited in the mature cellular product originally derived from genomic editing or modifications performed on an iPSC, precursor or de-differentiated cellular source.

[0120] "HLA-deficient," including HLA class I-deficient, HLA class II-deficient, or both, refers to cells that lack or no longer maintain or have reduced levels of surface expression of complete MHC complexes comprising HLA class I protein heterodimers and / or HLA class II heterodimers, the reduced or decreased levels being lower than those naturally detectable by other cells or synthetic methods.

[0121] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that are further modified by introducing genes expressing proteins associated with improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, including, 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, 4-1BB, DAP10, DAP12, CD24, CD3zeta, 4-1BBL, CD47, CD113, and PDL1. "Modified HLA-deficient" cells also include cells other than iPSCs.

[0122] The term "ligand" refers to a substance that forms a complex with a target molecule and generates a signal by binding to a site on the target. A ligand may be a natural or artificial substance that can specifically bind to a target. A ligand may be in the form of a protein, peptide, antibody, antibody complex, conjugate, nucleic acid, lipid, polysaccharide, monosaccharide, small molecule, nanoparticle, ion, neurotransmitter, or any other molecular entity that can specifically bind to a target. The target to which the ligand binds may be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and changes its function, inducing a signaling response, is called "agonistic" or "agonist". A ligand that binds to a target and blocks or reduces a signaling response is "antagonistic" or "antagonist".

[0123] The term "antibody" is used herein in the broadest sense and generally refers to an immune response generating molecule that contains at least one binding site that specifically binds to a target, which may be an antigen or a receptor that can interact with a particular antibody. For example, NK cells can be activated by the binding of an antibody or the Fc region of an antibody to its Fc-gamma receptor (FcγR), thereby triggering ADCC (antibody-dependent cellular cytotoxicity)-mediated effector cell activation. The particular fragment or portion of an antigen or receptor that an antibody binds to, or generally the target, is known as an epitope or antigenic determinant. The term "antibody" includes, but is not limited to, antibody mimetics that mimic the structure and / or function of an antibody or a particular fragment or portion thereof, including natural antibodies and variants thereof, fragments of natural antibodies and variants thereof, peptibodies and variants thereof, and single chain antibodies and fragments thereof. The antibody may be a murine antibody, a human antibody, a humanized antibody, a camelid IgG, a single variable new antigen receptor (VNAR), a shark heavy chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single domain antibody (dAb), an anti-idiotypic antibody, a bispecific, multispecific, or multimeric antibody, or an antibody fragment thereof. An anti-idiotypic antibody is specific for binding to the idiotope of another antibody, an idiotope being an antigenic determinant of an antibody. A bispecific antibody may be a BiTE (bispecific T cell engager) or BiKE (bispecific killer cell engager), and a multispecific antibody may be a TriKE (trispecific killer cell engager).Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fabc, pFc, Fd, single chain fragment variable (scFv), tandem scFv (scFv)2, single chain Fab (scFab), disulfide stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen binding fragments (sdAb), camelid heavy chain IgG and Nanobody® fragments, recombinant heavy-chain-only antibodies (VHH), and other antibody fragments which maintain the binding specificity of the antibody.

[0124] "Fc receptors", abbreviated as FcR, are classified based on the type of antibody they recognize. For example, those that bind the most common class of antibody IgG are called Fc-gamma receptors (FcγR), those that bind IgA are called Fc-alpha receptors (FcαR), and those that bind IgE are called Fc-epsilon receptors (FcεR). Classes of FcR are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and therefore different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0125] "Chimeric receptor" is a general term used to describe engineered artificial or hybrid receptor protein molecules that are made to contain two or more portions of amino acid sequence from at least two different proteins. Chimeric receptor proteins are engineered to confer the cell the ability to initiate signal transduction and perform downstream functions upon binding of an agonist ligand to the receptor. Exemplary "chimeric receptors" include, but are not limited to, chimeric antigen receptors (CARs), chimeric fusion receptors (CFRs), chimeric Fc receptors (CFcRs), and fusions of two or more receptors.

[0126] "Chimeric Fc receptor", abbreviated as CFcR, is a term used to describe engineered Fc receptors in which the native transmembrane domain and / or intracellular signaling domain have been modified or replaced with a non-native transmembrane domain and / or intracellular signaling domain. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane domain and the signaling domain being non-native, one or more stimulatory domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance cell activation, proliferation, and function upon receptor triggering. Unlike chimeric antigen receptors (CARs), which contain an antigen-binding domain for a target antigen, chimeric Fc receptors bind to Fc fragments, or Fc regions of antibodies, or Fc regions contained in engager or binding molecules, and bind to the molecule to activate cell function, with or without bringing the target cell into close proximity. For example, Fcγ receptors can be engineered to contain selected transmembrane domains, stimulatory domains, and / or signaling domains in the intracellular region that respond to the binding of IgG at the extracellular domain, thereby generating CFcR. In one example, CFcR is produced by engineering the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domains. 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 CFcRs that include a high affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or the extracellular domain of the receptor is replaced to be non-cleavable, i.e., not subject to shedding, thereby obtaining hnCD16-based CFcR.

[0127] Two isoforms of the FcγR receptor CD16 have been identified: Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high affinity CD16," "non-cleavable CD16," or "high affinity non-cleavable CD16" (abbreviated as hnCD16) refers to natural or non-natural variants of CD16. Wild-type CD16 has low affinity and upon NK cell activation undergoes ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane proximal region (positions 189-212) has been altered or eliminated are not subject to shedding. The cleavage site and membrane proximal region are described in detail in WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high affinity and non-cleavable. Another exemplary high affinity and non-cleavable CD16 (hnCD16) variant is an engineered CD16 that contains an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0128] "T cell receptor", abbreviated as "TCR", generally refers to a protein complex found on the surface of T cells and is responsible for the recognition of fragments of antigenic peptides bound to major histocompatibility complex (MHC) molecules. Binding of the TCR to an antigenic peptide initiates TCR-CD3 intracellular activation, recruitment of multiple signaling molecules, and branching and integration of signaling pathways, leading to the recruitment of transcription factors important for gene expression and typical T cell proliferation and acquisition of function. A typical TCR contains two highly variable protein chains (α and β), each of which contains a constant region adjacent to the cell membrane and a variable region (i.e., a binding domain) that binds peptide / MHC.

[0129] I. Cells and Compositions Useful for Adoptive Cell Therapy with Enhanced Properties To avoid the problem of allogeneic rejection, it is believed that multiple HLA class I and class II proteins must be matched for histocompatibility of the allogeneic recipient. One approach that has been investigated in allogeneic adoptive cell therapy without MHC matching is to eliminate or substantially reduce the expression of both HLA class I and HLA class II proteins. 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, the TAP1 gene, the TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers. B2M-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 activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-negative cells are HLA-II deficient. Thus, the present application provides iPSCs and derived cells therefrom that contain HLA-I and / or HLA-II deficiency, e.g., due to lack of B2M and / or CIITA expression, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching to avoid recognition and killing by the host's (allogeneic) T cells.

[0130] However, lack of HLA class I expression increases susceptibility to lysis by NK cells. Moreover, deficiencies in both HLA-I and HLA-II still do not prevent allorejection mediated by alloantigens other than MHC of allogeneic adoptive cells. Furthermore, HLA-I-dependent NK cell education processes, such as licensing, arming, or disarming, are thought to affect innate immune responsiveness to allogeneic cells, which may lead to reactive or partial reactive recipient NK cells to allogeneic donor cells even when the allogeneic donor cells are HLA-I sufficient.

[0131] To overcome this "loss of self" response, HLA-E, HLA-G, or other non-classical HLA-I proteins can be optionally knocked in to avoid NK cell recognition and killing of HLA-I-deficient effector cells derived from engineered iPSCs. In one embodiment, the HLA-I-deficient iPSCs and derived cells thereof provided further comprise an HLA-G knock-in. Alternatively, in one embodiment, the HLA-I-deficient iPSCs and derived cells thereof provided further comprise one or both of a CD58 knockout and a CD54 knockout.

[0132] On the other hand, lymphatic conditioning chemotherapy, such as Cy / Flu (cyclophosphamide / fludarabine), commonly administered with patient-specific CAR-T cell therapy, suppresses the patient's immune system, potentially creating an adequate therapeutic window for allogeneic cell therapy to elicit a clinical response. However, prolonged lymphatic conditioning is associated with insufficient immune reconstitution, increased susceptibility to opportunistic infections, and a reduced rate of host immune contribution to the development of sustained antitumor efficacy. Therefore, alternative approaches need to be investigated to enable effective treatment with allogeneic cell therapy.

[0133] However, the present application instead provides a strategy that maintains HLA-I or HLA-II or both intact in allogeneic effector cells (i.e., including HLA-I wild type and HLA-II wild type) and does not require lymphatic conditioning, while avoiding allorejection of allogeneic effector cells by eliminating or reducing activated T cells, B cells, and NK cells in the adoptive cell therapy recipient. The stealth strategy provided is comprehensive in that it engages multiple immune recognition pathways to enable pre-made allogeneic effector cells to not only circumvent the alloreactivity of host immune cells, but also to enhance their functional persistence, thereby enabling effective adoptive cell-based cancer therapy to be achieved.

[0134] 1. Alloimmune defense receptor (ADR) expression Provided herein is a strategy to systematically manipulate multiple regulatory circuits of clonal iPSCs without affecting the differentiation potential and cell developmental biology of iPSCs and their derived cells while enhancing the therapeutic properties of derived cells differentiated from iPSCs. The 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. The manipulation strategies and components are also suitable for modifying primary T cells or primary NK cells for similar purposes.

[0135] To control pathogenic conditions resulting from unwanted activation of the immune system, in various embodiments, the present application provides immune cells, iPSCs, and iPSC-derived effector cells genetically engineered to contain allogeneic immune defense receptors (ADRs) for effector cell expansion and selective depletion of alloreactive host T or NK cells, including pathogenic T cells, while sparing resting cells in the recipient, among other edits contemplated and described herein. As provided in the present application, expression of allogeneic immune defense receptors (ADRs) has the unique ability to eliminate alloreactive T and NK cells and evade host immune cells without the need for deletion of class I HLA.

[0136] In some embodiments, the ADR is specific for 4-1BB (CD137, also referred to as "41BB"), and the ADR comprises an extracellular domain targeting 4-1BB, which is upregulated in host T cells or NK cells upon activation, and a signaling domain that promotes effector cell activation. For example, the 41BB-ADR extracellular domain may comprise any suitable ligand for 4-1BB, including 4-1BBL, an antibody (or functional fragment thereof) targeting 4-1BB, a fusion of Fc with 4-1BBL, or a functional derivative or fragment thereof. Additional upregulated surface proteins of host activated immune cells include, but are not limited to, CD38, CD25, CD69, CD44, OX40, and CD40L, based on which the ADR may be constructed to comprise binding domains specific for CD38, CD25, CD69, CD44, OX40, and CD40L, respectively.

[0137] In some embodiments of the ADR specific for 4-1BB, the 41BB-ADR extracellular domain comprises 4-1BBL or a fragment thereof effective to bind 4-1BB. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 75. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 75. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 75. In some embodiments, the 41BB-ADR extracellular domain comprises the amino acid sequence of SEQ ID NO: 75.

[0138] SEQ ID NO:75 GLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

[0139] In one embodiment of a CD38-specific ADR, the CD38-ADR comprises a CD38 antibody binding domain or a fragment thereof. In some embodiments, the anti-CD38 antibody comprises a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a single variable novel antigen receptor (VNAR), a shark heavy chain only antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody binding domains or fragments thereof include Fab, Fab', F(ab')2, F(ab')3, Fv, single chain antigen binding fragments (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single domain antigen binding fragments (sdAb, nanobodies), recombinant heavy chain only antibodies (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody.

[0140] In some embodiments, the anti-CD38 antibody binding domain or fragment thereof comprised in CD38-ADR is selected from SEQ ID NOs:29 and 30, respectively, SEQ ID NOs:31 and 32, respectively, SEQ ID NOs:33 and 34, respectively, SEQ ID NOs:35 and 36, respectively, SEQ ID NOs:37 and 38, respectively, SEQ ID NOs:39 and 40, respectively, SEQ ID NOs:41 and 42, respectively, SEQ ID NOs:43 and 44, respectively, SEQ ID NOs:45 and 46, respectively, SEQ ID NOs:47 and 48, respectively, SEQ ID NOs:49 and 50, respectively, SEQ ID NOs:51 and 52, respectively, SEQ ID NOs: 53 and 54, respectively, 55 and 56, respectively, 57 and 58, respectively, 59 and 60, respectively, 61 and 62, respectively, 63 and 64, respectively, 65 and 66, respectively, 67 and 68, respectively, 69 and 70, respectively, or 71 and 72, respectively. Selected VH and VL sequences of exemplary CD38 antibodies are provided in Table 1 as numbered pairs 1-23. In some embodiments, the CD38-ADR extracellular domain comprises an amino acid sequence having at least about 90% sequence identity to the VH and / or VL sequence of any of pairs 1-23 in Table 1. In some embodiments, the CD38-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity to the VH and / or VL sequence of any of pairs 1-23 in Table 1. In some embodiments, the CD38-ADR extracellular domain comprises the amino acid sequence of the VH and / or VL sequence of any of pairs 1-23 in Table 1.

[0141] [Table 1-1]

[0142] [Table 1-2]

[0143] In some embodiments, the extracellular domain of a 41BB-ADR, CD38-ADR, CD25-ADR, CD69-ADR, CD44-ADR, OX40-ADR, or CD40L-ADR may be operably linked to one or more signaling domains that mediate downstream signaling when an effector cell is activated upon binding to 4-1BB, CD38, CD25, CD69, CD44, OX40, or CD40L (respectively) of an alloreactive host immune cell. In some embodiments, the ADR comprises CD3ζ represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:1 or a functional fragment thereof, or comprises a CD3ζ derivative (e.g., CD3ζ1XX represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:2 or a functional fragment thereof). In some embodiments, CD3ζ comprises an amino acid sequence of at least about 90% sequence identity to SEQ ID NO:1 or 2. In some embodiments, CD3ζ comprises an amino acid sequence of at least about 95% sequence identity to SEQ ID NO:1 or 2. In some embodiments, CD3ζ comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, CD3ζ comprises the amino acid sequence of SEQ ID NO:2. CD3ζ mediates downstream ITAM-derived signaling during effector T or NK cell activation. Other ITAM-containing signaling domains can include those derived from DAP12, Fc receptors, and other CD3 subunits. 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. Comparison of sequences and determination of percent identity between two sequences can be performed using mathematical algorithms recognized in the art.

[0144] SEQ ID NO:1 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD3ζ) SEQ ID NO:2 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (CD3ζ1XX containing two mutations in ITAM1)

[0145] In some embodiments, the intracellular domain of the ADR comprising the signaling domain further comprises one, two, three or more costimulatory domains that enhance cytokine production from an effector cell expressing the ADR. The costimulatory domain may be derived from the intracellular signaling domain of a costimulatory protein, including, but not limited to, CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, CD40, and the like. In some embodiments, the ADR comprising CD3ζ further comprises a costimulatory domain derived from the 4-1BB endodomain and is represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:73 or a functional fragment thereof. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:73. In some embodiments, the 4-1BB endodomain comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:73. In some embodiments, the 4-1BB endodomain comprises the amino acid sequence of SEQ ID NO: 73. In one embodiment, when the ADR comprises 4-1BBL in its extracellular domain, the costimulatory domain of the ADR is not derived from 4-1BB.

[0146] [Table 2]

[0147] The intracellular domain of the ADR may be non-covalently linked to the extracellular domain of the ADR via a transmembrane domain. In some embodiments, the ADR includes a transmembrane domain, which may be of any type, so long as it allows the CD3ζ component of the ADR to be located intracellularly and the extracellular domain targeting 4-1BB, CD38, CD25, CD69, CD44, OX40, or CD40L to be located extracellularly. In other cases, the ADR is a soluble protein that can promote cytotoxicity by binding to the respective ligand on activated T cells and crosslinking the TCR (e.g., ADR-CD3 T cell engager protein). If the extracellular domain is derived from a surface protein (e.g., CD40) that has a transmembrane domain, the ADR may include a transmembrane domain from its corresponding endogenous molecule. In some embodiments in which the ADR molecule includes one or more costimulatory domains, the transmembrane domain (transmembrane domain, TM) may be derived from the same endogenous molecule that has the costimulatory domain. Non-limiting examples of TM include those derived from CD3, CD8a, CD27, CD28, 4-1BB, OX40, and CD4.

[0148] In some embodiments, the ADR comprises a spacer between the extracellular protein and the transmembrane domain. In some embodiments, the spacer may comprise a sequence that is inactive or contributes substantially little or nothing to any function that the ADR may have, while in other cases, the spacer comprises a sequence that enhances the function of the ADR and / or makes it detectable and / or can be targeted for inhibition. In certain embodiments, the spacer comprises an encoded protein sequence that facilitates detection of cells expressing the ADR. For example, the spacer may encode an Fc region or a fragment thereof that allows for surface detection of cells expressing the ADR, such as by using an anti-Fc antibody. In certain embodiments, the spacer provides a separation between the ligand-binding extracellular domain and the membrane to avoid potential steric hindrance. As will be appreciated by those skilled in the art, the spacer may be of various sequences, lengths, whether or not a function other than physical separation is intended.

[0149] Exemplary spacers that may be included in the ADR are generally known in the art and include, but are not limited to, IgG4 spacers, CD28 spacers, CD8 spacers, or a combination of two or more spacers. The length of the spacer may also vary from about 15 amino acids (aa) to about 300 amino acids or more. Non-limiting exemplary spacer peptides include those represented by an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO:3 or 4. In some embodiments, the spacer peptide comprises an amino acid sequence of at least about 90% sequence identity to SEQ ID NO:3 or 4. In some embodiments, the spacer peptide comprises an amino acid sequence of at least about 95% sequence identity to SEQ ID NO:3 or 4. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO:4.

[0150] SEQ ID NO:3 ESKYGPPCPPCPGGGSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL (88 amino acids) SEQ ID NO:4 ESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKDPK (123 amino acid IgG4 hinge-IgG1 CH3)

[0151] In one embodiment of a 4-1BB specific ADR, the 41BB-ADR is represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any of SEQ ID NOs: 5-8, with the signal peptide, spacer, and transmembrane (TM) domain sequences varying. In some embodiments, the 41BB-ADR comprises an amino acid sequence of at least about 90% sequence identity to any of SEQ ID NOs: 5-8. In some embodiments, the 41BB-ADR comprises an amino acid sequence of at least about 95% sequence identity to any of SEQ ID NOs: 5-8. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 8.

[0152] [Table 3]

[0153] [Table 4]

[0154] [Table 5]

[0155] [Table 6]

[0156] In one embodiment of a CD38-specific ADR, the CD38-ADR is represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 74. In some embodiments, the CD38-ADR comprises an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 74. In some embodiments, the CD38-ADR comprises an amino acid sequence of at least about 95% sequence identity to SEQ ID NO: 74. In some embodiments, the CD38-ADR comprises the amino acid sequence of SEQ ID NO: 74.

[0157] [Table 7]

[0158] Thus, in some embodiments, the application provides immune cells, iPSCs, and iPSC-derived effector cells that comprise polynucleotides encoding ADRs specific for 4-1BB, CD38, CD25, CD69, CD44, OX40, or CD40L, and optionally other selected gene edits as described herein, to confer upon effector cells, including T cells and NK cells, the ability to selectively deplete activated host immune cells, such that the engineered effector cells are resistant to host immune alloreactivity associated with the allogeneic use of effector cells for the treatment of tumors and infectious diseases in patients. Further provided in the present application is a master cell bank comprising sorted single cells and expanded clonal engineered iPSCs having at least one phenotype provided herein, including ADRs specific for 4-1BB, CD38, CD25, CD69, CD44, OX40, or CD40L, which provides a platform for further iPSC manipulation 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 compositionally defined and uniform and can be cost-effectively mass-produced at substantial scale.

[0159] 2. Endogenous TCR knockout The alpha-beta T cell receptor (TCRαβ) is an antigen-specific receptor typically considered essential for immune responses and is present on the cell surface of αβ T lymphocytes. Binding of TCRαβ to the peptide-major histocompatibility complex (pMHC) initiates TCR-CD3 intracellular activation, recruitment of numerous signaling molecules, and branching and integration of signaling pathways, leading to recruitment of transcription factors important for gene expression and T cell proliferation and acquisition of function. Disrupting the constant region of endogenous TCR alpha or TCR beta (TRAC or TRBC) through direct editing of T cells or through genomic iPSC editing and differentiation as a source to obtain modified derived T lineage cells allows the expression of TCR alpha or TCR beta. negFor example, insertion of a 2A sequence at a preselected location in TRAC or TRBC, operably linked to either an endogenous promoter of TRAC or TRBC, or to an exogenous promoter, results in the disruption (or in this example, truncation) of TRAC or TRBC and the generation of TCR-negative cells (TCR neg 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 location of cleavage or disruption, and expression of the integrated genes can be driven by an operably linked exogenous promoter or by the endogenous promoter of TCR alpha or TCR beta upon integration, resulting in a TRAC or TRBC knockout, and thus a TCR knockout (TCR KO )

[0160] In certain embodiments, the TCR neg The cell is an iPSC. neg The cells are NK lineage cells. As used herein, "TCR negative" or "TCR neg The term "TCR gene disruption" refers to disruption of TCR gene expression (TCR KO It refers to the lack of endogenous TCR expression, either due to lack of TCR expression (such as in T lineage cells: primary or iPSC-derived T lineage cells) or due to the absence of natural expression of the TCR gene 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 homogeneous populations thereof, without TCR expression.

[0161] iPSC-derived TCR-negative effector cells obtained using this approach (with or without exogenous gene integration) can, when used in allogeneic adoptive cell therapy, not require HLA matching, have reduced alloreactivity, and prevent GvHD (Graft versus Host Disease).

[0162] Thus, in some embodiments, the present invention relates to a method for the treatment of 4-1BB-specific ADRs comprising administering to a subject an exogenous polynucleotide encoding an ADR specific for 4-1BB and, optionally, an endogenous TCR knockout (TCR KO ), and derived cells therefrom, including derived T cells and derived NK cells, which are useful for overcoming alloreactivity in immunocompetent host systems. In some embodiments, the iPSCs and derived cells thereof can be expressed in a variety of ways, including by expressing an exogenous polynucleotide encoding a 41BB-ADR as described herein and optionally a TCR, without adversely affecting the differentiation potential of the iPSCs and the function of derived effector cells, including derived T cells and derived NK cells. KO and one or more additional genome edits. ADR and optionally TCR specific for 4-1BB. KO Further provided in the present application is a master cell bank comprising sorted single cells and expanded clonal engineered iPSCs having at least one phenotype provided herein, comprising:

[0163] 3.CD38 knockout The cell surface molecule CD38 is highly upregulated in multiple hematological malignancies of both lymphoid and myeloid origin, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated cancer cell depletion usually results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cell-mediated cytotoxicity). In addition to ADCC, immune effector mechanisms in conjunction with therapeutic antibodies may also include antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0164] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells, as well as on NK cells, activated T cells, and B cells. During hematopoiesis, CD38 is expressed in association with CD34 + It is expressed in stem cells and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, as well as in the final stages of maturation that continue up to the plasma cell stage. As a type II transmembrane glycoprotein, CD38 serves cellular functions both as a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 mediates the synthesis and upregulation of NAD + CD38 catalyzes the hydrolysis of ribosomal acetylcholine to ADP-ribose, thereby generating the second messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which are important for calcium-dependent cell adhesion processes. CD38 recognizes CD31 as a receptor and regulates cytokine release and cytotoxicity of activated NK cells. CD38 associates with cell surface proteins in lipid rafts and mediates the release of cytoplasmic Ca. 2+ It has also been reported to regulate flux and mediate signaling in lymphoid and myeloid cells.

[0165] In the treatment of malignancies, the systemic use of T cells transduced with the CD38 antigen-binding receptor has been shown to inhibit the expression of CD34 +CD38 on hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells + Fraction lysis and impaired immune effector cell function in the recipient lead to incomplete therapeutic responses and reduced or eliminated efficacy. In addition, 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 and B cells were unaffected despite CD38 expression (Casneuf et al., Blood Advances. 2017; 1(23): 2105-2114).

[0166] Without being bound by theory, the present application provides a strategy to exploit the full potential of CD38-targeted cancer therapy by overcoming the depletion or reduction of effector cells through fratricide induced by CD38-specific antibodies and / or CD38 antigen-binding domains. In addition, since CD38 is upregulated on activated lymphocytes such as T cells or B cells, suppressing the activation of these recipient lymphocytes using CD38-specific antibodies such as daratumumab in recipients of allogeneic effector cells reduces and / or prevents host allo-rejection of these effector cells, thereby increasing the survival and persistence of effector cells. Thus, CD38-specific antibodies, secreted CD38-specific engagers, or CD38-CARs (chimeric antigen receptors) for activation of recipient T, Treg, NK, and / or B cells can be used as an alternative to lymphodepletion using chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine) prior to adoptive cell transfer.

[0167] In addition, CD38 is inhibited in the presence of a CD38 antagonist, including, but not limited to, an anti-CD38 antibody, CD38-ADR, or a CD38 inhibitor. - Effector cells were used to identify CD38 + For targeting T and pbNK cells, CD38 + When alloreactive cells are depleted, NAD +(nicotinamide adenine dinucleotide, a substrate for CD38) availability increases, and NAD + Consumption-associated cell death is reduced, which, among other benefits, boosts effector cell responses in the immunosuppressive tumor microenvironment and supports cellular rejuvenation in aging, degenerative, or inflammatory diseases.

[0168] Furthermore, in various embodiments, the strategies provided herein for knocking out CD38 are compatible with other components and processes contemplated herein, thereby providing CD38 knockout (CD38 KO ), master cell banks containing sorted single cells and expanded clonal CD38-negative iPSCs were generated, and CD38-negative (CD38 neg ) derived effector cells are obtained that, among other benefits, are protected from fratricide and allo-rejection when a CD38-targeted therapeutic moiety is used in conjunction with the effector cells. 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, and is an example of a process termed "CD38 conditioning" when administered for purposes including, but not limited to, lymphodepletion. Thus, in some embodiments, CD38 KO Combining effector cells with anti-CD38 monoclonal antibodies (i) reduces or prevents host cell alloreactivity against allogeneic effector cells, (ii) eliminates or reduces the number of alloreactive host cells, (iii) extends the survival and persistence of allogeneic effector cells, (iv) delays reconstitution of host immunity, and / or (v) prevents leakage of allogeneic effector cell protection against host cell alloreactivity via overexpression of HLA-G or HLA-E. negThe derived cells have the ability to resist CD38 antibody-mediated depletion and can be effectively administered in combination with anti-CD38 antibodies, CD38-CARs, or CD38-ADRs without the use of toxic conditioning agents, thus reducing and / or replacing chemotherapy-based lymphodepletion.

[0169] In one embodiment provided herein, the CD38 knockout in the iPSC line is a biallelic knockout. As disclosed herein, in some embodiments, the provided CD38 negative iPSC line comprises at least 41BB-ADR or CD38-ADR and optionally TCR. KO and optionally one or more additional engineered modalities described herein and shown in Table 2, wherein the iPSCs are mesodermal cells with definitive hemogenic endothelium (HE) potential, embryonic HE, CD34 + Directed differentiation can be performed to produce functional derived hematopoietic cells, including, but not limited to, immune effector cells, including hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, common myeloid progenitors, common lymphoid progenitors, erythrocytes, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells with one or more functional characteristics not present in primary NK cells, T cells, and / or NKT cells. In some embodiments, CD38 antibodies are used to induce ADCC or anti-CD38 CARs, or CD38-ADRs are used for targeted cell killing. negThe iPSCs and / or their derived effector cells are not eliminated by anti-CD38 antibodies, anti-CD38 CARs, or CD38-ADRs, or recipient activated T or B cells, thereby increasing the persistence and / or viability 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 viability in vivo in the presence of and / or after exposure to such therapeutic moieties.

[0170] A polynucleotide encoding an ADR specific for 4-1BB or CD38 and optionally a TCR KO and CD38 KO Further provided in the present application is a master cell bank comprising sorted single cells and expanded clonal engineered iPSCs having at least one phenotype provided herein, including one or both of the following:

[0171] 4.CD16 knock-in CD16 has been identified as two isoforms, the Fc receptors FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is expressed exclusively by human neutrophils. As used herein, "high affinity CD16," "non-cleavable CD16," or "high affinity non-cleavable CD16" refers to various CD16 variants. Wild-type CD16 has low affinity and is subject to downregulation upon NK cell activation, including ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V (also referred to as F158V in some publications) is an exemplary CD16 polymorphic allele / variant with high affinity, while the S197P variant is an example of an engineered non-cleavable version of CD16. Engineered CD16 variants including both F176V and S197P have high affinity and are non-cleavable, as described in more detail in WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. In addition, a chimeric CD16 receptor in which the ectodomain of CD16 is essentially replaced with at least a portion of the ectodomain of CD64 can also achieve the desired high affinity and non-cleavable properties of a CD16 receptor 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 an isoform or polymorphic variant thereof).

[0172] Thus, various embodiments of exogenous CD16 introduced into cells include functional CD16 variants and their chimeric receptors. In some embodiments, the functional CD16 variant is a high affinity non-cleavable CD16 receptor (hnCD16). In some embodiments, hnCD16 includes both F176V and S197P, and in some embodiments, includes F176V, eliminating the cleavage region. In some other embodiments, hnCD16 includes 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 SEQ ID NOs: 9, 10, and 11, which are exemplary sequences each including at least a portion of the CD64 ectodomain.

[0173] [Table 8]

[0174] [Table 9]

[0175] [Table 10]

[0176] Thus, among the editing contemplated and described herein, effector cells or iPSCs are provided herein that are genetically engineered to contain exogenous CD16 or variants thereof, where the effector cells are cells from a primary source or cells derived from iPSC differentiation, or the genetically engineered iPSCs can differentiate into derived effector cells that contain exogenous CD16 or variants thereof introduced into the iPSC. In some embodiments, the derived effector cells that contain hnCD16 are NK cells. In some embodiments, the exogenous CD16 is a high affinity non-cleavable CD16 (hnCD16) receptor. In some embodiments, the hnCD16 comprises a full-length or partial-length extracellular domain of CD64. In some embodiments, the exogenous CD16 is in the form of a CD16-based chimeric Fc receptor (CFcR) that contains a transmembrane domain, a stimulatory domain, and / or a signaling domain that is not derived from CD16.

[0177] In some embodiments, the primary origin or derived effector cells comprising exogenous CD16 or variants thereof are NK lineage cells. In some embodiments, the primary origin or derived effector cells comprising exogenous CD16 or variants thereof are T lineage cells. The exogenous CD16 or functional variants thereof contained in the iPSCs or effector cells have high affinity for binding to a ligand that triggers downstream signaling upon such binding. Non-limiting examples of ligands that bind to exogenous CD16 or functional variants thereof include ADCC antibodies or fragments thereof, as well as bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domains of CD16 or CD64 of the exogenous CD16. Examples of bispecific, trispecific, or multispecific engagers or binders are further described below in this application. Thus, at least one of the aspects of the present application provides derived effector cells or cell populations thereof pre-loaded with one or more preselected ADCC antibodies via exogenous CD16 expressed on the derived effector cells, wherein the exogenous CD16 comprises the extracellular binding domain of CD64, or of CD16 having F176V and S197P, in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection as further described herein.

[0178] In some other embodiments, the exogenous CD16 comprises a CFcR based on CD16 or a variant thereof. Chimeric Fc receptors (CFcRs) are produced to include a non-native transmembrane domain, a non-native stimulatory domain, and / or a non-native signaling domain by modifying or replacing the native CD16 transmembrane domain and / or intracellular domain. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor than the receptor that provides the extracellular domain. Exemplary herein, 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 CD16-based CFcR comprises a non-native transmembrane domain derived from CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor polypeptide. In some embodiments, the exogenous CD16-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 CD16-based CFcR comprises a non-native signaling domain derived from a CD3zeta, 2B4, DAP10, DAP12, DNAM1, CD137(4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of a CD16-based CFcR, a chimeric Fc receptor is provided that comprises a transmembrane domain and a signaling domain that are both derived from one of an IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide.One specific exemplary embodiment of a CD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, where the extracellular domain of CFcR is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, where the extracellular domain of CD16 comprises F176V and S197P. Another exemplary embodiment of a CD16-based chimeric Fc receptor comprises the transmembrane domain and signaling domain of CD3ζ, where the extracellular domain of CFcR is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, where the extracellular domain of CD16 comprises F176V and S197P.

[0179] Various embodiments of the CD16-based chimeric Fc receptor as described above can bind with high affinity to the Fc region of an antibody or fragment thereof, or to a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory and / or signaling domains of the chimeric receptor allow for effector cell activation and cytokine secretion, as well as killing of tumor cells targeted by the antibody, or by the bispecific, trispecific, or multispecific engager or binder having a tumor antigen binding moiety and an Fc region. Without being limited by theory, through the non-native transmembrane, stimulatory, and / or signaling domains of the CD16-based chimeric Fc receptor, or through the binding of the engager to the ectodomain, the CFcR can contribute to the killing capacity of the effector cells, increasing the likelihood of effector cell proliferation and / or expansion. Antibodies and engagers 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 CD16-based CFcR in attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0180] Unlike endogenous CD16 expressed by primary NK cells, which is cleaved from the cell surface following NK cell activation, the various non-cleavable versions of CD16 on derived NK cells avoid CD16 shedding and maintain constant expression. In derived NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cell-mediated cytotoxicity (ADCC) and the binding of bi-, tri-, or multi-specific engagers. ADCC is a mechanism of NK cell-mediated lysis via binding of CD16 to antibody-coated target cells. The additional high affinity properties of hnCD16 introduced into derived NK cells also allow in vitro loading of ADCC antibodies via hnCD16 prior to administration of the cells to a subject in need of cell therapy. As provided herein, hnCD16 may in some embodiments comprise F176V and S197P, or may comprise a full-length or partial-length ectodomain originating from CD64 as exemplified by SEQ ID NO: 9, 10, or 11, 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 described herein.

[0181] 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 previously unexpected that iPSCs with expressed exogenous uncleavable CD16 can differentiate into functional derived T lineage cells that not only express exogenous CD16 but can also perform functions through acquired ADCC mechanisms without compromising the developmental biology of T cells. This acquired ADCC in derived T lineage cells can further be used as an approach to rescue antigen escape, which often occurs in dual targeting and / or CAR-T cell therapy, where tumors relapse with reduced or lost expression of CAR-T target antigens, or expression of mutated antigens that avoid recognition by CAR. If the derived T lineage cells contain ADCC acquired via exogenous CD16 (including functional variants and CD16-based CFcR) 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 relapse of the targeted tumor commonly seen with CAR-T therapy. Such strategies to reduce and / or prevent antigen escape while achieving dual targeting are similarly applicable to NK cells expressing one or more CARs.

[0182] Thus, embodiments of the present invention, as provided herein, comprise a 41BB-ADR plus exogenous CD16, and optionally CD38 KO and TCR KOIn some embodiments, the CD16 in the derived effector cells is hnCD16 that comprises a CD16 ectodomain that comprises F176V and S197P. In some other embodiments, the hnCD16 in the derived effector cells may comprise a full-length or partial-length ectodomain originating from CD64, as exemplified by SEQ ID NO: 9, 10, or 11, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As described herein, such derived effector cells have an acquired mechanism for targeting tumors with monoclonal antibodies mediated by ADCC to enhance the therapeutic effect of the antibody. As disclosed, the present application also provides derived effector cells or cell populations thereof preloaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as described in more detail below.

[0183] As disclosed herein, an exogenous polynucleotide encoding 41BB-ADR and optionally CD16, CD38 KO , and endogenous TCR KO and wherein such cells may further comprise one or more of the additional engineered modalities described herein and shown in Table 2, the iPSCs include mesodermal cells with embryonic hemogenic endothelial (HE) potential, embryonic HE, CD34 + They can be directed to differentiate to produce functional derived hematopoietic cells, including, but not limited to, immune effector cells, including, but not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, common myeloid progenitors, common lymphoid progenitors, erythrocytes, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional characteristics not present in primary NK cells, T cells, and / or NKT cells.

[0184] 41BB-ADR, and optionally CD38 KO , endogenous TCR KO Further provided in the present application is a master cell bank comprising sorted single cells and expanded clonally engineered iPSCs having at least one phenotype provided herein, including, but not limited to, one or more of: exogenous CD16, and exogenous CD17, which provides a platform for further iPSC manipulation and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products, including, but not limited to, derived NK and T cells, that are compositionally defined and uniform and can be cost-effectively mass-produced at significant scale.

[0185] 5. Chimeric Antigen Receptor (CAR) Expression Applicable to engineered iPSCs and their derived effector cells can be any CAR design known in the art. CARs are fusion proteins that generally include an ectodomain, a transmembrane domain, and an endodomain, including an antigen recognition region. In some embodiments, the ectodomain can further include a signal peptide or leader sequence and / or a spacer. In some embodiments, the endodomain can further include a signaling domain, the signaling domain being derived from a cytoplasmic domain of a signaling protein specific for T cell and / or NK cell activation or function. In some embodiments, the antigen recognition domain can specifically bind to an antigen. In some embodiments, the antigen recognition domain can specifically bind to an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, and the tumor can be a liquid tumor or a solid tumor. In some embodiments, the CAR is suitable for activating either T lineage cells or NK lineage cells that express the CAR. In some embodiments, the CAR is NK cell specific, including NK specific signaling components. In certain embodiments, the NK cells are derived from iPSCs that include a polynucleotide encoding the CAR. In some embodiments, the CAR is T cell specific by including T cell specific signaling components. In certain embodiments, the T cells are derived from iPSCs comprising a polynucleotide encoding a CAR, and the derived T lineage 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.

[0186] In certain embodiments, the antigen recognition domain comprises a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a single variable novel antigen receptor (VNAR), a shark heavy chain only antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single chain antigen binding fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single domain antigen binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody. In some embodiments, the antigen recognition region of the CAR is derived from the binding domain of a T cell receptor (TCR) that targets a tumor associated antigen (TAA).

[0187] Non-limiting examples of antigens that can be targeted by a CAR include ADGRE2, B7H3, 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, cytometrium, and the like. Antigen of gallovirus (CMV)-infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 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 A1 (MAGE-A1), MICA / B, MR1, mucin 1 (Muc-1), mucin 16 (Muc- 16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, NYESO1, oncofetal antigen (h5T4), PDL1, PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.In yet some other embodiments, the CAR-T cells comprising an exogenous TCR complex further comprise a CAR specific for one of MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Muc1, Muc16, CD19, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1.

[0188] In some embodiments, the present specification provides a CAR (referred to as a "CD19-CAR") that includes an antigen recognition region that targets CD19. Non-limiting examples of CARs are described in WO 2019 / 133969, which is incorporated by reference herein. In one embodiment, the CD19-CAR includes a CD19 binding domain that includes a heavy chain variable region represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO:24, and a light chain variable region represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO:25. In one embodiment, the CD19-CAR comprises a single chain variable fragment (scFV) having an amino acid sequence at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO:26, where the linker and / or signal peptide are exemplary and can be replaced.

[0189] SEQ ID NO:24 EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS (CD19 scFv heavy chain (HC) of 122 amino acids) SEQ ID NO:25 DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR (CD19 scFv light chain (LC) of 108 amino acids)

[0190] [Table 11]

[0191] In some embodiments, a spacer / hinge is present between the antigen recognition region and the transmembrane domain of the CAR, while in some other embodiments, such a spacer / hinge is not required. Exemplary spacers that may be included in a CAR are generally known in the art and include, but are not limited to, an IgG4 spacer, a CD28 spacer, a CD8 spacer, or a combination of two or more spacers. The length of the spacer may also vary from about 15 amino acids to about 300 amino acids or more. In this application, for ease of explanation, a spacer less than about 80 amino acids, e.g., 10-80 amino acids, is considered short. A spacer of about 80-180 amino acids is considered medium, and a spacer of more than 180 amino acids is considered long. Non-limiting exemplary spacer peptides include those represented by an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of SEQ ID NOs: 3, 20, 21, 22, or 23.

[0192] SEQ ID NO:20 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (39 amino acids) SEQ ID NO:3 ESKYGPPCPPCPGGGSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL (88 amino acids) SEQ ID NO:21 ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLT (89 amino acids) SEQ ID NO:22 ESKYGPPCPPCPGGGSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (129 amino acids) SEQ ID NO:23 ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (229 amino acids)

[0193] 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 CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERI gamma, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or T cell receptor polypeptide.

[0194] In some embodiments, the signaling peptide of the internal domain (or intracellular domain) is selected from the group consisting of 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain; SEQ ID NO:27), CD3ζ1 XX (CD3ζ variant; SEQ ID NO: 28), DAP10 (hematopoietic cell signaling receptor), DAP12 (TYRO protein tyrosine kinase binding protein), DNAM1 (CD226 antigen), FcERIγ (high affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta), IL2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D CARs include the full length or at least a portion of the polypeptides of NKp30 (natural cytotoxicity triggering receptor 3), NKp44 (natural cytotoxicity triggering receptor 2), NKp46 (natural cytotoxicity triggering receptor 1), CS1 (SLAM family member 7), and CD8 (T-cell surface glycoprotein CD8 alpha chain). A description of exemplary signaling proteins, including transmembrane and cytoplasmic sequences of proteins suitable for incorporation into a CAR can be found in WO 2021 / 077117, the entire disclosures of each of which are incorporated herein by reference).

[0195] SEQ ID NO:27 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD3ζ cytoplasmic sequence of 113 amino acids) SEQ ID NO:28 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (CD3ζ1XX of 112 amino acids; contains two mutations in ITAM1)

[0196] In certain embodiments, the endodomain further comprises at least one costimulatory signaling region, which can include a 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.

[0197] In one embodiment, a CAR applicable to the cells provided in the present application comprises a costimulatory domain derived from CD28 and a signaling domain comprising a native or modified ITAM1 of CD3ζ represented by an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 12. In a further embodiment, the CAR comprising a costimulatory domain derived from CD28 and a native or modified ITAM1 of CD3ζ also comprises a hinge domain and a transmembrane domain derived from CD28, and the scFv can be connected to the transmembrane domain via the hinge, and the CAR comprises an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 13.

[0198] SEQ ID NO:12 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (153 amino acid CD28 costimulatory + CD3ζ ITAM)

[0199] [Table 12]

[0200] In another embodiment, a CAR applicable to the cells provided in the present application comprises a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3ζ 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: 14. 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, the amino acid sequence of such structure being at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 15.

[0201] [Table 13]

[0202] [Table 14]

[0203] Non-limiting CAR strategies include heterodimeric conditionally activated CARs through dimerization of a pair of intracellular domains (see, e.g., U.S. Pat. No. 9,587,020); homologous recombination of antigen-binding, hinge, and endodomains to generate split CARs (see, e.g., U.S. Pat. App. Pub. No. 2017 / 0183407); multi-chain CARs that allow for non-covalent association between two transmembrane domains connected to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Pat. App. Pub. No. 2014 / 0134142); CARs with bispecific antigen-binding domains (see, e.g., U.S. Pat. No. 9,447,194), or CARs with a pair of antigen-binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Pat. No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Invest. 2016;126(8):3036-3052); inducible CARs (see, e.g., U.S. Patent Application Publication Nos. 2016 / 0046700, 2016 / 0058857, and 2017 / 0166877); switchable CARs (see, e.g., U.S. Patent Application Publication No. 2014 / 0219975); and any other designs known in the art.

[0204] In a further embodiment, a CAR, 41BB-ADR, and optionally CD38 KO The iPSCs and derived effector cells have a CAR inserted into the TCR constant region and are capable of expressing endogenous TCR knockout (TCR KO ), placing CAR expression under the control of the endogenous TCR promoter. Disruption of the constant region of TCRα or TCRβ (TRAC or TRBC) results in the expression of the TCR negIn some other embodiments, the CAR inserted into the TCR constant region is specific for a tumor antigen comprising at least one of MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Muc1, Muc16, CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, GD2, MSLN, VEGF-R2, PSMA, and PDL1. Additional CAR insertion sites include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0205] In addition, TCR expression is negative in NK lineage effector cells differentiated from iPSCs. neg The cells do not require HLA matching and when used in allogeneic adoptive cell therapy, alloreactivity can be reduced and GvHD (graft versus host disease) can be prevented. Additional insertion sites for CARs include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. In another embodiment, the immune cells, iPSCs and derived NK cells thereof comprise a CAR, which when inserted into the NKG2A or NKG2D locus results in an NKG2A or NKG2D knockout, thereby placing CAR expression under the control of the endogenous NKG2A or NKG2D promoter.

[0206] Thus, in addition to the genetically engineered immune cells comprising the functional modalities provided herein, an aspect of the invention is derived cells obtained from differentiating genomically engineered iPSCs, wherein the iPSCs are expressing an exogenous polynucleotide encoding 41BB-ADR, and optionally exogenous CD16, CD38, KO , and endogenous TCR KO and wherein the iPSCs further comprise one or more CARs, optionally with the additional modification modalities discussed herein. KO , endogenous TCR KO Further provided in the present application is a master cell bank comprising sorted single cells and expanded clonally engineered iPSCs having at least one or more of the following:

[0207] 6. Exogenously introduced cytokine signaling complexes By avoiding systemic administration of clinically relevant cytokines at high doses, the risk of dose-limiting toxicity from such actions is reduced and cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, a cytokine signaling complex comprising one or more partial or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective corresponding receptors can be introduced into cells to allow cytokine signaling with or without expression of the cytokine itself, thereby maintaining or improving cell growth, proliferation, amplification, and / or effector function with reduced risk of cytokine toxicity. In some embodiments, the introduced cytokine and / or its respective native or modified receptor (signaling complex) for cytokine signaling is 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. In some embodiments, transient / transient expression of cell surface cytokine / cytokine receptor is via retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA-borne expression constructs, including mRNA.

[0208] Various construct designs are provided herein for introducing protein complexes into cells for signaling one, two or more cytokines, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18 and IL21. For example, in embodiments where the signaling complex is for IL15, the transmembrane (TM) domain can be native to the IL15 receptor or can be modified or replaced with the transmembrane domain of any other membrane-bound protein. In various embodiments, the cytokine signaling complex comprises an IL15 receptor fusion (IL15RF) comprising full-length or partial-length IL15 and full-length or partial-length IL15 receptor (IL15R). In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics the trans-presentation of IL15 without eliminating the cis-presentation of IL15. In another embodiment, IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without eliminating cis-presentation of IL15, but also ensuring that IL15 is membrane-bound. In another embodiment, IL15Rα with a truncated intracellular domain is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation of IL15, maintaining membrane-bound of IL15, and additionally eliminating cis-presentation and / or any other possible signaling pathways mediated by normal IL15R via its intracellular domain. In another embodiment, IL15Rα is fused to IL15 without the intracellular domain (IL15Δ), as described in WO 2019 / 191495 and WO 2019 / 126748, the disclosures of each of which are incorporated herein by reference in their entirety.

[0209] In various embodiments, such truncated constructs comprise an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 16. In one embodiment of truncated IL15 / IL15Rα, the construct does not include the last four amino acid residues (KSRQ) of SEQ ID NO: 16 and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17. In some embodiments, the construct comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct comprises the amino acid sequence of SEQ ID NO: 16.

[0210] [Table 15]

[0211] [Table 16]

[0212] In yet other embodiments, the cytoplasmic domain of IL15Rα can be omitted without adversely affecting the autonomous properties of IL15-equipped effector cells. In other embodiments, essentially the entire IL15Rα is removed, with the exception of the Sushi domain, which is fused to IL15 at one end and to a transmembrane domain at the other end (mb-Sushi), with optional linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of any membrane-bound protein. Unwanted signaling through IL15Rα, including cis-presentation, is eliminated, while only the desired trans-presentation of IL15 is retained. In some embodiments, the 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: 18.

[0213] [Table 17]

[0214] In other embodiments, native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to allow constitutive signaling and maintain IL15 membrane binding and trans presentation. In other embodiments, native or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling and membrane-bound trans presentation of cytokines. 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 IL2, IL4, IL7, IL9, IL15, and IL21 receptors. In other embodiments, engineered IL15Rβ, which forms homodimers in the absence of IL15, is useful for generating constitutive signaling of cytokines.

[0215] In various other embodiments, the cytokine signaling complex comprises an IL7 receptor fusion (IL7RF) comprising full length or partial length IL7 and full length or partial length IL7 receptor. The transmembrane (TM) domain may be native to the IL7 receptor or may be modified or replaced with the transmembrane domain of any other membrane-bound protein. In some embodiments, a native (or wild type) or modified IL7R may be fused to IL7 at the C-terminus via a linker (also referred to herein as "IL7RF"), allowing for constitutive signaling and maintaining membrane-bound IL7. In some embodiments, such constructs comprise an amino acid sequence at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 19, with the transmembrane domain, signal peptide, and linker being flexible and varying in length and / or sequence. In some embodiments, the construct comprises the amino acid sequence of SEQ ID NO: 19.

[0216] [Table 18]

[0217] In one embodiment, a native or engineered common receptor γC is fused to IL7 at the C-terminus via a linker for constitutive and membrane-bound cytokine signaling complexes. 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 IL2, IL4, IL7, IL9, and IL21 receptors. In another embodiment, engineered IL7R, which forms homodimers in the absence of IL7, is also useful for generating constitutive cytokine signaling.

[0218] Those skilled in the art will understand that the signal peptides and linker sequences described above are exemplary and in no way limit the variations thereof suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence can be substituted with another sequence without altering the activity of the functional peptide led by the signal peptide or linked by the linker.

[0219] In iPSCs and derived cells containing both CAR and exogenous cytokine and / or cytokine receptor signaling (cytokine signaling complex or "IL" in Table 2), the CAR and IL may be expressed in separate constructs or may be co-expressed in a bicistronic construct containing both the CAR and IL. In some further embodiments, the signaling complex may be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence. Thus, the IL signaling complex (e.g., IL7 signaling complex) and the CAR may be in a single open reading frame (ORF). In one embodiment, the signaling complex is included in a CAR-2A-IL or IL-2A-CAR construct. When CAR-2A-IL or IL-2A-CAR is expressed, the self-cleaving 2A peptide allows the expressed CAR and IL to dissociate, and the dissociated IL may then be presented on the cell surface with the transmembrane domain anchored to the cell membrane. The bicistronic design of CAR-2A-IL or IL-2A-CAR allows for coordinated expression of the CAR and IL signaling complexes, both in timing and quantity, under identical regulatory mechanisms that can be selected to incorporate, such as, for example, inducible promoters for expression of a single ORF or promoters with temporal or spatial specificity.Self-cleaving peptides are found in members of the picornavirus family, including the aphthovirus genus, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-I) (Donnelly, ML, et al, J. Gen. Virol, 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virol, 72, 2727-2732 (2001)), and the cardiovirus genus, such as Theilovirus (e.g., Theiler's murine encephalomyelitis virus) and encephalomyocarditis virus. The 2A peptides from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively.

[0220] The bicistronic CAR-2A-IL or IL-2A-CAR disclosed herein also contemplates expression of any other cytokine or cytokine signaling complex provided herein, such as IL2, IL4, IL6, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, the bicistronic CAR-2A-IL or IL-2A-CAR is for expression of one or more of IL2, IL4, IL7, IL9, IL15, and IL21.

[0221] In some embodiments, iPSCs and derived effector cells comprising any one of the genotypes in Table 2 contain a disruption 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 a disruption of at least one of HLA-E, 4-1BBL, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D, E, F, F, G, H, H, I, I, M, I, N, O, P, P, P, R, P, P, T, P, R, P, T ... 2AThe invention may further include the introduction of at least one of a surface triggering receptor for coupling to an R, a TCR, an Fc receptor, an antibody, and a bispecific, multispecific, or universal engager.

[0222] Thus, in various embodiments, the cytokines IL15 or IL7 and / or their receptors may be introduced into iPSCs using one or more of the above construct designs, and into their derived cells upon iPSC differentiation. In addition, polynucleotides encoding 41BB-ADR and, optionally, CAR, exogenous CD16, cytokine signaling complex, CD38, etc., are described herein. KO , and endogenous TCR KO The present invention provides an induced pluripotent cell (iPSC), a clonal iPSC, a clonal iPS cell line, or an iPSC-derived cell, comprising at least one or more of the following: KO , endogenous TCR KO Also provided is a master cell bank comprising sorted single cells and expanded clonally engineered iPSCs having at least a cytokine signaling complex, as described in this section, wherein the cytokine signaling complex comprises a partial or complete peptide of a cell surface expressed exogenous cytokine and / or its receptor, wherein 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 compositionally defined and uniform and can be cost-effectively mass-produced at significant scale.

[0223] 6. CD58 and / or CD54 knockout CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and promote migration of cells, including immune cells. CD58 knockout has a higher efficiency in reducing allogeneic NK cell activation than CD54 knockout, while double knockout of both CD58 and CD54 has been shown to have the most enhanced reduction of NK cell activation. In some observations, CD58 and CD54 double knockout is more effective than HLA-G overexpression on HLA-I deficient cells in overcoming the "loss of self" effect.

[0224] As provided herein, in some embodiments, 41BB-ADR and optionally CD38 KO and TCR KO In yet some other embodiments, the iPSCs and derived cells thereof that contain one or both of the exogenous polynucleotide encoding 41BB-ADR and, optionally, CD38 are CD54 negative or CD58 negative. KO and TCR KO The iPSCs and derived cells thereof containing one or both of 41BB-ADR and CD38 are CD54-negative and CD58-negative, and have improved resistance to host immune alloreactivity compared to cells not containing an exogenous polynucleotide. KO In some embodiments of iPSCs and derived cells comprising exogenous CD16 or a variant thereof, the cells are CD54 negative and / or CD58 negative. KO , T.C.R. KO In yet some other embodiments of iPSCs and derived cells comprising exogenous CD16 or a variant thereof, an IL, and a CAR, the cells are CD58 negative and / or CD54 negative.

[0225] In some embodiments, surface proteins that are upregulated in activated recipient immune cells include, but are not limited to, CD38, CD25, CD69, CD44, 4-1BB, OX40, or CD40L. When cells express such inactivated CARs, it is preferred that the cells do not express or have a knockout of the same surface protein targeted by the CAR. In some embodiments, the inactivated CAR comprises at least one of CD38-CAR, CD25-CAR, CD69-CAR, CD44-CAR, 41BB-CAR, OX40-CAR, and CD40L-CAR.

[0226] 41BB-ADR, and optionally exogenous CD16, CD38 KO , endogenous TCR KO Further provided in the present application is a master cell bank that contains sorted single cells and expanded clonal engineered iPSCs having at least one phenotype provided herein, including but not limited to one or more of the following: , CAR, and is CD58 negative and / or CD54 negative, providing a platform for further iPSC manipulation 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 compositionally defined and uniform and can be cost-effectively mass-produced at significant scale.

[0227] 7. Genetically engineered iPSC lines and derived cells provided herein In view of the above, the present application provides iPSCs, iPS cell line cells, or populations thereof, and derived effector cells obtained from differentiating iPSCs, each cell comprising a polynucleotide encoding 41BB-ADR and optionally a CD38 KO , T.C.R. KO, endogenous CD16, CAR, cytokine signaling complexes, and are CD58-negative and / or CD54-negative, wherein the cells are eukaryotic cells, animal cells, human cells, induced pluripotent cells (iPSCs), iPSC-derived effector cells, immune cells, or feeder cells. Also provided are master cell banks comprising sorted single cells and expanded clonal engineered iPSCs with the phenotypes described herein, which provide a renewable source for the production of off-the-shelf engineered homogenous cell therapy products that are compositionally defined and uniform and can be mass-produced cost-effectively at substantial scales. In some embodiments, the iPSC-derived cells are mesodermal cells with embryonic hemogenic endothelial (HE) potential, embryonic HE, CD34 + Hematopoietic cells include, but are not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, myeloid cells, neutrophil precursors, and / or share characteristics with T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, functional derived hematopoietic cells include effector cells that have one or more functional characteristics not present in the corresponding primary T cells, NK cells, NKT cells, and / or B cells.

[0228] Since 4-1BB is upregulated in activated effector cells, including alloreactive NK cells and T cells, iPSC-derived effector cells containing 41BB-ADRs are useful for overcoming alloreactivity in immunocompetent host systems. In some embodiments, activated T cells with upregulated 4-1BB expression are targeted, as opposed to non-activated T cells. To target these activated T cells, any suitable ligand for 4-1BB, including 4-1BBL, an antibody (or functional fragment thereof) targeting 4-1BB, a fusion of Fc with 4-1BBL, or a functional derivative or fragment thereof may be utilized in the ADR.

[0229] In addition, the present invention provides iPSCs and derived effector cells comprising a polynucleotide encoding 41BB-ADR, comprising a TCR KO In some embodiments, such cells have reduced alloreactivity and can prevent GvHD (graft versus host disease) when used in allogeneic adoptive cell therapy.

[0230] Further, the present invention provides iPSCs and derived effector cells comprising a polynucleotide encoding 41BB-ADR, KO and optionally TCR KO and exogenous CD16. KO Effector cells comprising CD38 have increased persistence and / or survival in vivo in the presence of an anti-CD38 therapeutic agent, which may be an anti-CD38 antibody. In some embodiments, the effector cells comprise T cells. KO and TCR KO iPSC-derived T cells containing CD38 undergo reduced cell depletion and acquire ADCC in the presence of anti-CD38 antibodies, providing an additional mechanism for T cell-mediated tumor killing. In some embodiments, effector cells include NK cells. KO and exogenous CD16, iPSC-derived NK cells in the presence of anti-CD38 antibodies have enhanced cytotoxicity and reduced NK cell fratricide. In some embodiments, when anti-CD38 antibodies are used to induce CD16-mediated enhanced ADCC, iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing effector cell elimination, i.e., reduction or depletion of CD38-expressing effector cells, thereby increasing the persistence and / or survival of iPSCs and their effector cells and delaying reconstitution of host immunity.

[0231] In addition, a polynucleotide encoding 41BB-ADR and, optionally, CD38 knockout, TCR KOand one or more of a polynucleotide encoding at least one cytokine signaling complex (IL) that enables cytokine signaling that contributes to cell viability, persistence, and / or proliferation, wherein the iPSC line can be directed differentiated to produce functional derived hematopoietic cells with improved viability, persistence, expansion, and effector function. In various embodiments, the exogenously introduced cytokine signaling includes signaling for any one, two, or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced cytokine signaling complex 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 cell surface cytokine / cytokine receptor is via retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA including mRNA. In some embodiments, the exogenous cell surface cytokine signaling complex comprised within the iPSC or derived cells described herein allows for IL7 signaling. In some embodiments, the exogenous cell surface cytokine signaling complex comprised within the iPSC or derived cells described herein allows for IL10 signaling. In some embodiments, the exogenous cell surface cytokine signaling complex comprised within the iPSC or derived cells described herein allows for IL15 signaling. Thus, the iPSC and derived cells according to some embodiments 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, the 41BB-ADR and optionally CD38 KO , T.C.R. KOiPSCs and their derived effector cells, comprising any of the genetic modalities described herein, are HLA-I and / or HLA-II intact and have synergistically increased persistence and / or survival in the presence of activated recipient T cells, B cells, and NK cells.

[0232] Also included are exogenous polynucleotides encoding 41BB-ADR, and optionally CD38. KO , T.C.R. KO Also provided is an iPSC comprising the CD38-CAR / CD38 ... - / - HLA-I WT or HLA WT iPSCs and their derived effector cells can be used with anti-CD38 antibodies to induce ADCC without effector cell elimination or allorejection by activated recipient T, B, and NK cells, thereby increasing the persistence and / or survival of iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or survival in vivo.

[0233] Another aspect provided herein is a polynucleotide encoding 41BB-ADR, and optionally CD38. KO , T.C.R. KO, and any other genetic modalities described herein, including iPSCs or iPSC-derived cells comprising a truncated fusion protein of IL15 and IL15Rα, the fusion protein not comprising an intracellular domain. Also referred to as "IL15Rα(ΔICD) fusion" and "IL5 / mb-Sushi", these embodiments are further collectively abbreviated as "IL15Δ" in Table 2 and 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: 16, 17, or 18. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 16. 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:18. In yet some other embodiments, iPSCs or iPSC-derived cells comprising a truncated IL15 / IL15Rα fusion protein lacking the intracellular domain (IL15Δ) further comprise one or more of 41BB-ADR, TCR knockout, CD38 knockout, exogenous CD16, exogenous cytokine signaling complex, and CAR, and the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, including, but not limited to, mesoderm cells with embryonic hemogenic endothelial (HE) potential, embryonic HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent precursors (MPPs), T cell precursors, NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0234] Thus, the present application provides iPSCs and their functional derived effector cells comprising any one of the following genotypes in Table 2. Unless specified as IL15Δ, which is a truncated fusion protein of IL15 and IL15Rα but does not have the intracellular domain, the "IL" provided in Table 2 represents any one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which particular cytokine / receptor expression is selected. Furthermore, when the iPSC and its functional derived effector cells have a genotype that includes both CAR and IL, the CAR and IL can be included in a bicistronic expression cassette that includes the 2A sequence. In contrast, in some other embodiments, the CAR and IL are in separate expression cassettes included in the iPSC and its functional derived hematopoietic cells. In one particular embodiment, the iPSCs and their functional derived effector cells comprise both a CAR and an IL, where the IL is IL15, and the IL15 construct is included in an expression cassette together with the CAR or separately from the CAR.

[0235] [Table 19]

[0236] 8. Additional Modifications In some embodiments, the genetic modification modality comprises one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or survival of iPSCs or derived cells thereof. In some embodiments, the genetically modified iPSCs and derived cells thereof comprise a genotype listed in Table 2. In some embodiments, the iPSCs and derived effector cells thereof comprising any one of the genotypes in Table 2 comprise a disruption of at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or a disruption of at least one of CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D, D, E, F, F, G, H, I, I, M, M, M, N, N, N, O ... 2A The antibody may further comprise introduction of at least one of an R, an antigen-specific TCR, an Fc receptor, an engager, and a surface triggering receptor for coupling with a bispecific, multispecific, or universal engager.

[0237] Engagers are fusion proteins consisting of two or more single chain variable fragments (scFv) of different antibodies, where at least one scFv binds to an effector cell surface molecule or a surface triggering receptor, and at least another scFv binds to a target cell via a target cell specific surface molecule. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs), multispecific killer cell engagers, or universal engagers that can be compatible with multiple immune cell types. Thus, engagers can be bispecific or multispecific. Such bispecific or multispecific engagers can direct effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils) to tumor cells and activate immune effector cells, showing great potential to maximize the benefits of CAR-T cell therapy.

[0238] In some embodiments, engagers are used in combination with a population of effector cells described herein, by simultaneous or sequential administration, where the effector cells comprise a surface molecule or surface triggering receptor that is recognized by the engager. In some other embodiments, the engager is a bispecific antibody expressed by derived effector cells through genetic engineering of iPSCs as described herein and directed differentiation of the engineered iPSCs. Exemplary effector cell surface molecules or surface triggering receptors that can be used for bispecific or multispecific engager recognition or coupling include, but are not limited to, CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, and chimeric Fc receptors disclosed herein. As described herein, in some embodiments, the exogenous CD16 expressed on the surface of the derived effector cells for engager recognition is hnCD16 comprising CD16 (including F176V and optionally S197P) or CD64 extracellular domain, and native or non-native transmembrane, stimulatory and / or signaling domains, as described herein. In some embodiments, the exogenous CD16 expressed on the surface of the effector cells for engager recognition is a CD16-based chimeric Fc receptor (CFcR). In some embodiments, the CD16-based CFcR comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, and the extracellular domain of the exogenous CD16 is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and optionally S197P.

[0239] In some embodiments, the target cells for the engager are tumor cells. Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. In one embodiment, the bispecific engager is a bispecific antibody specific for CD3 and CD19 (CD3-CD19). In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In another embodiment, the bispecific antibody is CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody is CD3-CD33.

[0240] In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and the tumor cell antigen binding domain. For example, modified IL15 can be used as a linker for effector NK cells to promote effector cell proliferation (referred to in some publications as TriKE, or trispecific killer engager). 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 ("2C1533"). In addition to IL15, cytokines suitable for inclusion in TriKE include, but are not limited to, IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21.

[0241] In some embodiments, the surface triggering receptor for a bispecific or multispecific engager may be endogenous to the effector cell, sometimes depending on the cell type. In some other embodiments, one or more exogenous surface triggering receptors can be introduced into the effector cell using the methods and compositions provided herein, i.e., by further manipulating iPSCs containing the genotypes listed in Table 2, and then directing the differentiation of the iPSCs into T cells, NK cells, or any other effector cells containing the same genotype and surface triggering receptor as the source iPSCs.

[0242] 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 targeting antigens associated with a condition, disease, or indication can be used in combination therapy with these effector cells. In some embodiments, antibodies are used in combination with a population of effector cells described herein by simultaneous or sequential administration to a subject. In other embodiments, such antibodies or fragments thereof can be expressed by effector cells by genetically engineering iPSCs with an exogenous polynucleotide sequence encoding the antibody or fragment thereof and directing differentiation of the engineered iPSCs. In some embodiments, the effector cells express an exogenous CD16 variant and the cytotoxicity of the effector cells is enhanced by the antibody via ADCC.

[0243] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus specific antigen activates the administered iPSC-derived effector cells to enhance their killing capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to the 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-CD5 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, ibritumomab, ocreliz ...HER2 (trastuzumab, pertuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-HER2 (trast 2 (alemtuzumab), anti-EGFR (cetuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc-engineered variants or fragments thereof, or functional equivalents and biosimilars thereof. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells further include bispecific or multispecific antibodies that target two or more antigens or epitopes on the target cell, or that target the target cell while recruiting effector cells (e.g., T cells, NK cells, or macrophage cells) to the target cell. Such bispecific or multispecific antibodies show great potential to attract effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils) to tumor cells and function as engagers that can activate immune effector cells, maximizing the benefits of antibody therapy.

[0244] 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 two or more antigens or epitopes on target cells or recruit effector cells (e.g., T cells, NK cells, or macrophage cells) toward target cells while targeting the target cells. Such bispecific or multispecific antibodies show great potential to maximize the benefit of antibody therapy by directing effector cells, whether bystander immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils in the recipient of the therapy) to tumor cells and functioning as engagers that can activate immune effector cells upon tumor antigen binding. The engager is specific for at least one tumor antigen and specific for at least one surface triggering receptor of immune effector cells, which may provide a multi-targeting approach for the engineered cells disclosed herein to address tumor antigen escape and tumor heterogeneity. Examples of engagers include, but are not limited to, a bi-specific T cell engager (BiTE), a bi-specific killer cell engager (BiKE), a tri-specific killer cell engager (TriKE), or a multispecific killer cell engager, or a universal engager that is compatible with multiple immune cell types.

[0245] 10. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate immune responses if not inhibited. It has become evident that tumors exploit certain immune checkpoint pathways as a major mechanism of immune resistance, particularly to T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can reduce the expression of checkpoint genes or gene products or reduce the activity of checkpoint molecules, thereby blocking inhibitory checkpoints and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, with these agents resulting in long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. Thus, one aspect of the present application provides a therapeutic approach to overcome CI resistance by including genomically engineered functional iPSC-derived cells as provided herein in a combination therapy with CI. In one embodiment of the combination therapy described herein, the iPSC-derived cells are NK cells. In another embodiment of the combination therapy described herein, the iPSC-derived cells are T cells. In addition to exhibiting direct anti-tumor 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, the tumor infiltration of T cells promoted by functionally potent genomically engineered derived NK cells indicates that the NK cells can synergize with T cell-targeted immunotherapy, including checkpoint inhibitors, to alleviate local immune suppression and reduce tumor burden.

[0246] In some embodiments of the combination therapy, a checkpoint inhibitor is used in combination with a population of effector cells described herein by simultaneous or sequential administration thereof to a subject. In some other embodiments, the checkpoint inhibitor is expressed by the effector cells by genetically engineering iPSCs with an exogenous polynucleotide sequence encoding the checkpoint inhibitor or a fragment or variant thereof and directing differentiation of the engineered iPSCs. Some embodiments of the combination therapy with effector cells described herein include at least one checkpoint inhibitor for targeting at least one checkpoint molecule, and the effector cells have a genotype listed in Table 2.

[0247] In another embodiment, iPSC-derived effector cells for checkpoint inhibitor combination therapy comprise 41BB-ADR and, optionally, TCR KO In some embodiments, the derived T cells comprise one or more of the following genotypes: TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or a deletion, disruption, or reduced expression of at least one of CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, or B. 2A Further comprising induced or increased expression of at least one of a surface triggering receptor for coupling to an R, a CAR, a TCR, an Fc receptor, an engager, and a bispecific, multispecific, or universal engager.

[0248] In various embodiments, the derived effector cells contain a polynucleotide encoding a 41BB-ADR, and optionally a TCR. KOIn some embodiments, the iPSC or iPS clone cell line cells further comprise a deletion, disruption, or reduced expression of at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or a deletion, disruption, or reduced expression of at least one of CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, or a combination of ... 2A Further comprising the introduction, or introduced or increased expression, of at least one of an R, a CAR, a TCR, an Fc receptor, an engager, and a surface triggering receptor for coupling with a bispecific, multispecific, or universal engager.

[0249] Suitable checkpoint inhibitors for combination therapy with derived NK or T cells provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (CD223), CTLA-4 (CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A 2A These include, but are not limited to, antagonists of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0250] In some embodiments, the antagonist that inhibits any of the above-mentioned checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a single variable novel antigen receptor (VNAR), a shark heavy chain only antibody (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), minibodies, diabodies, triabodies, tetrabodies, single domain antigen binding fragments (sdAb, nanobodies), recombinant heavy chain only antibodies (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be more cost-effective to produce, easier to use, or more sensitive than whole antibodies. In some embodiments, the one or two or three or more checkpoint inhibitors comprise at least one of atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR antibody), IPH43 (anti-MICA antibody), IPH33 (anti-TLR3 antibody), lilimumab (anti-KIR antibody), monalizumab (anti-NKG2A antibody), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and any derivatives, functional equivalents, or biosimilars thereof.

[0251] In some embodiments, the antagonists that inhibit any of the above-mentioned checkpoint molecules are microRNA-based, as many miRNAs are found as regulators that control the expression of immune checkpoints (Dragomir et al., Cancer Biol Med. 2018, 15(2): 103-115). In some embodiments, checkpoint antagonistic miRNAs include, but are not limited to, miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197, miR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c.

[0252] Some embodiments of combination therapies with derived NK cells or T cells provided include at least one checkpoint inhibitor that targets at least one checkpoint molecule, and the derived cells have a genotype listed in Table 2. Some other embodiments of combination therapies with derived NK cells or T cells provided include two, three or more checkpoint inhibitors, such that two, three or more checkpoint molecules are targeted. In some embodiments of combination therapies including at least one checkpoint inhibitor and derived cells having a genotype listed in Table 2, 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 derived cells by expressing an exogenous polynucleotide sequence encoding the antibody, or a fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the checkpoint-inhibiting antibody, fragment or variant thereof is co-expressed with the CAR, either in a separate construct or in a bicistronic construct that includes both the CAR and the sequence encoding the antibody or fragment thereof. In some further embodiments, the antibody or fragment thereof coding sequence can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence, exemplified as, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences for the checkpoint inhibitor and the CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered and expressed and secreted as a payload by derived effector cells that can infiltrate the tumor microenvironment (TME), it counteracts inhibitory checkpoint molecules upon binding to the TME and activates modalities such as CAR or activates effector cells by activating receptors.In some embodiments, the checkpoint inhibitor co-expressed with the CAR is selected from the group consisting of checkpoint molecules: PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A. 2A Inhibits at least one of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-l, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some embodiments, the checkpoint inhibitor co-expressed with the CAR in the derivative cells having the genotypes listed in Table 2 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 other 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 other 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.

[0253] In some other embodiments of the combination therapy comprising iPSC-derived cells and at least one antibody that inhibits a checkpoint molecule provided herein, the antibody is not produced by or within the iPSC-derived cells and is further administered prior to, concurrently with, or after administration of derived cells having a genotype listed in Table 2. In some embodiments, the administration of one, two, three or more checkpoint inhibitors in the combination therapy with the provided derived effector 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 2, 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 2, the checkpoint inhibitor included in the treatment is atezolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising derived NK cells or T cells having a genotype listed in Table 2, the checkpoint inhibitor included in the treatment is nivolumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising derived NK cells or T cells having a genotype listed in Table 2, the checkpoint inhibitor included in the treatment is pembrolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0254] 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 target cell. Targeted genome editing (interchangeably "targeted genome editing" or "targeted gene editing") allows for insertion, deletion, or replacement at a preselected site in 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 knocked down by sequence deletion. Thus, targeted editing can also be used to precisely disrupt endogenous gene expression. The term "targeted integration," as also used herein, refers to a process that involves the insertion of one or more exogenous sequences, with or without the deletion of endogenous sequences at the insertion site. In contrast, randomly integrated genes are susceptible to position effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomeric regions are particularly prone to transgene silencing. The newly integrated genes may affect the surrounding endogenous genes and chromatin, altering cell behavior or promoting cell transformation. Therefore, inserting exogenous DNA into preselected loci such as safe harbor loci or genomic safe harbor (GSH) is important for safety, efficiency, copy number control, and reliable gene response regulation.

[0255] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach, in which homologous recombination is guided by homologous sequences flanking the exogenous polynucleotide to be inserted via the host cell's enzymatic machinery.

[0256] Alternatively, targeted editing can be achieved at higher frequencies by specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing exploits DNA repair mechanisms including non-homologous end joining (NHEJ) that occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in random insertion or deletion (indels) of a small number of endogenous nucleotides. In contrast, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homology arms, exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in "targeted integration". In some situations, the targeted integration site is intended to be within the coding region of a selected gene, and thus targeted integration can disrupt gene expression and result in simultaneous knock-in and knock-out (KI / KO) in one single editing step.

[0257] One or more transgenes can be inserted into selected positions in a gene locus of interest (GOI) to achieve simultaneous gene knockout. Suitable loci for simultaneous knock-in and knockout (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, 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 the site or under the exogenous promoter included in the construct. When two or more transgenes are inserted into a selected position of the CD38 locus, a linker sequence, e.g., a 2A linker or an IRES, is placed between any two transgenes. The 2A linker encodes, for example, a self-cleaving peptide (referred to as "F2A", "E2A", "P2A" and "T2A", respectively) derived from FMDV, ERAV, PTV-I or TaV, 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. In various embodiments, the exogenous promoter can be CAG or other constitutive, inducible, time-specific, tissue-specific or cell type-specific promoter, including but not limited to CMV, EF1α, PGK and UBC.

[0258] Available endonucleases that can introduce specific targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems. In addition, homing endonucleases and the DICE (dual integrase cassette exchange) system, which utilizes phiC31 and Bxb1 integrases, are also promising tools for targeted integration.

[0259] ZFNs are targeted nucleases that include a nuclease fused to a zinc finger DNA binding domain. "Zinc finger DNA binding domain" or "zinc finger DNA binding domain, ZFBD" refers to a polypeptide domain that binds to DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, the C2H2 zinc finger, the C3H zinc finger, and the C4 zinc finger. A "designed" zinc finger domain is a domain that does not occur in nature, whose design / construction results primarily from rational criteria, such as the application of substitution rules and computerized algorithms to process information from databases that store information on existing ZFP designs and binding data. See, e.g., U.S. Pat. Nos. 6,140,081, 6,453,242, and 6,534,261. See also WO 98 / 53058, WO 98 / 53059, WO 98 / 53060, WO 02 / 016536 and WO 03 / 016496. A "selected" zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trapping, or hybrid selection. ZFNs are described in detail in U.S. Pat. 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.

[0260] TALENs are targeted nucleases that contain a nuclease fused to a TAL effector DNA binding domain. "Transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" refers to the polypeptide domain of a TAL effector protein that is responsible for 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 the transactivation domain. The specificity of the TAL effector DNA binding domain depends on the effector variable number of imperfect 34 amino acid repeats that contain polymorphisms at selected repeat positions called repeat variable-diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application Publication No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of a FokI nuclease to a TAL effector DNA binding domain.

[0261] Another example of a targeted nuclease for use in the subject methods is a targeted Spo11 nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain having specificity for a DNA sequence of interest, e.g., a zinc finger DNA-binding domain, a TAL effector DNA-binding domain, etc.

[0262] Further examples of targeted nucleases suitable for embodiments of the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used individually or in combination.

[0263] 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.

[0264] Using Cas9 as an example, CRISPR / Cas9 requires two major components: (1) Cas9 endonuclease and (2) crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to a target DNA sequence containing a PAM and a PAM-proximal seeding region. 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 then be delivered into mammalian cells via transfection or transduction.

[0265] DICE-mediated insertion uses a pair of recombinases, e.g., phiC31 and Bxb1, to provide unidirectional integration of exogenous DNA that is tightly restricted by each enzyme's own small attB and attP recognition sites. These targeted att sites do not naturally occur in mammalian genomes and must first be introduced into the genome at the desired integration site. See, e.g., U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0266] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct into a cell to allow site-specific homologous recombination by a cellular host enzyme machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to allow ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to allow TALEN-mediated insertion. In another embodiment, a method of targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a gRNA comprising a Cas9 expression cassette and a guide sequence specific for a desired integration site into the cell to allow insertion via Cas9. In yet another embodiment, a method of targeted integration in a cell includes introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to allow targeted integration via DICE.

[0267] Potential sites for targeted integration include, but are not limited to, safe harbor loci, or genomic safe harbors (GSH), which are intragenic or extragenic regions of the human genome that, in theory, can accommodate predictable expression of the newly integrated DNA without adverse effects on the host cell or organism. A useful safe harbor should allow sufficient transgene expression to produce the desired levels of vector-encoded protein or non-coding RNA. A safe harbor should also not predispose cells to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it should ideally meet criteria including, but not limited to: absence of disruption of regulatory elements or genes as judged by sequence annotation, being an intergenic region within a gene-dense region or a convergent position between two genes transcribed in opposite directions, maintaining a distance that minimizes the possibility of long-range interactions between vector-encoded transcriptional activators and the promoters of adjacent genes, particularly cancer-associated and microRNA genes, and having apparently ubiquitous transcriptional activity as reflected by widespread spatial and temporal expressed sequence tag (EST) expression patterns that indicate ubiquitous transcriptional activity. This latter attribute 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 favorable for exogenous insertion, the precise locus selected for insertion should be devoid of repetitive elements and conserved sequences, allowing easy design of primers for the amplification of homology arms.

[0268] 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 orthologue of the mouse ROSA26 locus. In addition, the human orthologue of the mouse H11 locus may also be a suitable site for insertion using the compositions and methods of targeted integration disclosed herein. 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, especially in stem cells, for certain integration events, and optimization of the insertion strategy, such as promoter selection, exogenous gene sequence and positioning, and construct design, is often required.

[0269] For targeted indels, the editing site is often contained in the endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted indel is associated with immune response regulation and modulation. In some other embodiments, the endogenous gene containing the targeted indel is associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response regulation and modification, or proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival rate of stem and / or progenitor cells and cells derived therefrom.

[0270] Thus, another aspect of the invention provides a method of 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 method of targeted integration 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 of a genomic safe harbor. In one embodiment, a method of targeted integration into a cell comprising introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing a construct comprising a pair of homologous arms and one or more exogenous sequences specific to a desired integration site to enable site-specific homologous recombination by the cellular host enzymatic machinery, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1, or other locus that meets the criteria for genomic safe harbor. Further integration sites include endogenous loci intended for disruption, e.g., reduction or knockout, including B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0271] In another embodiment, a method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site into the cell to allow for ZFN-mediated insertion, the desired integration site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method of targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site into the cell to allow for TALEN-mediated insertion, the desired integration site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.In another embodiment, a method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a Cas9 expression cassette specific for a desired integration site and a gRNA comprising a guide sequence into the cell to allow for Cas9 mediated insertion, the desired integration site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinase 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, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0272] Further, as provided herein, the above-mentioned methods for targeted integration with safe harbors are used to insert polynucleotides of interest, such as polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, and proteins that promote stem cell and / or progenitor cell engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival. In some other embodiments, the constructs comprising one or more exogenous polynucleotides further comprise one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the 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 can eliminate them upon administration of the mAb rituximab. Furthermore, modified EGFR containing an epitope recognized by cetuximab can be used to deplete genetically engineered cells when the cells are exposed to cetuximab. Thus, one aspect of the present invention provides a method for targeted integration of one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0273] In some embodiments, one or more exogenous polynucleotides integrated by the methods described 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 disclosed herein include, but are not limited to, cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0274] The exogenous polynucleotides integrated by the methods described herein may be driven by endogenous promoters in the host genome at the integration site. In one embodiment, the methods described herein 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 described herein 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 described herein 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 described herein 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 method described herein is used for targeted integration at the HTRP locus in the genome of cells.In one embodiment, at least one integrated polynucleotide is driven by endogenous HTRP promoter.Theoretically, only correct insertion at desired position will allow gene expression of exogenous gene driven by endogenous promoter.

[0275] In some embodiments, one or more exogenous polynucleotides included in the construct for the method of targeted integration are driven by one promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to increase the physical separation between the moieties and maximize access to the enzymatic machinery. The linker peptide of the linker sequence may be composed of amino acids selected to make the physical separation between the moieties (exogenous polynucleotides and / or proteins or peptides encoded therefrom) more flexible or more rigid, depending on the function involved. The linker sequence may be protease cleavable or chemically cleavable to generate separate moieties. Examples of enzymatic cleavage sites in the linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is one that is naturally produced by the host or is exogenously introduced. Alternatively, the cleavage site in the linker may be one that can be cleaved upon exposure to a selected chemical, for example, 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 in order for the moiety to function properly. The linker may also be a simple amino acid sequence of sufficient length to prevent any steric hindrance between the moieties. In addition, the linker sequence may provide for post-translational modifications, including, but not limited to, phosphorylation sites, biotinylation sites, sulfation sites, gamma-carboxylation sites, and the like. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single undesired conformation. The linker may be comprised primarily of amino acids with small side chains, such as glycine, alanine, and serine, to provide flexibility. In some embodiments, about 80-90 percent or more of the linker sequence includes glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, a G4S linker peptide separates the terminal processing and endonuclease domains of the fusion protein. In other embodiments, a 2A linker sequence allows two separate proteins to be produced from a single translation. Suitable linker sequences can be easily identified empirically. In addition, suitable sizes and sequences of linker sequences can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry site (IRES). In some embodiments, any two consecutive linker sequences are different.

[0276] The method of introducing a construct containing an exogenous polynucleotide for targeted integration into a cell can be achieved using methods of gene transfer into cells known per se. In one embodiment, the construct comprises the backbone of a viral vector, such as an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a Sendai viral vector. In some embodiments, a plasmid vector is used to deliver and / or express the exogenous polynucleotide into the target cell (e.g., pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.). In some other embodiments, an episomal vector is used to deliver the exogenous polynucleotide into the target cell. In some embodiments, recombinant adeno-associated viruses (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV do not integrate into the host genome. In addition, episomal rAAV vectors mediate a much higher rate of homologous gene targeting compared to transfection of conventional targeting plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the genome of iPSCs. In some embodiments, the genomically modified iPSCs and their derived cells obtained using the methods and compositions described herein comprise at least one genotype listed in Table 2.

[0277] III. Methods for Obtaining and Maintaining Genome-Engineered iPSCs In one aspect, the invention also provides a method of obtaining and maintaining genomically engineered iPSCs that contain one or more targeted edits (e.g., multiplexed genome engineering) at one or more desired sites, where the one or more targeted edits remain intact and functional at each selected edit site in the expanded genomically engineered iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions (i.e., targeted integrations and / or indels at selected sites) into the genome of the iPSCs and derived cells. The many advantages of obtaining genomically engineered iPSC-derived effector cells through editing and differentiation of iPSCs provided herein compared to direct manipulation of patient-derived peripheral blood-derived primary effector cells include, but are not limited to, an unlimited source of engineered effector cells, no need for repeated manipulation of effector cells, especially when multiple engineered modalities are involved, the resulting effector cells have extended telomeres and are younger due to less attrition, and the effector cell population is homogenous with respect to editing sites, copy number, and lack of allelic variation, random mutations, and expression variegation, primarily due to the ability to perform clonal selection in the engineered iPSCs provided herein.

[0278] In some embodiments, genomically engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells for extended periods in cell maintenance culture medium (FMM), where the iPSCs retain the targeted edits and functional modifications at the selected sites. iPSCs cultured in FMM have been shown to continue to maintain their undifferentiated, basal or naive profile; provide genomic stability without the need for culture washing or selection; and readily give rise to all three somatic lineages, in vitro differentiation via embryoid bodies or monolayers (without formation of embryoid bodies), and in vivo differentiation by teratoma formation. See, e.g., WO 2015 / 134652, the disclosure of which is incorporated herein by reference.

[0279] In some embodiments, genomically engineered iPSCs containing one or more targeted integrations and / or indels are maintained, passaged, and expanded in medium containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM), and free or essentially free of a TGFβ receptor / ALK5 inhibitor, and the iPSCs retain intact and functional targeted edits at the selected sites.

[0280] Another aspect of the invention provides a method of generating genomically engineered iPSCs through targeted editing of iPSCs or by first generating genomically engineered non-pluripotent cells by targeted editing and then reprogramming the selected / isolated genomically engineered non-pluripotent cells to obtain iPSCs containing the same targeted edit as the non-pluripotent cells. A further aspect of the invention provides genomically engineered non-pluripotent cells undergoing simultaneous reprogramming by introducing targeted integrations and / or targeted indels into the cells, the contacted non-pluripotent cells being under conditions sufficient for reprogramming, the reprogramming conditions comprising contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genomic engineering and reprogramming, the targeted integrations and / or targeted indels can be introduced into the non-pluripotent cells prior to initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally one or more small molecules, or essentially simultaneously.

[0281] In some embodiments, for simultaneous genomic engineering and reprogramming of non-pluripotent cells, targeted integrations and / or indels may also be introduced into non-pluripotent cells after the multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and vectors carrying the constructs are introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes, including, but not limited to, SSEA4, Tra181 and CD30.

[0282] In some embodiments, reprogramming is initiated by contacting the non-pluripotent cells with at least one reprogramming factor, and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. In some embodiments, iPSCs genomically engineered through 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.

[0283] In some embodiments of the method of producing genomically engineered iPSCs, the method comprises genomically engineering iPSCs by introducing one or more targeted integrations and / or indels into the iPSCs to obtain genomically engineered iPSCs having a genotype as provided herein. Alternatively, the method of generating genomically engineered iPSCs comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genomically engineered non-pluripotent cell comprising a targeted integration and / or indel at a selected site, and (b) contacting the genomically engineered non-pluripotent cell with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, to obtain a genomically engineered iPSC comprising a targeted integration and / or indel at a selected site. Alternatively, a method of generating genomically engineered iPSCs includes: (a) contacting a non-pluripotent cell with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cell; (b) introducing one or more targeted integrations and / or indels into the reprogrammed non-pluripotent cell for genome engineering; and (c) obtaining a clonal genomically engineered iPSC that contains the targeted integrations and / or indels at the selected site. Any of the above methods may further include single cell sorting of the genomically engineered iPSCs to obtain clonal iPSCs, and / or screening for off-target editing and abnormal karyotypes in the genomically engineered iPSCs. Through clonal expansion of the genomically engineered iPSCs, a master cell bank is generated to include single cell sorted and expanded clonally engineered iPSCs having at least one phenotype as provided herein. The master cell bank is subsequently cryopreserved, providing a platform for further iPSC manipulation and a renewable source for the production of off-the-shelf engineered homogenous cell therapy products that are compositionally defined and uniform and can be cost-effectively mass-produced at substantial scale.

[0284] The reprogramming factor is selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in WO 2015 / 134652 and WO 2017 / 066634, the disclosures of which are incorporated herein by reference. The one or more reprogramming factors may be in the form of a polypeptide. The reprogramming factor may also be in the form of a polynucleotide encoding the reprogramming factor, and thus may be introduced into the non-pluripotent cell by vectors such as retrovirus, Sendai virus, adenovirus, episomes, plasmids, and minicircles. In certain embodiments, the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, the one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by plasmid combination. For example, see International Publication No. WO2019 / 075057(A1), the disclosure of which is incorporated herein by reference.

[0285] In some embodiments, non-pluripotent cells are transfected with multiple constructs comprising different exogenous polynucleotides and / or different promoters by multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNAs, including, but not limited to, siRNAs, shRNAs, miRNAs, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue-specific or cell type-specific promoters. Thus, the polynucleotides are expressible under conditions that activate the promoter, for example, in the presence of an inducer, or in a specific differentiated cell type. In some embodiments, the polynucleotides are expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, the one or more suicide genes are driven by a constitutive promoter, e.g., capase-9 driven by CAG. These constructs, comprising different exogenous polynucleotides and / or different promoters, can be transferred simultaneously or sequentially into non-pluripotent cells. Non-pluripotent cells subjected to targeted integration of multiple constructs can be contacted with one or more reprogramming factors simultaneously to initiate reprogramming simultaneously with genome manipulation, thereby obtaining genome-engineered iPSCs containing multiple targeted integrations in the same pool of cells. Thus, this robust method allows for the derivation of clonal genome-engineered iPSCs with multiple modalities integrated into one or more selected target sites by simultaneous reprogramming and manipulation strategies.

[0286] IV. Methods for obtaining genetically engineered effector cells by differentiating genomically engineered iPSCsFurther aspects of the invention provide methods of in vivo differentiation of genomically engineered iPSCs by teratoma formation, where differentiated cells derived in vivo from genomically engineered iPSCs retain intact and functional targeted edits, including targeted integrations and / or indels, at desired sites. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma formation contain one or more inducible suicide genes integrated at one or more desired sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, CD38, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of the genomic safe harbor. In some other embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma formation contain polynucleotides encoding targeting modalities or polynucleotides encoding proteins that promote trafficking, homing, viability, self-renewal, persistence, and / or survival of stem and / or progenitor cells. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma formation containing one or more inducible suicide genes further comprise one or more indels in endogenous genes associated with regulating and mediating immune responses. In some embodiments, the indels are comprised in one or more endogenous checkpoint genes. In some embodiments, the indels are comprised in one or more endogenous T cell receptor genes. In some embodiments, the indels are comprised in one or more endogenous MHC class I suppressor genes. In some embodiments, the indels are comprised in one or more endogenous genes associated with the major histocompatibility complex.In some embodiments, the indels are in one or more endogenous genes, including, but not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0287] In some embodiments, genomically engineered iPSCs containing one or more genetic modifications provided herein are used to derive hematopoietic cell lineages or any other specific cell type in vitro, and the induced non-pluripotent cells retain the functional genetic modification including the targeted edit at the selected site. In some embodiments, the genomically engineered iPSCs used to derive hematopoietic cell lineages or any other specific cell type in vitro are master cell bank cells that are cryopreserved and thawed immediately prior to their use. In one embodiment, the genomically engineered iPSC-derived cells are mesodermal cells with embryonic hemogenic endothelial (HE) potential, embryonic HE, CD34 + Cells derived from genomically engineered iPSCs, including but not limited to hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, retain the functional genetic modification including the targeted edit at the desired site.

[0288] Applicable differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lineages include, for example, those set forth in WO 2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, methods and compositions for generating hematopoietic cell lineages through definitive hemogenic endothelium (HE) derived from pluripotent stem cells, including iPSCs, in a culture platform that is scalable and does not require monolayer EB formation under serum-free, feeder-free, and / or stroma-free conditions. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated and transdifferentiated cells, and to cells of various lineages that have transitioned directly to a hematopoietic fate without passing through a pluripotent intermediate. Similarly, cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0289] A method for differentiating and expanding cells of hematopoietic lineage from pluripotent stem cells in monolayer culture includes contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then subjected to contact with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with definitive hemogenic endothelial (HE) potential without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF, and optionally with a ROCK inhibitor, and / or a WNT pathway activator, differentiates the mesodermal cells with definitive HE potential into definitive HE cells that are also expanded during differentiation.

[0290] The methods for obtaining cells of hematopoietic lineage provided herein are superior to EB-mediated pluripotent stem cell differentiation because EB formation results in moderate to minimal cell proliferation, does not allow for monolayer culture, which is important for many applications requiring uniform proliferation, and uniform differentiation of cells within a population, making it laborious and less efficient.

[0291] The provided monolayer differentiation platform promotes differentiation into definitive hemogenic endothelium resulting in the induction of hematopoietic stem cells and differentiated progeny such as T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion, enabling the delivery of therapeutically relevant numbers of pluripotent stem cell-derived hematopoietic cells for a variety of therapeutic applications. Furthermore, monolayer culture using the methods provided herein results in functional hematopoietic lineage cells that enable a full range of in vitro differentiation, in vivo modification, and in vivo long-term hematopoietic self-renewal, reconstitution, and engraftment. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, embryonic somatic hemogenic endothelium, hematopoietic pluripotent progenitor cells, hematopoietic stem cells and progenitors, T cell precursors, NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0292] A method for directing differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator, and optionally bFGF, to initiate differentiation and proliferation of mesodermal cells from the pluripotent stem cells; and (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to initiate differentiation and proliferation of mesodermal cells having definitive HE potential from the mesodermal cells, the composition optionally comprising a TGFβ receptor / ALK inhibitor. and (iii) contacting mesodermal cells having definitive HE potential with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator, to initiate differentiation and proliferation of definitive hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having definitive hemogenic endothelial potential, wherein the composition optionally does not comprise a TGFβ receptor / ALK inhibitor.

[0293] In some embodiments, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and no TGFβ receptor / ALK inhibitor, to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs comprising one or more genetic imprints, and the one or more genetic imprints comprised in the iPSC are retained in hematopoietic cells differentiated therefrom. In some embodiments of the method for directing differentiation of pluripotent stem cells into cells of a hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of a hematopoietic lineage is in a monolayer culture form without the generation of embryoid bodies.

[0294] In some embodiments of the above-described methods, the obtained pluripotent stem cell-derived definitive hemogenic endothelial cells are CD34 + In some embodiments, the obtained definitive hemogenic endothelial cells are CD34 + CD43 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CD43 - CXCR4 - CD73 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CXCR4 - CD73 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CD43 - CD93 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CD93 - It is.

[0295] In some embodiments of the above-described method, the method further comprises: (i) contacting the pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, and optionally a BMP activator, to initiate differentiation of the definitive hemogenic endothelium into a pre-T cell precursor, and optionally (ii) contacting the pre-T cell precursor with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to initiate differentiation of the pre-T cell precursor into a T cell precursor or a T cell. In some embodiments of the method, the pluripotent stem cell-derived T cell precursor is CD34 + CD45 + CD7 + In some embodiments of this method, the pluripotent stem cell-derived T cell precursors are CD45 + CD7 + It is.

[0296] In some further embodiments of the above methods for directing differentiation of pluripotent stem cells into cells of a hematopoietic lineage, the method further comprises: (i) contacting the definitive hemogenic endothelium derived from the pluripotent stem cells with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator, to initiate differentiation of the definitive hemogenic endothelium into pre-NK cell precursors, and optionally (ii) contacting the pre-NK cell precursors derived from the pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, to initiate differentiation of the pre-NK cell precursors into NK cell precursors or NK cells, wherein the medium does not comprise one or more of VEGF, bFGF, TPO, BMP activators, and a ROCK inhibitor. In some embodiments, the pluripotent stem cell-derived NK precursors are CD3 - CD45 + CD56+ CD7 + In some embodiments, the pluripotent stem cell-derived NK cells are CD3 - CD45 + CD56 + and optionally, NKp46 + , CD57 + and CD16 + The present invention is further defined by:

[0297] In some embodiments, the genomically engineered iPSC-derived cells resulting from the above methods comprise one or more inducible suicide genes integrated at one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or other loci that meet the criteria for the genomic safe harbor. In some other embodiments, the genomically engineered iPSC-derived cells comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, potential drug targets, or proteins that promote stem and / or progenitor cell trafficking, homing, viability, self-renewal, persistence, and / or survival. In some embodiments, the genomically engineered iPSC-derived cells comprising one or more suicide genes further comprise one or more indels in one or more endogenous genes associated with immune response regulation and mediation, including, but not limited to, checkpoint genes, endogenous T cell receptor genes, and MHC class I suppressor genes. In one embodiment, the genomically engineered iPSC-derived cells comprising one or more suicide genes further comprise an indel in the B2M gene, where B2M is knocked out.

[0298] Additionally, applicable dedifferentiation methods and compositions for obtaining second fate genomically engineered hematopoietic cells from first fate genomically engineered hematopoietic cells include, for example, those set forth in WO 2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided therein allow for partial reprogramming of starting non-pluripotent cells into non-pluripotent intermediate cells by limiting expression of the endogenous Nanog gene during reprogramming, and subjecting the non-pluripotent intermediate cells to conditions for differentiation of the intermediate cells into the desired cell type.

[0299] V. Therapeutic Uses of Derived Immune Cells with Functional Modalities Differentiated from Genetically Engineered iPSCs The invention provides, in some embodiments, a composition comprising an isolated population or subpopulation of functionally enhanced derived immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSCs of the composition comprise one or more targeted gene edits disclosed herein that can be retained in the iPSC-derived effector cells, and the genetically engineered iPSCs and their derived cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells of the composition comprises iPSC-derived CD34 + In one embodiment, the isolated population or subpopulation of engineered effector cells of the composition comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of engineered effector cells of the composition comprises iPSC-derived pro-T cells or T cells. In one embodiment, the isolated population or subpopulation of engineered effector cells of the composition comprises iPSC-derived pro-NK cells or NK cells. In one embodiment, the isolated population or subpopulation of engineered effector cells of the composition comprises iPSC-derived immune modulatory cells or myeloid derived suppressor cells (MDSC).

[0300] In some embodiments of the composition, the iPSC-derived engineered effector cells are further conditioned ex vivo for improved therapeutic potential. In one embodiment of the composition, the isolated population or subpopulation of iPSC-derived engineered effector cells comprises an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment of the composition, the isolated population or subpopulation of iPSC-derived engineered effector cells comprises an increased number or proportion of type I NKT cells. In another embodiment of the composition, the isolated population or subpopulation of iPSC-derived engineered effector cells comprises an increased number or proportion of adaptive NK cells. In some embodiments of the composition, the iPSC-derived engineered CD34 T cells are further conditioned ex vivo for improved therapeutic potential. In one embodiment of the composition, the isolated population or subpopulation of iPSC-derived engineered effector cells comprises an increased number or proportion of type I NKT cells. In another embodiment of the composition, the isolated population or subpopulation of iPSC-derived engineered effector cells comprises an increased number or proportion of adaptive NK cells. + The isolated population or subpopulation of cells, HSC cells, T cells, NK cells, or myeloid-derived suppressor cells is allogeneic. In some other embodiments of the composition, the genetically engineered CD34 cells derived from iPSCs are + The isolated population or subpopulation of cells, HSC cells, T cells, NK cells, or MDSCs is autologous.

[0301] In some embodiments of the composition, the iPSCs for differentiation contain genetic imprints selected to convey desired therapeutic properties in derived effector cells, provided that cellular developmental biology is not disrupted during differentiation and that the genetic imprints are retained and functional in the iPSC-derived differentiated hematopoietic cells.

[0302] In some embodiments of the composition, the genetic imprint of the pluripotent stem cells comprises (i) one or more genetic modification modalities obtained through genomic insertion, deletion or substitution in the genome of the pluripotent cells during or after reprogramming of the non-pluripotent cells to iPSCs, or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor, disease, or therapeutic response specific, where the pluripotent cells are reprogrammed from source-specific immune cells and the iPSCs retain the source therapeutic property, which is also contained in the iPSC-derived hematopoietic lineage cells.

[0303] In some embodiments of the composition, the genetic modification modality comprises one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or survival rate of iPSCs or derived cells thereof. In some embodiments of the composition, the genetically modified iPSCs and derived cells thereof comprise a genotype listed in Table 2. In some other embodiments of the compositions, the genetically modified iPSCs and derived cells thereof comprising the genotypes listed in Table 2 further comprise: (1) disruption of one or more of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or TCR beta constant region (TRAC or TRBC), NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD54, CD56, CD58, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT; and / or (2) disruption of one or more of HLA-G, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D, E, F, G, H, I, I, M, I, N, O, P, P, R, P, S, T ... 2A The present invention further includes additional genetically modified modalities including introduction of one or more of an R, CAR, TCR, Fc receptor, or surface triggering receptor.

[0304] In yet some other embodiments of the composition, the iPSC-derived hematopoietic lineage cells have therapeutic properties including one or more of: (i) increased cytotoxicity, (ii) improved persistence and / or survival, (iii) enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site, (iv) improved tumor penetration, (v) enhanced ability to reduce tumor immune suppression, (vi) improved ability to rescue tumor antigen escape, (vii) controlled apoptosis, (viii) enhanced or acquired ADCC, and (ix) ability to avoid fratricide, compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue without the same gene edit. In some embodiments of the composition, the iPSC-derived hematopoietic lineage cells further comprise a therapeutic attribute of promoting homing or trafficking and retention of effector cells at the tumor site.

[0305] In some embodiments of the composition, the iPSC-derived hematopoietic cells comprising the genotypes listed in Table 2 express at least one cytokine signaling complex comprising all or a portion of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, or any modified protein thereof, and express at least a CAR. In some embodiments of the composition, the engineered expression of the cytokine and CAR is NK cell specific. In some other embodiments of the composition, the engineered expression of the cytokine and CAR is T cell specific. In some embodiments of the composition, the iPSC-derived hematopoietic effector cells are antigen specific. In some embodiments of the composition, the antigen-specific derived effector cells target liquid tumors. In some embodiments of the composition, the antigen-specific derived effector cells target solid tumors. In some embodiments of the composition, the antigen-specific iPSC-derived hematopoietic effector cells can rescue tumor antigen escape.

[0306] Additionally, the present application provides rationally designed effector cells that can synergize with tumor sensitization protocols that upregulate one or more tumor surface molecules, such as 4-1BB and / or CD38, to enhance effector cell tumor site homing, trafficking, and retention, and / or CD38 conditioning (i.e., CD38 upregulation in the presence of anti-CD38 antibodies). - This allows for combination therapeutic approaches by enhancing the use of effector cells, contributing to increased effector cell cytotoxicity and persistence.

[0307] Tumor sensitization can be utilized to overcome tumor resistance by fine-tuning underlying tumorigenic mechanisms (including, but not limited to, cell cycle progression, inflammation, proliferation, apoptosis, invasion, perfusion, metastasis, and angiogenesis) to make tumor cells more susceptible to the activity of another selective drug, such as an allogeneic effector cell with the desired engineered therapeutic attributes described herein, thereby enhancing the efficacy of the therapeutic effector cell targeting the tumor. Without being bound by theory, exemplary sensitizing agents useful in the compositions and methods disclosed herein can be radiotherapy, radiopharmaceuticals, or chemotherapeutic agents. Thus, the compositions discussed herein can further include a sensitizing agent as described above. In various embodiments, the sensitizing agent increases tumor cell surface expression of 4-1BB and / or CD38.

[0308] A variety of diseases can be ameliorated by introducing the derived effector cells and / or compositions disclosed herein into a subject suitable for adoptive cell therapy. In some embodiments, the iPSC-derived hematopoietic cells or compositions provided herein are for allogeneic adoptive cell therapy. Additionally, the present invention provides, in some embodiments, therapeutic uses of the immune cells and / or therapeutic compositions and / or combination therapies described above by introducing the cells or compositions into a subject suitable for adoptive cell therapy, the subject having an autoimmune disorder, hematological malignancy, solid tumor, or infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphomas, non-Hodgkin's lymphomas, and the like. Examples of solid cancers include, but are not limited to, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, breast and lung cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric / abdominal cancer, head and neck cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, metastatic cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, salivary gland cancer, skin cancer, testicular tumors, thyroid tumors, urothelial cancer, and uterine / endometrial cancer. Examples of various autoimmune disorders include alopecia areata, autoimmune hemolytic These include, but are not limited to, anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigoid / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease).Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpes virus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella zoster virus), adenovirus-, lentivirus-, and BK polyomavirus-associated diseases.

[0309] Treatment using the derived hematopoietic lineage cells of the embodiments disclosed herein or compositions provided herein can be performed upon presentation of symptoms or to prevent relapse. The terms "treating", "treatment" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of completely or partially preventing the disease, and / or therapeutic, in terms of partially or completely curing the disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses any intervention of a disease in a subject, including: preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having it, and inhibiting the disease, i.e., arresting its development, or relieving the disease, i.e., reversing the disease. Therapeutic agents and / or compositions can be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, in which the treatment stabilizes or reduces the patient's undesirable clinical symptoms, is also of particular importance. In certain embodiments, a subject in need of treatment has a disease, condition, and / or injury for which at least one associated symptom can be inhibited, ameliorated, and / or improved by cell therapy. Certain embodiments contemplate that subjects in need of cell therapy include, but are not limited to, bone marrow or stem cell transplant candidates, subjects undergoing chemotherapy or radiation therapy, subjects having or at risk of having a hyperproliferative disorder or cancer, e.g., a hyperproliferative disorder or cancer of the hematopoietic system, subjects having or at risk of developing a tumor, e.g., a solid tumor, subjects having or at risk of having a viral infection or a disease associated with a viral infection.

[0310] When assessing responsiveness to a treatment comprising the derived hematopoietic lineage cells of the embodiments disclosed herein,...

Claims

1. A cell or a group thereof, (i) The cells are (a) induced pluripotent cells (iPSCs), or (b) derived cells obtained by differentiating the iPSCs. (ii) The iPSC and derived cells are (a) Exogenous polynucleotides encoding alloprotective receptors (ADRs), and optionally, (b) CD38 knockout and / or endogenous TCR knockout, including, A cell or a group thereof.

2. The cells or population thereof according to claim 1, wherein the cells exhibit improved resistance to host immune alloreactivity compared to cells that do not contain the exogenous polynucleotide.

3. (i) The iPSC is a cloned iPSC, a single-cell dissociated iPSC, an iPSC cell line, or an iPSC master cell bank (MCB) cell, or (ii) The derived cell is a derived CD34 ＋ The cell or population thereof according to claim 1, comprising cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, or derived B lineage cells, or (iii) derived effector cells having one or more functional characteristics not present in the corresponding primary T, NK, NKT, and / or B cells.

4. The cell or population thereof according to claim 1, wherein the ADR is specific to 4-1BB or CD38.

5. The aforementioned ADR, (i) A 41BB-specific ligand operably linked to a signaling domain that promotes effector cell activation, or (ii) A CD38-binding domain operably linked to a signaling domain that promotes effector cell activation, A cell or population thereof according to claim 3, including the cell or population thereof.

6. The cell or population thereof according to claim 5, wherein the 41BB-specific ligand is an antibody or fragment thereof that targets 4-1BBL, 4-1BB, or a 4-1BBL-Fc fusion.

7. The cell or population thereof according to claim 5, wherein the signal transduction domain comprises CD3ζ derived from DAP12 or a functional fragment thereof, an Fc receptor, or a combination thereof.

8. The cell or population thereof according to any one of claims 1 to 7, wherein the ADR further comprises one, two, three, or more costimulatory domains.

9. The cell or population thereof according to claim 1, wherein the ADR contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:

8.

10. The aforementioned cells, (i) Chimeric antigen receptor (CAR), (ii) Exogenous CD16 or its variants, (iii) A cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine expressed on the cell surface and / or its receptor, (iv) At least one of the genotypes listed in Table 2, (v) The fewest of the following failures: TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (vi) CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ Introduction of at least one of R, antigen-specific TCR, Fc receptor, antibody or functional variant or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for coupling with an agonist. The cell or population thereof according to claim 1, further comprising one or more of the above.

11. The aforementioned cells are NK cell lineage cells or T cell lineage cells, (i) The NK lineage cells or the T lineage cells have improved infiltration and / or retention in the tumor site, (ii) The NK cell lineage can mobilize and / or migrate T cells to the tumor site, (iii) The NK lineage cells or the T lineage cells can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. The cells or population thereof as described in claim 1.

12. The aforementioned cells, (i) Exogenous polynucleotides encoding the allogeneic immune defense receptor (ADR), (ii) CD38 knockout, (iii) Exogenous polynucleotides encoding CD16 or its variants, and (iv) TCR knockout, The cells or population thereof according to claim 1, wherein the cells or population thereof have improved resistance to host immune alloreactivity compared to cells that do not have all of (i) to (iv).

13. The aforementioned cells, (i) Exogenous polynucleotides encoding the allogeneic immune defense receptor (ADR), (ii) CD38 knockout, (iii) Exogenous polynucleotides encoding CD16 or its variants, (iv) Exogenous polynucleotides encoding partial or complete peptides of IL-15 and partial or complete peptides of the IL-15 receptor, (v) CAR The cells or population thereof according to claim 1, wherein the cells or population thereof have improved resistance to host immune alloreactivity compared to cells that do not have all of (i) to (v).

14. A method for improving the resistance of effector cells to host immune alloreactivity, (i) Obtaining an engineered iPSC containing an exogenous polynucleotide encoding an allogeneic immune protective receptor (ADR), and optionally one or both of the following: (ii) Differentiating the iPSCs into effector cells, thereby producing effector cells in which resistance to host immune alloreactivity is improved compared to the corresponding cells that do not contain the exogenous polynucleotides, Methods that include...

15. A composition comprising cells or a population thereof as described in any one of claims 1 to 13.

16. The composition according to claim 15, further comprising one or more therapeutic agents.

17. The composition according to claim 15, further comprising a sensitizer.

18. Use of the composition according to any one of claims 15 to 17 in the manufacture of a pharmaceutical for the treatment of autoimmune disorders, hematological malignancies, solid tumors, cancer, or viral infections.

19. A master cell bank (MCB) comprising cloned iPSCs according to any one of claims 1 to 13.

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