Enhancement of the durability and effectiveness of effector cells in adoptive cell therapy

Abstract

Methods and compositions are provided for obtaining functionally enhanced derivative effector cells derived from the directed differentiation of genomically engineered iPSCs. Embodiments of the derivative cells provided herein have stable functional genomic editing that results in an improved or enhanced therapeutic effect. Also provided are therapeutic compositions and their use that include the functionally enhanced derivative effector cells alone or in combination therapy with an antibody or checkpoint inhibitor.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 367,596, filed on July 1, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation by Reference of Sequence Listing This application includes a sequence listing submitted electronically. The sequence listing, entitled 184143 - 644601_SL.xml, created on June 28, 2023, and having a size of 30,164 bytes, is incorporated herein by reference in its entirety.

[0003] The present disclosure broadly relates to the field of off - the - shelf immune cell products. More specifically, the present disclosure relates to strategies for developing multifunctional effector cells that can provide therapeutically relevant properties in vivo. The cell products developed under the present disclosure address significant limitations of patient - derived cell therapies.

Background Art

[0004] The field of adoptive cell therapy is currently focused on using patient - derived and donor - derived cells, making it particularly difficult to achieve consistent manufacturing of cancer immunotherapy and to provide therapy to all patients who could potentially benefit from it. There is also a need to improve the effectiveness and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. Lymphocytes such as T cells and natural killer (NK) cells are powerful anti - tumor effectors that play important roles in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains difficult, and there are unmet needs for improvement. Therefore, there remains an important opportunity to maximize the potential of T cells, NK cells, or other lymphocytes in adoptive immunotherapy.

Summary of the Invention

[0005] Functionally improved effector cells are needed to address various issues such as efficacy, cell consumption, loss of infused cells (survival rate and / or persistence), tumor escape due to loss of target or lineage conversion, accuracy of tumor targeting, off-target toxicity, off-tumor effects, effectiveness against solid tumors, i.e., tumor microenvironment and related immunosuppression, mobilization, transport, and infiltration.

[0006] An object of embodiments of the present invention is to provide a method and composition for generating derivative non-pluripotent cells differentiated from a single-cell-derived iPSC (induced pluripotent stem cell) clone strain, where this iPSC strain contains one or several gene modifications in its genome. The aforementioned one or several gene modifications include, in some embodiments, DNA insertions, deletions, and substitutions, and these modifications are retained and continue to function in the subsequent derived cells after differentiation, proliferation, passage, and / or transplantation.

[0007] The iPSC-derived non-pluripotent cells of the present application include, but are not limited to, CD34 cells, hematopoietic endothelial cells, HSC (hematopoietic stem and progenitor cells), hematopoietic multipotent 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 gene modifications in their genome through differentiation from iPSCs containing the same gene modification. In some embodiments, the engineered clonal iPSC differentiation strategy for obtaining genetically engineered derivative cells benefits from the fact that the developmental potential of iPSCs in directed differentiation is not significantly adversely affected by the engineered modality of the iPSCs, and that the engineered modality functions as intended in the derivative cells. Furthermore, this strategy overcomes the current barriers in engineering primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely, that such cells often result in cells that lack reproducibility and uniformity, exhibit poor cell persistence with high cell death and low cell proliferation, and are difficult to engineer. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations obtained in other ways using primary cell sources that are initially heterogeneous.

[0008] Accordingly, in one aspect, the present invention also provides a cell or a population thereof, wherein (i) the cell is (a) an immune cell, (b) an induced pluripotent stem cell (iPSC), a cloned iPSC, or an iPS cell line cell, or (c) a derivative cell obtained by differentiating the cell of (b), and (ii) the cell contains (a) an extracellular Fas-binding domain containing the extracellular domain (ECD) of the Fas receptor (FAS) or a partial or complete peptide of a variant or allele thereof, and (b) a cytoplasmic signaling domain containing a partial or complete peptide of the intracellular domain (ICD) of one or more co-stimulatory molecules, and contains an exogenous polynucleotide encoding a signal transduction redirector receptor, and the signal transduction redirector receptor is a Fas redirector that redirects Fas signaling upon binding to a Fas agonist, thereby providing improved apoptosis resistance and / or exhaustion resistance to the cell or derivative cell. In various embodiments, (i) the Fas redirector further contains a transmembrane region containing the transmembrane domain of a transmembrane protein or a part thereof, (ii) one or more co-stimulatory molecules include CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof, (iii) one or more co-stimulatory molecules do not include 41BB, (iv) the Fas agonist includes Fas ligand (FasL), or (v) the cell further contains one or more of (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), (b) an exogenous polynucleotide encoding CD16 or a variant thereof, (c) a CD38 knockout, and (d) an exogenous polynucleotide encoding a cytokine signaling complex containing a partial or complete peptide of an extracellular cytokine and / or its receptor.In some embodiments, the transmembrane region of the Fas redirector comprises (i) the full-length or at least a portion of the native or modified transmembrane region of FAS, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, 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, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide, or (ii) the full-length or partial length of the transmembrane domain of FAS.

[0009] In some embodiments of a cell or population thereof, (i) the extracellular binding domain of the Fas redirector comprises a sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 1, (ii) the cytoplasmic signaling domain of the Fas redirector comprises (a) the full-length or partial length of the intracellular domain (ICD) of MyD88 and CD40 represented by SEQ ID NO: 2, or (b) the full-length or partial length of the intracellular domain (ICD) of CD27 represented by SEQ ID NO: 3, or (c) the full-length or partial length of the intracellular domain (ICD) of CD28 represented by SEQ ID NO: 4, or (d) the full-length or partial length of the intracellular domain (ICD) of OX40 represented by SEQ ID NO: 5, or (e) the full-length or partial length of the intracellular domain (ICD) of IL12Rβ2 represented by SEQ ID NO: 6, or (f) the full-length or partial length of the intracellular domain (ICD) of IL18R1 represented by SEQ ID NO: 7, or (g) the full-length or partial length of the intracellular domain (ICD) of IL21R represented by SEQ ID NO: 8, or (iii) the Fas redirector comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 9-15.

[0010] In various embodiments of the cell or population thereof, the cell comprises (i) at least one of the genotypes listed in Table 1, (ii) a deletion of HLA-I and / or a deletion of HLA-II, (iii) introduction of HLA-G or non-cleavable HLA-G, or knockout of one or both of CD58 and CD54, (iv) deletion or disruption of at least one of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (v) HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or a functional variant or fragment thereof, checkpoint inhibitor, engager, and introduction of at least one of a surface trigger receptor for coupling with a bispecific or multispecific or universal engager. In various embodiments of the cell or population thereof, the cell comprises a deletion of HLA-I and / or a deletion of HLA-II, and optionally, the cell comprises an exogenous polynucleotide encoding HLA-G, HLA-E, or a variant thereof, and / or a deletion or disruption of one or both of CD54 and CD58. In some embodiments, the HLA-I deletion comprises a deletion or disruption of at least one of B2M, TAP1, TAP2, and tapasin, or the HLA-II deletion comprises a deletion or disruption of at least one of CIITA, RFX5, RFXAP, and RFXANK.

[0011] In various embodiments of the cells or populations thereof, the derived cells include (a) derived CD34+ cells, derived hematopoietic stem progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional properties not present in the corresponding primary T, NK, NKT, and / or B cells, or are allogeneic effector cells that, compared to their natural corresponding cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, have at least one of the following characteristics: (i) improved persistence and / or survival rate, (ii) increased resistance to activated recipient immune cells, (iii) increased cytotoxicity, (iv) improved tumor penetration, (v) enhanced or acquired ADCC, (vi) enhanced ability to migrate tumor site, activate and / or mobilize bystander immune cells, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability to rescue tumor antigen escape, and (ix) decreased apoptosis and / or fratricide, and are derived NK cells or derived T cells.

[0012] In some embodiments of a cell or a population thereof that comprises an exogenous polynucleotide encoding CD16 or a variant thereof, the exogenous CD16 comprises at least one of: (a) high-affinity non-cleavable CD16 (hnCD16) or a variant thereof, (b) F176V and S197P in the extracellular domain of CD16, (c) a complete or partial extracellular domain 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, a signaling domain, and a stimulatory domain that are not derived from CD16 and are derived from the same or different polypeptides. In some embodiments, (a) the non-native transmembrane domain is derived from CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor (TCR) polypeptide, (b) the non-native stimulatory domain is derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or an NKG2D polypeptide, (c) the non-native signaling domain is derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or an NKG2D polypeptide, or (d) the non-native transmembrane domain is derived from NKG2D, the non-native stimulatory domain is derived from 2B4, and the non-native signaling domain is derived from CD3ζ.

[0013] In some embodiments of a cell or population of cells comprising an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), the CAR is (i) T cell-specific or NK cell-specific, (ii) a bispecific antigen-binding CAR, (iii) a switchable CAR, (iv) a dimerized CAR, (v) a split CAR, (vi) a multi-chain CAR, (vii) an inducible CAR, (viii) optionally co-expressed with a cytokine signaling complex comprising a partial or full peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, either in a separate construct or in a bicistronic construct, (ix) optionally co-expressed with a checkpoint inhibitor, either in a separate construct or in a bicistronic construct, and / or (x) optionally, (1) inserted into the TRAC or TRBC locus and / or driven by the endogenous promoter of the TCR and / or the TCR is knocked out by the CAR insertion, (2) inserted into a safe harbor locus, or (3) inserted into a locus intended for disruption.In some embodiments, the CAR is specific for at least one of (i) CD19, BCMA, B7H3, CD20, CD22, CD38, CD52, CD79b, CD123, EGFR, EGP2 / EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MR1, MSLN, Muc1, Muc16, NYESO1, VEGF-R2, PSMA, and PDL1 and / or (ii) ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, 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 kinases erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (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), mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NYESO-1, tumor fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and any one of pathogen antigens.

[0014] In some embodiments of a cell or a population thereof that contains an exogenous polynucleotide encoding a cytokine signaling complex, the cytokine signaling complex is (a) a cell surface-expressed exogenous cytokine that includes at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors and / or a partial or complete peptide of the receptor, or (b) (i) co-expression of IL15 and IL15Rα sandwiching 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 shortened (IL15Δ), (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and common receptor γC, where the common receptor γC is natural or modified, and (vii) a homodimer of IL15Rβ, where at least one of (b)(i)-(vii) can be co-expressed with a CAR in a separate construct or in a bicistronic construct, or (c) (i) a fusion protein of IL7 and IL7Rα, (ii) a fusion protein of IL7 and common receptor γC, where the common receptor γC is natural or modified, and (iii) a homodimer of IL7Rβ, where at least one of (c)(i)-(iii) is optionally co-expressed with a CAR in a separate construct or in a bicistronic construct, and optionally, (d) is transiently expressed.

[0015] In various embodiments of the cell or population thereof, the derived cell is a derived NK cell or a derived T cell, the derived NK cell can recruit and / or migrate T cells to the tumor site, and the derived NK cell or the derived T cell can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. In some embodiments, the one or more checkpoint inhibitors are antagonists to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2A 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, retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or an inhibitory KIR. In some embodiments, the one or more checkpoint inhibitors include (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0016] In various embodiments of the cell or population thereof, the cell comprises (i) one or more exogenous polynucleotides integrated into one safe harbor locus or a locus intended for disruption, or (ii) three or more exogenous polynucleotides integrated into different safe harbor loci or loci intended for disruption. In some embodiments, the safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1, or the locus intended for disruption comprises at least one of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or TCRβ constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0017] In some embodiments of the cell or population thereof, the cytoplasmic signaling domain of the Fas redirector in the cell comprises at least one of the full-length or partial-length intracellular domains (ICDs) of MyD88 and CD40 represented by SEQ ID NO: 2, or the full-length or partial-length intracellular domain (ICD) of CD27 represented by SEQ ID NO: 3, or the full-length or partial-length intracellular domain (ICD) of OX40 represented by SEQ ID NO: 5. In some other embodiments, the cell or population thereof comprising the cytoplasmic signaling domain further comprises an exogenous polynucleotide encoding a fusion protein of a partial or full peptide of IL7 and a partial or full peptide of IL7Rα.

[0018] In another aspect, the present invention provides a composition comprising the cells or a population thereof described herein. In various embodiments, the composition further comprises one or more therapeutic agents. In some embodiments, the one or more therapeutic agents are a peptide, cytokine, checkpoint inhibitor, mitogen, growth factor, small molecule RNA, dsRNA (double-stranded RNA), mononuclear cell, feeder cell, feeder cell component or its replenishing factor, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In some embodiments where the therapeutic agent is a checkpoint inhibitor, (i) the checkpoint inhibitor is (a) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AOne or more antagonist 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, retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR, (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or (ii) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide. In some embodiments where the therapeutic agent is an antibody, the antibody comprises (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, or an anti-PDL1 antibody, or (b) rituximab, belzutifan, ofatumumab, ublituximab, ocrelizumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daclizumab, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, vibativumab, RG7356, G44-26, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof and functional equivalents and biosimilars thereof, one or more of them.In some embodiments where the therapeutic agent is an engager, the engager comprises (i) a bispecific T cell engager (BiTE), (ii) a bispecific killer cell engager (BiKE), or (iii) a tri-specific killer cell engager (TriKE), or the engager comprises (a) a first binding domain that recognizes the extracellular portion of CD3, CD5, CD16, CD28, CD32, CD33, CD64, CD89, NKG2C, NKG2D, or any functional variant thereof of a cell or bystander immune effector cell, and (b) a second binding domain specific for an antigen comprising any one of B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, 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, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROR1, or VEGF-R2.

[0019] In another aspect, the invention provides a therapeutic use of the compositions provided herein by introducing the compositions into a subject in need of adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection.

[0020] In another aspect, the invention provides a master cell bank (MCB) comprising the clonal iPSCs provided herein.

[0021] In another aspect, the present invention provides a method of manufacturing the derivative cells provided herein, the method comprising differentiating genetically engineered iPSCs, wherein the derivative cells are effector cells and the genetically engineered iPSCs comprise an exogenous polynucleotide encoding a Fas redirector that redirects Fas signaling upon binding to a FAS agonist, thereby providing the effector cells with improved apoptosis resistance and / or exhaustion resistance. In some embodiments of the manufacturing method, the Fas redirector comprises (a) an extracellular Fas binding domain comprising the extracellular domain (ECD) of the Fas receptor (FAS) or a partial or complete peptide of a variant or allele thereof, and (b) a cytoplasmic signaling domain comprising a partial or complete peptide of the intracellular domain (ICD) of one or more costimulatory molecules, and the genetically engineered iPSCs are single cells, clonal cells, or cell line cells. In some embodiments of the manufacturing method, (i) the Fas redirector further comprises a transmembrane region comprising the transmembrane domain of a transmembrane protein or a portion thereof, (ii) the one or more costimulatory molecules comprise CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof, (iii) the one or more costimulatory molecules do not comprise 41BB, (iv) the FAS agonist comprises Fas ligand (FasL), or (v) the genetically engineered iPSCs comprising the Fas redirector further comprise one or more of (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), (b) an exogenous polynucleotide encoding CD16 or a variant thereof, (c) a CD38 knockout, and (d) an exogenous polynucleotide encoding a cytokine signaling complex comprising a partial or complete peptide of an extracellular cytokine and / or its receptor.

[0022] In some embodiments of the manufacturing method, the transmembrane region of the Fas redirector comprises (i) the full length or at least a part of the transmembrane region of a native or modified transmembrane region of FAS, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, 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, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide, or (ii) the full length or partial length of the transmembrane domain of FAS. In some embodiments, (i) the Fas redirector further comprises a transmembrane region comprising the transmembrane domain of a transmembrane protein or a part thereof, (ii) one or more co-stimulatory molecules comprise CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof, (iii) one or more co-stimulatory molecules do not comprise 41BB, (iv) the FAS agonist comprises Fas ligand (FasL), or (v) the genetically engineered iPSC comprising the Fas redirector further comprises one or more of (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), (b) an exogenous polynucleotide encoding CD16 or a variant thereof, (c) a CD38 knockout, and (d) an exogenous polynucleotide encoding a cytokine signaling complex comprising a cell surface-expressed exogenous cytokine and / or a partial or full peptide of its receptor.In some embodiments, the transmembrane region of the Fas redirector comprises (i) the full length or at least a portion of the native or modified transmembrane region of FAS, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, 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, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide, or (ii) the full length or partial length of the transmembrane domain of FAS. In some embodiments, (i) the extracellular binding domain of the Fas redirector comprises a sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 1, (ii) the cytoplasmic signaling domain of the Fas redirector comprises (a) the full length or partial length of the intracellular domain (ICD) of MyD88 and CD40 represented by SEQ ID NO: 2, or (b) the full length or partial length of the intracellular domain (ICD) of CD27 represented by SEQ ID NO: 3, or (c) the full length or partial length of the intracellular domain (ICD) of CD28 represented by SEQ ID NO: 4, or (d) the full length or partial length of the intracellular domain (ICD) of OX40 represented by SEQ ID NO: 5, or (e) the full length or partial length of the intracellular domain (ICD) of IL12Rβ2 represented by SEQ ID NO: 6, or (f) the full length or partial length of the intracellular domain (ICD) of IL18R1 represented by SEQ ID NO: 7, or (g) the full length or partial length of the intracellular domain (ICD) of IL21R represented by SEQ ID NO: 8, or (iii) the Fas redirector comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 9-15.

[0023] In various embodiments of the manufacturing method, the genetically engineered iPSCs are (i) at least one of the genotypes listed in Table 1, (ii) a deficiency of HLA-I and / or a deficiency of HLA-II, (iii) the introduction of HLA-G or non-cleavable HLA-G, or the knockout of one or both of CD58 and CD54, (iv) the disruption of at least one of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, and / or (v) HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or its functional variant or fragment, checkpoint inhibitor, engager, and the introduction of at least one of surface trigger receptors for coupling with bispecific or multispecific or universal engagers. In some embodiments, the cells include an HLA-I deficiency and / or an HLA-II deficiency, and optionally, the cells include an exogenous polynucleotide encoding HLA-G, HLA-E, or a variant thereof, or include a deletion or disruption of one or both of CD54 and CD58.

[0024] In various embodiments of the method of manufacture, the method further comprises genome engineering of the iPSCs to knock in (a) a polynucleotide encoding a signal transduction redirector receptor, optionally (b) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), and optionally (c) an exogenous polynucleotide encoding CD16 or a variant thereof, and optionally, (i) to knock out CD38, (ii) to knock out one or both of B2M and CIITA, (iii) to knock out one or both of CD58 and CD54, and / or (iv) to introduce a signal transduction complex comprising HLA-G or non-cleavable HLA-G, and / or a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, further comprising genome engineering of the iPSCs. In some embodiments, the genome engineering comprises targeted editing. In some embodiments, the targeted editing comprises deletions, insertions, or indels, and the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0025] In another aspect, the invention provides a method of improving the durability of effector cells or preventing cell death of effector cells in adoptive cell therapy provided to a subject in need thereof by obtaining the effector cells provided herein.

[0026] In another aspect, the present invention provides a method for improving the effectiveness of adoptive cell therapy provided to a subject in need of adoptive cell therapy, the method comprising administering effector cells to the subject, the effector cells comprising the derived cells or a population thereof provided herein. In some embodiments, the method further comprises administering to the subject one or more therapeutic agents. In some embodiments, the one or more therapeutic agents comprise a peptide, cytokine, checkpoint inhibitor, mitogen, growth factor, small molecule RNA, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replenishing factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In some embodiments where the therapeutic agent is a checkpoint inhibitor, (i) the checkpoint inhibitor is (a) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AOne or more antagonist 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, retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR, (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or (ii) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide. In some embodiments where the therapeutic agent is an antibody, the antibody comprises (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, or an anti-PDL1 antibody, or (b) one or more of rituximab, belzutifan, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daclizumab, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, vibativumab, RG7356, G44-26, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof and functional equivalents thereof and biosimilars thereof.In some embodiments where the therapeutic agent is an engager, the engager comprises (i) a bispecific T cell engager (BiTE), (ii) a bispecific killer cell engager (BiKE), or (iii) a tri-specific killer cell engager (TriKE), or the engager comprises (a) a first binding domain that recognizes the extracellular portion of CD3, CD5, CD16, CD28, CD32, CD33, CD64, CD89, NKG2C, NKG2D, or any functional variant thereof of a cell or bystander immune effector cell, and (b) a second binding domain specific for an antigen comprising any one of B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, 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, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROR1, or VEGF-R2.

[0027] In another aspect, the present invention provides a method of treating a subject in need of adoptive cell therapy, the method comprising injecting effector cells into the subject, wherein the effector cells comprise the derived cells or a population thereof provided herein in combination with an exogenous cytokine.

[0028] The various objects and advantages of the compositions and methods provided herein will become apparent from the following description in conjunction with the accompanying drawings, which illustrate specific embodiments of the invention by way of example and illustration. BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

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Mode for Carrying Out the Invention

[0030] Genome modification of iPSCs (induced pluripotent stem cells) includes insertion, deletion, and substitution of polynucleotides. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types derived from genome-engineered iPSCs. On the other hand, directly manipulating primary immune cells such as T cells or NK cells is difficult and poses an obstacle to the preparation and delivery of engineered immune cells for adoptive cell therapy. In various embodiments, the present invention provides an efficient and reliable targeting approach for stably integrating one or more exogenous genes including suicide genes and other functional modalities, which provides improved therapeutic properties related to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, lifespan, self-renewal, persistence, and / or survival rate to iPSC-derived cells including, but not limited to, HSCs (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, and NK cells.

[0031] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars.

[0032] It is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein and may, therefore, vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined only by the claims.

[0033] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0034] The use of “or” (e.g., “alternatively”) is to be understood to mean either one, both, or any combination of them.

[0035] The term “and / or” is to be understood to mean either one or both of the alternatives.

[0036] As used herein, the terms “about” or “approximately” refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by an amount of 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms “about” or “approximately” refer to a range of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths that are ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0037] As used herein, the terms “substantially” or “essentially” refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more as compared to a referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms “substantially the same” or “essentially the same” refer to a range of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths that are substantially identical to a referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0038] As used herein, the terms "substantially free of" and "essentially free of" are used interchangeably and, when used to describe a composition such as a cell population or a culture medium, refer to a composition that is free of a particular substance or source thereof, such as 95%, 96%, 97%, 98%, 99% or more free of, or undetectable by conventional means of measurement. The term "free of" or "essentially free of" a particular component or substance in a composition also means that such component or substance is (1) not present in the composition at any concentration, or (2) present in the composition at a low concentration that is functionally inert. 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.

[0039] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be interpreted as including the stated step, or element, or group of steps or elements but not excluding any other step, or element, or group of steps or elements. In certain embodiments, the terms "include", "have", "contain" and "comprise" are used synonymously.

[0040] "Consisting of" means including, and limited to, all that follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are necessary or essential and that no other elements may be present.

[0041] "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activities or operations specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but other elements are not optional and may or may not be present depending on whether they affect the activities or operations of the listed elements.

[0042] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "specific embodiments", "additional embodiments", or "further embodiments" 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 the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0043] The term "ex vivo" generally refers to activities performed outside the living body, such as experiments or measurements performed in or on living tissue within an artificial environment outside the body, preferably with minimal changes to natural conditions. In certain embodiments, "ex vivo" procedures include living cells or tissues that are taken from a living body and cultured in an experimental apparatus under normal aseptic conditions, typically for up to several hours or about 24 hours (but including up to 48 hours or 72 hours or more depending on the circumstances). In certain particular embodiments, such tissues or cells may be collected and frozen and later thawed for ex vivo processing. Tissue culture experiments or procedures that use living cells or tissues and last longer than a few days are typically considered "in vitro", but in certain embodiments, this term may be used interchangeably with ex vivo.

[0044] The term "in vivo" generally refers to activities that occur within a living body.

[0045] As used herein, the terms "reprogramming," "dedifferentiation," "increase in cell potency," or "increase in developmental potential" refer to methods of increasing the potency of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an un-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in an un-reprogrammed state.

[0046] As used herein, the term "differentiation" is the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or differentiation-induced cells are cells that occupy a more specialized ("committed") position within the cell lineage. The term "committed," when applied to the process of differentiation, refers to cells that have progressed along a differentiation pathway to the point where they will continue to differentiate into a particular cell type or subset of cell types under normal circumstances and, under normal circumstances, cannot return to a less differentiated cell type that can differentiate into different cell types. As used herein, the term "pluripotency" refers to the ability of cells (i.e., the embryo itself) to form all lineages of a living organism or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency encompasses a range of developmental potential from less primitive and less pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0047] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a stem cell that has been induced or modified, i.e., reprogrammed, from a differentiated adult, neonatal, or fetal cell to a cell that can be differentiated into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm, using a driving method by reprogramming factors and / or small molecule chemicals, and is produced in vitro. The produced iPSCs do not refer to naturally occurring cells.

[0048] As used herein, the term "embryonic stem cell" refers to a naturally occurring pluripotent stem cell of the inner cell mass of a blastocyst. Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers, i.e., ectoderm, endoderm, and mesoderm, during development. They do not contribute to the extraembryonic membranes or placenta (i.e., they are not totipotent).

[0049] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Thus, pluripotent cells can also be referred to as "partially differentiated cells". Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells such as hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell 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 having a lower degree of developmental potential than totipotency and pluripotency.

[0050] Pluripotency can be determined in part by evaluating the pluripotency characteristics of cells. The characteristics of pluripotency include, but are not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm and endoderm), (v) teratoma formation consisting of the three somatic cell lineages, and (vi) the formation of embryoid bodies consisting of cells from the three somatic cell lineages, but not limited to these.

[0051] Two types of pluripotency have been described so far, namely, "primed" or "quasi-stable" pluripotency similar to the epiblast stem cell (EpiSC) of the late blastocyst epiblast, and "naive" or "basal" pluripotency similar to the inner cell mass of the early / pre-implantation blastocyst. While both pluripotent states exhibit the characteristics as described above, the naive or basal state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival rate in single cell culture, (iii) overall reduction of DNA methylation, (iv) reduction of deposition of the H3K27me3 repressive chromatin mark on developmental regulatory gene promoters, and (v) reduced expression of differentiation markers compared to primed state pluripotent cells. The standard methodology of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, generally appears to have the characteristics of the primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the primed state and basal state characteristics are observed unless exogenous transgene expression is maintained.

[0052] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. The morphology of normal embryonic stem cells is characterized by a high nucleus-to-cytoplasm ratio, prominent nucleoli, and a typical intercellular spacing, with a round and small shape.

[0053] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0054] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0055] "Culture" or "cell culture" refers to the maintenance, proliferation, and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (singular in each case is "medium"), "supplemental component", and "medium supplement component" refer to the nutrient compositions for culturing cell cultures.

[0056] "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 a medium as a source of nutrients, hormones, and / or other factors useful for the growth and / or maintenance of cells.

[0057] As used herein, the term "mesoderm" refers to one of the three germ layers that appears during early embryonic development and gives rise to various specialized cell types including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0058] As used herein, the terms "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial-hematopoietic transition. Hematopoietic cell development in the embryo proceeds sequentially from the lateral plate mesoderm through angioblasts to definitive hemogenic endothelial cells and hematopoietic precursors.

[0059] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation and include pluripotent hematopoietic stem cells (blood cells), myeloid precursors, megakaryocyte precursors, erythroid precursors, and lymphocyte precursors. Hematopoietic stem and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid (T cells, B cells, NK cells). As used herein, the term "secondary hematopoietic stem cells" refers to CD34 + hematopoietic cells that can give rise to both mature myeloid and lymphoid cell types, including T-lineage cells, NK-lineage cells, and B-lineage cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0060] 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 various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and inactivation of other immune cells in an MHC class I-restricted manner. The T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or cultured T cell lines, e.g., T cells from Jurkat, SupT1, etc., or T cells obtained from a mammal. The T cells can be CD3 + cells. The T cells can be CD4 + / CD8 + double positive T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD8 +Any type of T cell, 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 (γδT cells), etc., can be of any developmental stage. 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 cell" 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 can also be differentiated from stem cells 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 can have a T cell lineage in some respects, but at the same time have one or more functional properties 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".

[0061] "CD4 +"Cells" refers to a subset of T cells that express CD4 on their surface and are associated with the cellular immune response. They are characterized by their secretion profile after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. The "CD4" molecule is a 55kD glycoprotein originally defined as a differentiation antigen of T lymphocytes, but is also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as an associated recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, it defines the helper / inducer subset.

[0062] "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 an associated recognition element in major histocompatibility complex class I-restricted interactions.

[0063] As used herein, the terms "NK cell" or "natural killer cell" refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). NK cells can be any NK cell, such as 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 cell" and "memory NK cell" are interchangeable and phenotypically CD3 - and CD56 +and refers to a subset of NK cells that express at least one of NKG2C and CD57, and optionally CD16, but lack the expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, CD56 + An isolated subpopulation of NK cells comprises the expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and / or DNAM-1. CD56 + can be either weakly positive (dim) or strongly positive (bright) expression. NK cells or NK cell-like effector cells can be differentiated from stem cells 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 can have an NK cell lineage in some respects, but at the same time have one or more functional traits that are not present in primary NK cells. In the present 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”.

[0064] As used herein, the terms "NKT cell" or "natural killer T cell" or "NKT lineage cell" refer to CD1d-restricted T cells that express a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire, namely 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, exhibits more heterogeneous TCRαβ usage. Type I NKT cells are thought to be suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of one or more of the markers TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0065] The term "effector cell" generally applies to certain cells in the immune system that perform specific activities in response to stimulation and / or activation, or cells that result in a specific function upon activation. As used herein, the term "effector cell" includes immune cells, "differentiated immune cells", and primary or differentiated cells that have been engineered and / or regulated to perform specific activities in response to stimulation and / or activation, and in some contexts is interchangeable with them. Non-limiting examples of effector cells include primary- or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0066] As used herein, terms such as "isolated" refer to cells or a population of cells that have been separated from their original environment, i.e., the environment of the isolated cells is substantially free of at least one component found in the environment in which the "non-isolated" reference cells are present. This term includes cells that are removed from some or all components when found in their natural environment, e.g., when isolated from a tissue or biopsy sample. This term also includes cells that are removed from at least one, some, or all components when found in a non-natural environment, e.g., when isolated from a cell culture or cell suspension. Thus, an "isolated cell" is partially or completely separated from at least one component including other substances, cells, or cell populations, whether found in nature or growing, stored, or persisting in a non-natural environment. Specific examples of isolated cells include a partially pure cell composition, a substantially pure cell composition, and cells cultured in a non-naturally occurring medium. Isolated cells can be obtained by separating the desired cells 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.

[0067] As used herein, terms such as "purify" refer to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0068] As used herein, the term "encoding" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that functions as a template for the synthesis of other polymers and macromolecules in the defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or the defined sequence of amino acids and biological processes arising therefrom. Thus, when transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system, that gene encodes the protein. Both the nucleotide sequence that is identical to the mRNA sequence, usually the coding strand described in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0069] "Construct" refers to a macromolecular or molecular complex comprising a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct that can induce delivery or transfer of foreign genetic material into a target cell and can replicate and / or express in the target cell. Thus, the term "vector" includes the construct being delivered. A vector can be a linear or circular molecule. A vector may or may not be integrated. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0070] "Integration" means that one or more nucleotides of a construct are stably inserted into the cell genome, i.e., covalently bound to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted into the chromosomal or mitochondrial DNA of the cell at a preselected site or "integration site". As used herein, the term "integration" further refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of the endogenous sequence or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" may further include replacement of the deleted nucleotides with the endogenous sequence or one or more inserted nucleotides.

[0071] As used herein, the term "exogenous" is intended to mean that a reference molecule or activity is introduced into the host cell or is non-native to the host cell. A molecule can be introduced, for example, by integration into the host's chromosome or by introducing the coding nucleic acid into the host genetic material as non-chromosomal genetic material such as a plasmid. Thus, the term used with respect to the expression of a coding nucleic acid refers to introducing the coding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a reference molecule or activity present in the host cell. Similarly, when this term is used with respect to the expression of a coding nucleic acid, it refers to the expression of a coding nucleic acid that is contained within the cell and has not been exogenously introduced.

[0072] As used herein, the "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and optionally translated into a polypeptide in vivo. The gene or polynucleotide of interest can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, the gene of interest can encode miRNA, shRNA, a native polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, a variant polypeptide (i.e., a variant of a native polypeptide having less than 100% sequence identity to the native polypeptide) or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, a contrast marker, a selectable marker, etc.

[0073] As used herein, the term "polynucleotide" refers to a nucleotide in polymeric form 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) in the case of DNA, and thymine is uracil (U) in the case of RNA being a polynucleotide. 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.

[0074] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, which in the art are generally also referred to as peptides, oligopeptides, and oligomers, and long chains, which are generally referred to as polypeptides or proteins in the art. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0075] As used herein, the term "subunit" refers to each individual polypeptide chain of a protein complex when used herein, and each individual polypeptide chain can form a stable folded structure by itself. Many protein molecules are composed of two or more subunits, where the amino acid sequences can be 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, consecutive portions of the polypeptide chain often fold into compact, local semi-independent units called "domains." Many protein domains can further contain independent "structural subunits," also called subdomains, that contribute to the common function of the domain. Thus, as used herein, the term "subdomain" refers to a protein domain within a larger domain, such as a binding domain within the extracellular domain of a cell surface receptor, or a stimulatory or signaling domain within the intracellular domain of a cell surface receptor.

[0076] "Operably-linked" or "operatively linked" is interchangeable with "operably connected" or "operatively connected" and refers 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 (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter) if it can affect the expression of that coding sequence or functional RNA. The coding sequence can be operably linked to regulatory sequences in the sense or antisense direction. 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 converts a signal in response to that binding.

[0077] "Fusion protein" or "chimeric protein", as used herein, is a protein produced through genetic manipulation to join two or more partial or complete polynucleotides encoding sequences encoding separate proteins, the expression of these joined polynucleotides resulting 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 two adjacent polypeptides of different sources in a fusion protein.

[0078] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic properties of the source cell or iPSC and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can further differentiate into specific cell types, including any hematopoietic lineage cells. Source cell-derived iPSCs, and cells differentiated therefrom, may collectively be referred to as "derived" cells or "descendant" cells, depending on the context. For example, throughout this application, derived effector cells, or derived NK cells or derived T cells are cells differentiated from iPSCs when compared to their primary counterparts obtained from natural / native sources such as peripheral blood, cord blood, or other donor tissues. As used herein, the genetic imprint conferring preferential therapeutic properties is incorporated into iPSCs by reprogramming selected source cells that are specific to a donor, disease, or treatment response, or by introducing gene recombination modalities into iPSCs using genome editing. In the context of source cells obtained from specifically selected donors, diseases, or treatment situations, the genetic imprint contributing to preferential therapeutic attributes may include a heritable phenotype passed on to descendant cells of the selected source cell, i.e., a context-specific genetic or epigenetic modification representing a preferential therapeutic attribute, regardless of whether underlying molecular events have been identified. Source cells that are specific to a donor, disease, or treatment response may contain a genetic imprint that can be retained in iPSCs and derived hematopoietic lineage cells, which may include, for example, pre-arranged single-specific TCRs from virus-specific T cells or invariant natural killer T (iNKT) cells, traceable and desirable gene polymorphisms, such as homozygosity for a point mutation encoding a high-affinity CD16 receptor in a selected donor, and predetermined HLA requirements, i.e., selected HLA-matched donor cells that exhibit a haplotype in an increased population, but are not limited thereto.As used herein, preferred therapeutic properties include improved engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of the immune response, survival rate, and cytotoxicity of the source cells. Preferred therapeutic properties also include antigen-targeted receptor expression, HLA presentation or absence thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modification, improved on-target specificity with reduced off-tumor effects, and resistance to treatments such as chemotherapy. When iPSCs incorporating a genetic imprint conferring preferred therapeutic properties are differentiated to obtain derivative cells having one or more therapeutic properties, such derivative 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 as compared to their primary counterparts obtained from natural / native sources such as peripheral blood, cord blood, or other donor tissues. In some embodiments, the synthetic cells have one or more non-natural cell functions when compared to their closest corresponding primary cells.

[0079] As used herein, the term "enhanced therapeutic property" refers to the therapeutic properties of a cell that are enhanced as compared to typical immune cells of the same general cell type. For example, NK cells having "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties as compared to typical unmodified and / or naturally occurring NK cells. The therapeutic properties of immune cells can include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, control and regulation of the immune response, survival rate, and cytotoxicity. The therapeutic properties of immune cells are also related to antigen-targeted receptor expression, HLA presentation or absence thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modification, improved on-target specificity with a reduction in off-tumor effects, and / or resistance to treatments such as chemotherapy.

[0080] As used herein, the term "engager" refers to a molecule, such as a fusion polypeptide, that can form a linkage between an immune cell (e.g., T cell, NK cell, NKT cell, B cell, macrophage, neutrophil) and a tumor cell and can activate the immune cell. Examples of engagers include, but are not limited to, bi-specific T cell engager (BiTE), bi-specific killer cell engager (BiKE), tri-specific killer cell engager (TriKE), or multi-specific killer cell engager, or a universal engager that is compatible with multiple immune cell types.

[0081] As used herein, the term "surface trigger receptor" refers to a receptor that can induce or initiate an immune response, such as a cytotoxic response. The surface trigger receptor can be engineered and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, the surface trigger receptor facilitates the binding of a bispecific or multispecific antibody between an effector cell and a specific target cell (e.g., a tumor cell), independent of the natural receptor and cell type of the effector cell. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and differentiated into a population of various effector cell types expressing the universal surface trigger receptor. "Universal" means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind or ligate to an engager recognizable by the surface trigger receptor, regardless of the tumor-binding specificity of the engager. In some embodiments, an engager having the same tumor targeting specificity is used to bind to the universal surface trigger receptor. In some embodiments, an engager having different tumor targeting specificities is used to bind to the universal surface trigger receptor. Thus, one or more effector cell types may engage to kill one specific type of tumor cell, or may kill two or more types of tumors. The surface trigger receptor generally includes a co-stimulatory domain for activation of the effector cell and an anti-epitope specific for the epitope of the engager. The bispecific engager is specific for the anti-epitope of the surface trigger receptor at one end and specific for the tumor antigen at the other end.

[0082] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or other adverse effects of cell therapy. In some examples, the expression of the safety switch protein is conditionally controlled to address concerns about the safety of transplanted and engineered cells that have permanently integrated the gene encoding the safety switch protein into the genome. This conditional regulation can vary and may include post-translational activation via small molecules and control by tissue-specific and / or transient transcriptional regulation. Safety switch proteins may 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, such as a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, the prodrug administered upon occurrence of an adverse event is activated by the suicide gene product to kill the transduced cells.

[0083] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. A pharmaceutically 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 pharmaceutically active protein also has preventive properties and is used to prevent the onset of a disease or to reduce the severity thereof if such a disease or pathological condition occurs. "Pharmaceutically active protein" includes the whole protein or peptide or a pharmaceutically active fragment thereof. This term also includes pharmaceutically active analogs of the protein or peptide or analogs of fragments of the protein or peptide. The term "pharmaceutically active protein" also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0084] As used herein, the term "signaling molecule" refers to any molecule that modifies, participates in, inhibits, activates, reduces or increases cell signaling. "Cell signaling" refers to the transmission of molecular signals in the form of chemical modifications by the mobilization of protein complexes along a pathway that ultimately causes a biochemical event within a cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Toll-like receptor signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0085] As used herein, the term "targeting modality" refers to a molecule, e.g., a polypeptide, that is genetically incorporated into a cell and promotes antigen and / or epitope specificity, including, but not limited to, antigen specificity when associated with a native chimeric antigen receptor (CAR) or T cell receptor (TCR), engager specificity when associated with a monoclonal antibody or bispecific engager, targeting of transformed cells, targeting of cancer stem cells, and other targeting strategies in the absence of a specific antigen or surface molecule.

[0086] As used herein, the terms "specific" or "specificity" can be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as contrasted with non-specific or non-selective binding.

[0087] As used herein, the term "adoptive cell therapy" refers to a cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, regardless of whether the immune cells are isolated from a human donor, are effector cells obtained from in vitro differentiation of pluripotent cells, are genetically modified, or are primary donor cells that have been passaged, expanded, or immortalized ex vivo after isolation from the donor.

[0088] As used herein, "lymphodepletion" and "lymphodepletion conditioning" are used interchangeably to typically refer to the destruction of lymphocytes and T cells, typically prior to immunotherapy. The purpose of lymphodepletion conditioning prior to adoptive cell therapy is to promote the homeostatic expansion of effector cells and to eliminate regulatory immune cells and other competing elements of the immune system that compete for homeostatic cytokines. Thus, lymphodepletion conditioning is typically achieved by administering one or more chemotherapeutic agents to a subject prior to the first dose of adoptive cell therapy. In various embodiments, lymphodepletion conditioning is performed several hours to several days prior to the first dose of adoptive cell therapy. Exemplary chemotherapeutic agents useful for lymphodepletion conditioning include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, sufficient lymphodepletion by anti-CD38 mAb can provide an alternative conditioning process for this iNK cell therapy, without or with minimal need for a CY / FLU-based lymphodepletion conditioning procedure, as further described herein.

[0089] As used herein, "homing" or "transport" refers to the active navigation (migration) of cells to a target site (e.g., a cell, tissue (e.g., a tumor), or 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 with a target site. In some embodiments, a homing molecule is a chemokine receptor.

[0090] As used herein, "therapeutically sufficient amount" within the meaning thereof includes an amount that is non-toxic but sufficient and / or effective to provide the desired therapeutic effect of the particular therapeutic agent and / or pharmaceutical composition being referred to. The exact amount required will vary from subject to subject depending on factors such as the overall health of the patient, 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 alleviate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0091] Differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium and changes in the physical state of the cells. The most common strategy is to utilize the formation of embryoid bodies (EBs) as a common and important intermediate for initiating lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryonic development as it generates multiple lineages within a three-dimensional region. Typically, through a differentiation process that lasts from several hours to several days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and grow into cystic EBs, which typically takes several days to several weeks, at which point they are further processed to continue differentiating. EB formation is initiated by bringing pluripotent stem cells into proximity with each other in a three-dimensional multilayer cluster of cells. Typically, this is achieved by one of several methods, including sedimenting pluripotent cells in droplets, sedimenting cells in a "U" bottom well plate, or by mechanical agitation. Aggregates maintained in pluripotent culture maintenance medium do not form proper EBs, so in order to promote EB growth, aggregates of pluripotent stem cells require additional cues for differentiation. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cues for induction into the selected lineage. EB-based culture of pluripotent stem cells typically generates a moderately proliferating differentiated cell population within the EB cell cluster (i.e., the germ layers of ectoderm, mesoderm, and endoderm). EBs have been shown to promote cell differentiation, but they generate heterogeneous cells in various differentiation states because the exposure of the three-dimensional structured cells to cues for differentiation in the environment is inconsistent. In addition, EBs are cumbersome to create and maintain. Furthermore, cell differentiation by EB formation is accompanied by moderate cell proliferation, which also leads to a decrease in differentiation efficiency.

[0092] In contrast, "aggregate formation", unlike "EB formation", can be used to proliferate a population of pluripotent stem cell-derived cells. For example, during the proliferation of pluripotent stem cells based on aggregates, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally involves increasing the size of the aggregates to form larger aggregates, which can be mechanically or enzymatically dissociated into smaller aggregates to maintain cell proliferation in culture and increase the number of cells. Unlike EB culture, cells cultured within aggregates in the maintenance culture medium maintain pluripotency markers. Pluripotent stem cell aggregates require additional cues for differentiation to induce differentiation.

[0093] As used herein, "monolayer differentiation" is a term that refers to a differentiation method different from the differentiation across three-dimensional multi-layer clusters of cells, i.e., "EB formation". Monolayer differentiation, among other advantages disclosed herein, particularly avoids the need for EB formation to initiate differentiation. Since monolayer culture does not mimic embryogenesis as in the case of EB formation, differentiation into a specific lineage is considered minimal compared to the differentiation of all three germ layers in EB formation.

[0094] As used herein, "dissociated cells" or "single dissociated cells" refer to cells that are substantially separated from or purified to be separated from other cells or from a surface (e.g., the surface of a culture plate). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be dissociated from each other enzymatically or mechanically, 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 involves disrupting cell interactions with the extracellular matrix (ECM) and substrate (such as the culture surface), or disrupting the ECM between cells.

[0095] As used herein, "Master Cell Bank" or "MCB" refers to a clonal master-manipulated 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 the potential for contamination by reducing and / or eliminating the total number of times the iPSC line is passaged, thawed, or handled during the manufacturing process.

[0096] As used herein, "feeder cell" or "feeder" refers to a type of cell that, by providing stimuli, growth factors, nutrients, and supporting a second cell type, co-cultures with a second type of cell to provide an environment in which the second type of cell can grow, proliferate, or differentiate. 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 treatment with an anti-mitotic agent such as irradiation or mitomycin to prevent them from proliferating more than the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular type of feeder cell can be a human feeder such as human dermal fibroblasts. Another type of feeder cell can be a mouse embryonic fibroblast (MEF). Generally, various feeder cells can be used in part to maintain pluripotency, direct differentiation into specific lineages, enhance proliferation capacity, and promote maturation into specialized cell types such as effector cells.

[0097] As used herein, a "feeder-free" (FF) environment refers to an environment such as a culture condition, cell culture, or culture medium that is essentially free of feeder or stromal cells and / or has not been pretreated by culturing feeder cells. A "pretreated" medium refers to a medium taken after feeder cells have been cultured in the medium for a period such as at least one day. The pretreated medium contains many mediator substances, including growth factors and cytokines secreted from feeder cells cultured in the medium. In some embodiments, the feeder-free environment contains neither feeder cells nor stromal cells and has not been pretreated by culturing feeder cells.

[0098] "Functional", as used in connection with genome editing or modification of iPSCs and their differentiated non-pluripotent derivative cells, or genome editing or modification of non-pluripotent cells and their reprogrammed iPSC derivative cells, refers to (1) at the gene level, success of transgenic or controlled gene expression such as inducible or transient expression at a desired cell developmental stage achieved by knock-in, knockout, knockdown gene expression, direct genome editing or modification, or "passage" through differentiation from or reprogramming of a starting cell that has first been genomically engineered, or (2) at the cell level, (i) gene expression modification obtained in the cell through direct genome editing; (ii) gene expression modification maintained in the cell through "passage" through differentiation from or reprogramming of a starting cell that has first been genomically engineered; (iii) downstream gene control in the cell as a result of gene expression modification that appears only at an earlier developmental stage of the cell or only in the starting cell that gives rise to the cell through differentiation or reprogramming, or (iv) success of removal, addition, or modification of cell functions / characteristics due to enhanced or newly achieved cell functions or attributes shown in mature cell products initially derived from genome editing or modification performed on iPSCs, precursors, or dedifferentiated cell origins.

[0099] "HLA deficiency", including HLA class I deficiency, HLA class II deficiency, or both, refers to cells in which the surface expression level of the complete MHC complex containing the HLA class I protein heterodimer and / or the HLA class II heterodimer is insufficient, or no longer maintained, or reduced, and the reduced or decreased level is lower than the level naturally detectable by other cells or synthetic methods.

[0100] As used herein, "modified HLA-deficient iPSC" refers to HLA-deficient iPSCs that are further modified by introducing genes that express proteins related to, but not limited to, improved differentiation ability, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, co-stimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 4-1BB, DAP10, DAP12, CD24, CD3ζ, 4-1BBL, CD47, CD113, and PDL1. Cells with "modified HLA deficiency" include cells other than iPSCs.

[0101] 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. The ligand may be a natural or artificial substance that can specifically bind to the target. The 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 the target. The target to which the ligand binds can be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and changes its function to induce a signaling response is called "agonistic" or an "agonist". A ligand that binds to a target and blocks or reduces a signaling response is "antagonistic" or an "antagonist".

[0102] The term "antibody" is used in the broadest sense herein and generally refers to an immune response generating molecule that contains at least one binding site that specifically binds to a target, where the target can be an antigen or a receptor that can interact with a particular antibody. For example, NK cells are activated by the binding of an antibody or its Fc region to its Fc-gamma receptor (FcγR), thereby inducing antibody-dependent cellular cytotoxicity (ADCC)-mediated effector cell activation. The particular fragment or portion of the antigen or receptor to which the antibody binds, 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 their variants, fragments of natural antibodies and their variants, peptibodies and their variants, and single-chain antibodies and their fragments. Antibodies can be murine antibodies, human antibodies, humanized antibodies, camel IgG, single variable new antigen receptors (VNARs), shark heavy chain antibodies (Ig-NARs), chimeric antibodies, recombinant antibodies, single domain antibodies (dAbs), anti-idiotypic antibodies, bispecific, multispecific, or multimeric antibodies, or fragments thereof. Anti-idiotypic antibodies are specific for binding to the idiotype of another antibody, where the idiotype is the antigenic determinant of the antibody. Bispecific antibodies can be BiTEs (bispecific T cell engagers) or BiKEs (bispecific killer cell engagers), and multispecific antibodies can be TriKEs (trispecific killer cell engagers).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), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragment (sdAb), camel heavy chain IgG and Nanobody® fragments, heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of an antibody.

[0103] "Fc receptor" is abbreviated as FcR and is classified based on the type of antibody it recognizes. For example, those that bind to the most common class of antibody, IgG, are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). The classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signal transduction properties of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and thus different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0104] "Chimeric receptor" is a general term used to describe engineered artificial or hybrid receptor protein molecules that are created to contain two or more portions of amino acid sequences derived from at least two different proteins. Chimeric receptor proteins are engineered to confer upon cells 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.

[0105] The term "chimeric Fc receptor", abbreviated as CFcR, is used to describe an engineered Fc receptor in which the native transmembrane domain and / or intracellular signaling domain has been modified or replaced with a non-native transmembrane domain and / or intracellular signaling domain. In some embodiments of the chimeric Fc receptor, in addition to one or both of the transmembrane domain and the signaling domain being non-native, one or more stimulatory domains are introduced into the intracellular portion of the engineered Fc receptor such that upon receptor engagement, cell activation, proliferation, and function can be enhanced. Unlike a chimeric antigen receptor (CAR) that includes an antigen-binding domain for a target antigen, a chimeric Fc receptor binds to an Fc fragment, or the Fc region of an antibody, or an Fc region included in an engager or binding molecule, and can activate cell function by bringing target cells in proximity or by binding to the molecule without bringing the target cells in proximity. For example, an Fcγ receptor can be engineered to include a selected transmembrane domain, stimulatory domain, and / or signaling domain in the intracellular region that generates the CFcR in response to IgG binding at the extracellular domain. In one example, a CFcR is produced by engineering CD16, which is an Fcγ receptor, by replacing its transmembrane domain and / or intracellular domain. To further improve the binding affinity of the CD16-based CFcR, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of the CFcR that include the high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or replaced such that the extracellular domain of the receptor is non-cleavable, i.e., does not undergo shedding, thereby yielding an hnCD16-based CFcR.

[0106] It has been confirmed that CD16, an Fcγ receptor, has two isoforms, Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate 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 undergoes ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes when NK cells are activated. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane-proximal region (positions 189-212) is altered or eliminated do not undergo shedding. The cleavage site and the membrane-proximal region are described in detail in International Publication No. WO 2015 / 148926, the complete 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 have high affinity and are 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.

[0107] The "T cell receptor", abbreviated as "TCR", generally refers to a protein complex found on the surface of T cells and is involved in the recognition of fragments of antigen peptides bound to major histocompatibility complex (MHC) molecules. Binding of the TCR to the antigen peptide initiates TCR-CD3 intracellular activation, recruitment of numerous signaling molecules, and branching and integration of signaling pathways, leading to gene expression and recruitment of transcription factors important for typical T cell proliferation and acquisition of function. A typical TCR contains two highly variable protein chains (α and β), each chain containing a constant region proximal to the cell membrane and a variable region (i.e., binding domain) that binds to peptide / MHC.

[0108] I. Cells and Compositions Useful for Adoptive Cell Therapies with Enhanced Properties Provided herein is a strategy for systematically manipulating the regulatory circuitry of clonal iPSCs while enhancing the therapeutic properties of derivative cells differentiated from iPSCs without affecting the differentiation potential and cell developmental biology of iPSCs and their derivative cells. iPSC-derived cells are functionally improved, and combinations of select modalities are suitable for adoptive cell therapies after being introduced into cells at the iPSC level through genomic engineering. Previously, it was unclear whether modified iPSCs containing one or more provided gene edits retained the ability to enter cell development while maintaining modified activity and / or properties, and / or the ability to mature into functionally differentiated cells. Unexpected failures during cell differentiation directed from iPSCs are due to aspects including, but not limited to, specific gene expression or lack thereof at the developmental stage, requirements for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure differentiation protocols that allow for changes in cell phenotype and / or function. This application shows that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from the engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from individual or combined genomic modifications that are retained in the effector cells after iPSC differentiation.

[0109] 1. Exogenous FAS Signal Transducer The Fas receptor (Fas) is a death receptor that can induce apoptosis when bound to its respective ligand, Fas ligand (FasL). Multiple liquid and solid tumors, as well as activated peripheral B cells, T cells, and NK cells, express high levels of FasL. This elevated expression of FasL in the recipient represents a major obstacle to adoptive immune cell products. CAR-T and CAR-NK cell therapy products can be Fas positive either by standard expression or through FasL-induced activation of Fas, thereby being targeted and killed through FasL-induced apoptosis in the tumor environment or through fratricide by adjacent product cells expressing Fas ligand.

[0110] To provide genetic enhancement to adoptive cell products, several constructs encoding a Fas signal transducer receptor (also referred to as "Fas transducer" or "Fas signal transducer") were designed. The Fas signal transducer receptor includes an extracellular Fas-binding domain that contains the extracellular domain (ECD) of the Fas receptor or a partial or complete peptide of its variant or allele, and a cytoplasmic signaling domain that contains a partial or complete peptide of the intracellular domain (ICD) of one or more costimulatory molecules. The Fas signal transducer receptor redirects Fas signaling upon FasL binding to provide improved apoptosis resistance and / or exhaustion resistance to cells. The Fas transducers described herein further include a transmembrane region that includes the transmembrane domain of a transmembrane protein or a portion thereof. In some embodiments, both the transmembrane region and the extracellular binding domain of the Fas transducer are derived from the Fas receptor. In some embodiments, the transmembrane region of the Fas transducer is derived from a transmembrane protein other than the Fas receptor. In some embodiments, the transmembrane region and the cytoplasmic signaling domain of the Fas transducer are derived from the same protein or different proteins.

[0111] In some embodiments, the extracellular binding domain of the Fas redirector comprises a sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 1. In some embodiments, the extracellular binding domain of the Fas redirector comprises a sequence having at least about 90% identity to SEQ ID NO: 1. In some embodiments, the extracellular binding domain of the Fas redirector comprises a sequence having at least about 95% identity to SEQ ID NO: 1. In some embodiments, the extracellular binding domain of the Fas redirector comprises the amino acid sequence of SEQ ID NO: 1. 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 × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be performed using mathematical algorithms recognized in the art. SEQ ID NO: 1 QVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN

[0112] In some embodiments, the transmembrane region of the Fas redirector comprises the full-length or at least a portion of the transmembrane region of Fas, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, 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, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a native or modified transmembrane region of a T cell receptor polypeptide. In some embodiments, the transmembrane region of the Fas redirector is the full-length or partial length of the transmembrane domain of the Fas receptor.

[0113] In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof. In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector is not 41BB by itself.

[0114] In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full-length or partial-length intracellular domain (ICD) of MyD88 and CD40, represented by SEQ ID NO: 2. In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full-length or partial-length intracellular domain (ICD) of CD27, represented by SEQ ID NO: 3. In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full-length or partial-length intracellular domain (ICD) of CD28, represented by SEQ ID NO: 4. In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full-length or partial-length intracellular domain (ICD) of OX40, represented by SEQ ID NO: 5. In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full-length or partial-length intracellular domain (ICD) of IL12Rβ2, represented by SEQ ID NO: 6. In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full-length or partial-length intracellular domain (ICD) of IL18R1, represented by SEQ ID NO: 7. In some embodiments, the co-stimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full-length or partial-length intracellular domain (ICD) of IL21R, represented by SEQ ID NO: 8. SEQ ID NO: 2 MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ SEQ ID NO: 3 QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP SEQ ID NO: 4 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 5 ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 6 SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ SEQ ID NO: 7 YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES SEQ ID NO: 8 SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS

[0115] In some embodiments, a Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain that includes a partial or complete peptide of the ICD of MyD88 and CD40 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 9, and it is understood by those skilled in the art that its signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced with the signal peptide sequence and / or transmembrane domain sequence of a different protein. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 9. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 9. In some embodiments, the Fas redirector comprises the amino acid sequence of SEQ ID NO: 9.

[0116]

Table 1

[0117] In some embodiments, a Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain that includes a partial or complete peptide of the ICD of CD27 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 10, and it is understood by those skilled in the art that its signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced with the signal peptide sequence and / or transmembrane domain sequence of a different protein. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 10. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 10. In some embodiments, the Fas redirector comprises the amino acid sequence of SEQ ID NO: 10.

[0118]

Table 2

[0119] In some embodiments, a Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain that includes a partial or complete peptide of the ICD of CD28 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 11, and it will be understood by those skilled in the art that its signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced by the signal peptide sequence and / or transmembrane domain sequence of a different protein. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 11. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 11. In some embodiments, the Fas redirector comprises the amino acid sequence of SEQ ID NO: 11.

[0120]

Table 3

[0121] In some embodiments, a Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain that includes a partial or complete peptide of the ICD of OX40 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 12, and it is understood by those skilled in the art that its signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced with the signal peptide sequence and / or transmembrane domain sequence of a different protein. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 12. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 12. In some embodiments, the Fas redirector comprises the amino acid sequence of SEQ ID NO: 12.

[0122]

Table 4

[0123] In some embodiments, a Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain that includes a partial or complete peptide of the ICD of IL12Rβ2 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 13, and it is understood by those skilled in the art that its signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced with the signal peptide sequence and / or transmembrane domain sequence of a different protein. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 13. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 13. In some embodiments, the Fas redirector comprises the amino acid sequence of SEQ ID NO: 13.

[0124]

Table 5

[0125] In some embodiments, a Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain that includes a partial or complete peptide of the ICD of IL18R1 comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 14, and it is understood by those skilled in the art that its signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced with the signal peptide sequence and / or transmembrane domain sequence of a different protein. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 14. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 14. In some embodiments, the Fas redirector comprises the amino acid sequence of SEQ ID NO: 14.

[0126]

Table 6

[0127] In some embodiments, a Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain that includes a partial or complete peptide of the ICD of IL21R comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 15, and it is understood by those skilled in the art that its signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced with the signal peptide sequence and / or transmembrane domain sequence of a different protein. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 15. In some embodiments, the Fas redirector comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 15. In some embodiments, the Fas redirector comprises the amino acid sequence of SEQ ID NO: 15.

[0128]

Table 7

[0129] Accordingly, in various embodiments, the Fas redirectors provided herein may be introduced into induced pluripotent cells (iPSCs) using one or more of the above construct designs, or may be introduced into their derivative cells during iPSC differentiation. In addition to iPSCs, cloned iPSCs, cloned iPS cell lines, and iPSC-derived effector cells are provided that include at least one engineered modality disclosed herein. Also provided is a master cell bank comprising sorted single cells and expanded cloned engineered iPSCs that have at least an exogenous inducible Fas redirector as described in this section, the cell bank providing a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf engineered homogeneous cell therapy products that can be mass-produced on a large scale in a manner that is well-defined, uniform, and cost-effective.

[0130] Accordingly, in some embodiments, the invention provides iPSCs comprising an exogenous Fas redirector (Fas in Table 1) and derivative cells therefrom, and the cells comprising derivative T cells and derivative NK cells are useful for overcoming Fas-induced apoptosis or fratricide, and the Fas redirector comprises a partial or complete peptide of the extracellular domain (ECD) of the Fas receptor, or a variant or allele thereof, and a partial or complete peptide of the intracellular domain (ICD) of one or more costimulatory molecules. In some embodiments, the iPSCs and derivative cells therefrom comprise one or more additional genome edits described herein without adversely affecting the differentiation ability of the iPSCs and the function of the derivative effector cells comprising the derivative T cells and derivative NK cells.

[0131] 2. Chimeric antigen receptor (CAR) expression Those applicable to genetically engineered iPSCs and their derived effector cells can be any CAR design known in the art. A CAR is a fusion protein generally comprising an extracellular domain containing an antigen recognition domain, a transmembrane domain, and an intracellular domain. In some embodiments, the extracellular domain can further comprise a signal peptide or leader sequence and / or a spacer. In some embodiments, the intracellular domain can further comprise a signaling peptide that activates the effector cell expressing the CAR. In some embodiments, the intracellular domain can further comprise a signaling domain, which is derived from the cytoplasmic domain of a signaling protein specific for the activation or function of T cells and / or NK cells. 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 a T lineage cell or an NK lineage cell expressing the CAR. In some embodiments, the CAR is NK cell-specific and comprises an NK-specific signaling component. In certain embodiments, the T cell is derived from an iPSC containing the CAR, and the induced T lineage cell can comprise T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof. In certain embodiments, the NK cell is derived from an iPSC containing the CAR.

[0132] In certain embodiments, the antigen recognition region / domain comprises a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab′)2, F(ab′)3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody. 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).

[0133] 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, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (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, NYESO-1, tumor fetal 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.

[0134] Accordingly, in some embodiments, the genetically engineered iPSCs and their derivative cells comprise an exogenous polynucleotide encoding a CAR, and the CAR is specific for a target including, but not limited to, B7H3, CD19, BCMA, CD20, CD22, CD38, CD52, CD79b, CD123, EGFR, EGP2 / EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MR1, MSLN, Muc1, Muc16, NYESO1, VEGF-R2, PSMA, and PDL1.

[0135] In some embodiments, the transmembrane domain of the CAR comprises the full length or at least a portion of the transmembrane region of CD2, CD3δ, CD3ε, CD3γ, CD3ζ, 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, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a native or modified transmembrane region of a T cell receptor polypeptide.

[0136] In some embodiments, the signal transduction peptide of the internal domain (or intracellular domain) comprises the full length or at least a portion of the polypeptide 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), CD3ζ1XX (CD3ζ variant), DAP10 (hematopoietic cell signal transducer), 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), IL-2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D type II integral membrane protein), 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).

[0137] In some embodiments, the intracellular domain of the CAR further includes a second signaling domain and optionally a third signaling domain, and each of the first signaling domain, the second signaling domain, and the third signaling domain is different. In certain embodiments, the second signaling domain and / or the third signaling domain includes the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8.

[0138] In certain embodiments, the intracellular domain of the CAR further includes at least one co-stimulatory signaling region. The co-stimulatory signaling region can include the full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.

[0139] In some embodiments, the CAR applicable to the cells provided herein includes a co-stimulatory domain derived from CD28 and a signaling domain including a native or modified ITAM1 of CD3ζ represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 16. In some embodiments, the CAR includes an amino acid sequence having at least about 90% identity to SEQ ID NO: 16. In some embodiments, the CAR includes an amino acid sequence having at least about 95% identity to SEQ ID NO: 16. In some embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 16. In a further embodiment, the CAR including the co-stimulatory domain derived from CD28 and the native or modified ITAM1 of CD3ζ also includes a hinge domain and a transmembrane domain derived from CD28, the scFv can be connected to the transmembrane domain via the hinge, and the CAR includes an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 17. In some embodiments, the CAR includes an amino acid sequence having at least about 90% identity to SEQ ID NO: 17. In some embodiments, the CAR includes an amino acid sequence having at least about 95% identity to SEQ ID NO: 17. In some embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 16 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (153 - amino acid CD28 co - stimulation + CD3ζ ITAM)

[0140]

Table 8

[0141] In various embodiments, the CARs applicable to the cells provided herein include a transmembrane domain derived from NKG2D, a co-stimulatory domain derived from 2B4, and a signaling domain comprising a native or modified CD3ζ represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 18. In some embodiments, the CAR includes an amino acid sequence having at least about 90% identity to SEQ ID NO: 18. In some embodiments, the CAR includes an amino acid sequence having at least about 95% identity to SEQ ID NO: 18. In some embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 18. A CAR comprising a transmembrane domain derived from NKG2D, a co-stimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3ζ may further include a CD8 hinge, and the amino acid sequence of such a structure has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 19. In some embodiments, the CAR includes an amino acid sequence having at least about 90% identity to SEQ ID NO: 19. In some embodiments, the CAR includes an amino acid sequence having at least about 95% identity to SEQ ID NO: 19. In some embodiments, the CAR includes the amino acid sequence of SEQ ID NO: 19.

[0142]

Table 9

[0143] Non-limiting CAR strategies include heterodimeric conditional activation CARs by dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9,587,020); split CARs (homologous recombination of antigen binding, hinge, and internal domains to generate a CAR) (see, e.g., U.S. Patent Application Publication No. 2017 / 0183407); multi-chain CARs that allow non-covalent binding between two transmembrane domains, each connected to an antigen binding domain and a signaling domain (see, e.g., U.S. Patent Application Publication No. 2014 / 0134142); CARs with bispecific antigen binding domains (see, e.g., U.S. Patent 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. Patent No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Invest. 2016;126(8):3036-3052); inducible CARs (see, e.g., U.S. Patent 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 are further included.

[0144] In a further embodiment, iPSCs and their derived effector cells comprising a Fas redirector and optionally a CAR have a CAR inserted into the TCR constant region and an endogenous TCR knockout (TCR KO) results in and optionally places CAR expression under the control of the endogenous TCR promoter. In 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.

[0145] Accordingly, aspects of the invention provide genomically engineered iPSCs and derivative cells obtained from differentiating the genomically engineered iPSCs, wherein the iPSCs and derivative cells encode, optionally together with one or more additional modification modalities provided in Table 1, a Fas redirector and a CAR without adversely affecting the differentiation capacity of the iPSCs and the function of derivative effector cells including derivative T cells and derivative NK cells. Also provided is a master cell bank comprising sorted single cells and expanded clonal engineered iPSCs having at least an exogenous inducible Fas redirector and optionally a CAR, the cell bank providing a platform for further iPSC engineering and a renewable source for manufacturing a ready-to-use engineered homogeneous cell therapy product that can be mass-produced on a large scale in a manner that is compositionally defined, homogeneous, and cost-effective.

[0146] 3. CD16 Knock-in CD16 has been identified as two isoforms, the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate the NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high-affinity CD16", "non-cleavable CD16", or "high-affinity non-cleavable CD16" refers to various CD16 variants. Wild-type CD16 has low affinity and, when NK cells are activated, is subject to an ectodomain shedding proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V (also called F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is an example of a genetically engineered non-cleavable version of CD16. Engineered CD16 variants containing both F176V and S197P have high affinity and are non-cleavable, which is described in more detail in International Publication No. WO 2015 / 148926, the complete disclosure of which is incorporated herein by reference. In addition, chimeric CD16 receptors in which the extracellular domain of CD16 is essentially replaced by at least a portion of the extracellular domain of CD64 can also achieve the desired high affinity and non-cleavability properties of CD16 receptors capable of performing ADCC. In some embodiments, the replaced extracellular domain 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).

[0147] Unlike endogenous CD16, which is cleaved from the cell surface following NK cell activation in primary NK cells, various non-cleavable versions of CD16 in derived NK cells avoid shedding of CD16 and maintain a certain level of expression. In derived NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, which are indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cell-mediated cytotoxicity (ADCC), as well as the binding of bispecific, trispecific, or multispecific engagers. ADCC is the mechanism of NK cell-mediated lysis via the binding of CD16 to antibody-coated target cells. The additional high-affinity properties of hnCD16 introduced into derived NK cells also enable in vitro loading of ADCC antibodies onto NK cells via hnCD16 prior to administration of the cells to a subject in need of cell therapy. As presented herein, in some embodiments, hnCD16 may comprise F176V and S197P, or may comprise a full-length or partial-length extracellular domain derived from CD64, 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 a population of such cells pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further described below.

[0148] Unlike primary NK cells, mature T cells derived from a primary source (i.e., a natural / natural source such as peripheral blood, cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing exogenous non-cleaved CD16 could differentiate into functional derived T lineage cells that not only express exogenous CD16 but also execute functions through the acquired ADCC mechanism without impairing the developmental biology of T cells. This ADCC acquired in the derived T lineage cells can be additionally used as an approach to rescue antigen escape, where tumors relapse with reduced or lost CAR-T target antigen expression, which often occurs in dual targeting and / or CAR-T cell therapy, or the expression of mutant antigens that avoid recognition by the CAR (chimeric antigen receptor). When the derived T lineage cells contain ADCC acquired through the expression of exogenous CD16 (including functional variants and CD16-based CFcRs), and the antibody targets a tumor antigen different from the tumor antigen targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent the recurrence or relapse of the target tumor, which is often seen in CAR-T therapy. Such a strategy of reducing and / or preventing antigen escape while achieving dual targeting is similarly applicable to NK cells expressing one or more CARs.

[0149] Thus, various embodiments of exogenous CD16 introduced into cells include functional CD16 variants and chimeric receptors thereof. 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 and the cleavage region is excluded. In some other embodiments, hnCD16 has a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween when compared to any of SEQ ID NOs: 20, 21, and 22, each of which includes at least a portion of the CD64 extracellular domain, which is an exemplary sequence. In some embodiments, hnCD16 includes an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 20-22. In some embodiments, hnCD16 includes an amino acid sequence that is at least 95% identical to any of SEQ ID NOs: 20-22. In some embodiments, hnCD16 includes the amino acid sequence of SEQ ID NO: 20. In some embodiments, hnCD16 includes the amino acid sequence of SEQ ID NO: 21. In some embodiments, hnCD16 includes the amino acid sequence of SEQ ID NO: 22.

[0150]

Table 10

[0151] Thus, provided herein is exogenous CD16 (``CD16'' in Table 1), among other edits contemplated and described herein, in particular exo"), i.e., iPSCs genetically engineered to contain the high-affinity non-cleavable CD16 receptor (hnCD16), and the genetically engineered iPSCs can differentiate into effector cells containing hnCD16 introduced into the iPSCs. In some embodiments, the induced effector cells comprising a Fas redirector and optionally a CAR and / or exogenous CD16 are NK cells. In some embodiments, the induced effector cells comprising a Fas redirector and optionally a CAR and / or exogenous CD16 are T cells.

[0152] Exogenous hnCD16 expressed within the iPSC or its derivative cells exhibits high affinity not only for ADCC antibodies or fragments thereof, but also for binding to bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domains of CD16 or CD64 of hnCD16. Bispecific, trispecific, or multispecific engagers or binders are further described below in this application. Accordingly, this application provides a derivative effector cell or a cell population thereof, pre-loaded with one or more pre-selected ADCC antibodies via high-affinity binding to the extracellular domain of hnCD16 or a variant thereof expressed on the derivative effector cells, in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection described in more detail below. The hnCD16 comprises the extracellular binding domain of CD64 or CD16 having F176V and S197P. Accordingly, in some embodiments, the derivative NK cells are pre-loaded with antibodies. In some embodiments, the derivative NK cells are used in combination therapy with antibodies. In some embodiments, the antibody or the antibody pre-loaded on the derived effector cells in combination therapy specifically targets CD38. In some embodiments, the antibody or the antibody pre-loaded on the derived effector cells in combination therapy specifically targets an antigen different from CD38. In some embodiments, the anti-CD38 antibody is daratumumab.

[0153] In some other embodiments, the exogenous CD16 expressed in iPSCs or their derivative cells comprises a chimeric Fc receptor (CFcR) based on CD16 or a variant thereof. A chimeric Fc receptor (CFcR) is 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 other than the receptor that provides the extracellular domain. In the exemplification herein, the CD16-based CFcR 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 CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-native stimulatory / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-native signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In some embodiments of the CD16-based CFcR, the chimeric Fc receptor provided comprises a transmembrane domain and a signaling domain both derived from one of IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptides.A specific exemplary embodiment of the CD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, wherein the extracellular domain of the chimeric Fc receptor 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 S197P. Another exemplary embodiment of the CD16-based chimeric Fc receptor comprises the transmembrane domain and the signaling domain of CD3ζ, wherein the extracellular domain of the chimeric Fc receptor 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 S197P.

[0154] The 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 its fragment, or to a bispecific, trispecific, or multispecific engager or binder that recognizes the CD16 or CD64 extracellular binding domain of the chimeric Fc receptor. Upon binding, the stimulatory domain and / or signaling domain of the chimeric receptor enables activation of effector cells and cytokine secretion, as well as killing of tumor cells targeted by the antibody or a bispecific, trispecific, or multispecific engager or binder having a tumor antigen-binding component and an Fc region. Without being limited by theory, through the non-native transmembrane, stimulatory, and / or signaling domains of the CD16-based chimeric Fc receptor, or through binding of an engager to an external domain, the CFcR can contribute to the killing ability of effector cells and increase the likelihood of effector cell proliferation and / or expansion. Antibodies and engagers can bring tumor cells expressing an antigen into proximity with effector cells expressing the CFcR, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, multispecific engagers or binders include, but are not limited to, B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding effector cells expressing the CD16-based CFcR when attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0155] Accordingly, in some embodiments, the present invention provides iPSCs and derivative cells therefrom that include an exogenous Fas redirector and an exogenous CD16 or a variant thereof, and the cells including the derivative T cells and derivative NK cells are useful for overcoming Fas-induced apoptosis or fratricide. In some embodiments, the iPSCs and derivative cells therefrom include a Fas redirector, an exogenous CD16 or a variant thereof, and optionally a CAR. In some embodiments, the iPSCs and derivative cells therefrom include one or more additional genome edits described herein without adversely affecting the differentiation ability of the iPSCs and the function of the derivative effector cells including the derivative T cells and derivative NK cells. Also provided is a master cell bank including sorted single cells and expanded clonal engineered iPSCs having at least an exogenous inducible Fas redirector and an exogenous CD16 or a variant thereof, the cell bank providing a platform for further iPSC engineering and a renewable source for manufacturing ready-to-use engineered homogeneous cell therapy products that can be mass-produced on a large scale in a manner that is well-defined, homogeneous, and cost-effective.

[0156] 4. CD38 Knockout The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies, both lymphoid and myeloid, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated depletion of cancer cells typically 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 concert with therapeutic antibodies can also include antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0157] In addition to being highly expressed in malignant cells, CD38 is also expressed in plasma cells as well as NK cells, activated T cells, and B cells. During hematopoiesis, CD38 is expressed in CD34 + stem cells, and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, as well as at the final stage of maturation that continues to the plasma cell stage. As a type II transmembrane glycoprotein, CD38 functions in cells both as a receptor and as a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 catalyzes the hydrolysis of the reaction from synthesis and NAD + to ADP-ribose, thereby generating the second messengers CADPR and NAADP that stimulate the release of calcium from the endoplasmic reticulum and lysosomes, which is important for calcium-dependent cell adhesion processes. CD38 recognizes CD31 as a receptor and controls cytokine release and cytotoxicity of activated NK cells. CD38 has also been reported to associate with cell surface proteins in lipid rafts, control cytoplasmic Ca 2+ flux, and mediate signal transduction in lymphoid and myeloid cells.

[0158] However, in the treatment of malignancies, when T cells transduced with CD38 antigen-binding receptors are used systemically, CD34 + hematopoietic progenitor cells, monocytes, NK cells, T cells, and the CD38 + fraction of B cells are lysed, and the recipient's immune effector cell function is impaired, resulting in incomplete therapeutic efficacy and reduced or eliminated potency. In addition, in multiple myeloma patients treated with daratumumab, a CD38-specific antibody, a reduction in NK cells was observed in both the bone marrow and peripheral blood, but other immune cell types such as T cells and B cells were not affected despite the expression of CD38 (Casneuf et al., Blood Advances. 2017;1(23):2105-2114).

[0159] Although not bound by theory, the present application maximizes the potential of CD38-targeted cancer therapy by knocking out CD38 in effector cells, thereby providing a strategy to overcome the depletion or reduction of effector cells induced by CD38-specific antibodies and / or CD38 antigen-binding domains through fratricide. Additionally, since CD38 is upregulated in activated lymphocytes such as T cells or B cells, the use of CD38-specific antibodies such as daratumumab in recipients of allogeneic effector cells suppresses the activation of these recipient lymphocytes, thereby reducing and / or preventing host allogeneic rejection of these effector cells, thereby increasing the survival rate and persistence of effector cells. Thus, CD38-specific antibodies, secreted CD38-specific engagers, or CD38-CAR (chimeric antigen receptor) against the activation of recipient T, Treg, NK, and / or B cells can be used as an alternative to lymphocyte depletion using chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine) prior to adoptive cell transfer. Additionally, in the presence of an anti-CD38 antibody or CD38 inhibitor, hnCD16a + / CD38 - Using effector cells to target CD38 + When targeting T and pbNK cells, CD38 + Depletion of alloreactive cells increases NAD + (nicotinamide adenine dinucleotide, a substrate of CD38) availability and reduces NAD + consumption-related cell death, which, among other advantages, boosts effector cell responses in the immunosuppressive tumor microenvironment and supports cellular rejuvenation in aging, degenerative, or inflammatory diseases.

[0160] Thus, the strategies provided herein also include creating iPSC lines that include a Fas redirector and CD38 knockout, and optionally one or more of CAR and exogenous CD16 or variants thereof, and through directed differentiation of the engineered iPSC lines, a Fas redirector and CD38 null (CD38 - / -) and obtaining derived effector cells optionally comprising one or more of CAR and exogenous CD16 or variants thereof. In one embodiment, the CD38 knockout in the iPSC line is a biallelic knockout.

[0161] As disclosed herein, in some embodiments, the provided iPSC line comprising a Fas redirector and CD38 - / - , and optionally one or more of CAR and exogenous CD16, can be directed to differentiate into mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell precursors, NK cell precursors, myeloid cells, neutrophil precursors, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutrophils, dendritic cells, and functional derived hematopoietic cells including but not limited to macrophages. In some embodiments, when an anti-CD38 antibody is used to induce CD16-mediated enhanced ADCC or an anti-CD38 CAR is used for target cell killing, the Fas redirector and CD38 - / -iPSCs that include, optionally, one or more of a CAR and exogenous CD16 and / or its derivative 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 the iPSCs and their effector cells in the presence of and / or after exposure to such therapeutic agents. In some embodiments, the effector cells have increased persistence and / or survival in vivo in the presence of and / or after exposure to such anti-CD38 therapeutic agents such as anti-CD38 antibodies. In some embodiments, the anti-CD38 antibody is daratumumab, isatuximab, or MOR202. Additionally, since CD38 is upregulated on activated lymphocytes such as T cells or B cells, CD38-specific antibodies can be used for lymphocyte depletion, thereby eliminating their activated lymphocytes without fratricide in recipients of allogeneic effector cell therapy, overcoming allograft rejection, and increasing the survival and persistence of CD38-negative effector cells.

[0162] In some embodiments, the induced effector cells that include a Fas redirector, CD38 - / - and, optionally, one or more of a CAR and exogenous CD16 are NK cells derived from iPSCs. In some embodiments, the effector cells that include a Fas redirector, CD38 - / - and, optionally, one or more of a CAR and exogenous CD16 are T cells derived from iPSCs. In some embodiments, the iPSCs that include a Fas redirector and CD38 - / - and, optionally, one or more of a CAR and exogenous CD16 and / or derivative cells include one or more additional genome edits provided in Table 1 without adversely affecting the differentiation ability of the iPSCs and the function of the derivative effector cells including the derivative T cells and derivative NK cells. Also provided is an exogenous inducible Fas redirector and CD38 - / -A master cell bank comprising sorted single cells and expanded clonally engineered iPSCs having at least this, which cell bank provides a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf engineered homogeneous cell therapy products that can be mass-produced in large quantities in a manner that is clearly defined, uniform, and cost-effective.

[0163] 5. Exogenously introduced cytokines and / or cytokine signaling By avoiding systemic high-dose administration of clinically significant cytokines, the risk of dose-limiting toxicity from such an act 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 containing one or more partial-length or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective corresponding receptors is introduced into the cells to enable cytokine signaling with or without the expression of the cytokine itself, thereby maintaining or improving cell growth, proliferation, amplification, and / or effector function with a reduced risk of cytokine toxicity. In some embodiments, the introduced cytokines and / or their respective native or modified receptors (signaling complexes) for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or temporary. In some embodiments, transient / temporary expression of cell surface cytokines / cytokine receptors is via an expression construct carried by a retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA including mRNA.

[0164] Provided herein are various construct designs for introducing into cells protein complexes for the signaling of one, two, or more cytokines, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In embodiments where the cytokine 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 some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics trans-presentation of IL15 without eliminating cis-presentation of IL15. In other embodiments, IL15Rα is fused to IL15 at the C-terminus via a linker, which not only mimics trans-presentation without eliminating cis-presentation of IL15 but also ensures that IL15 is membrane-bound. In other embodiments, IL15Rα with a truncated intracellular domain is fused to IL15 at the C-terminus via a linker, mimics trans-presentation of IL15, maintains membrane-bound IL15, and in addition, eliminates cis-presentation and / or other potential signaling pathways mediated by the normal IL15R via its intracellular domain.

[0165] In various embodiments, such cleaved constructs comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 23. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 23. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 23. In some embodiments, IL15 / IL15Rα comprises the amino acid sequence of SEQ ID NO: 23. In one embodiment of the cleaved IL15 / IL15Rα, the construct does not include the last 4 amino acid residues (KSRQ) of SEQ ID NO: 23 and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 24. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 24. In some embodiments, IL15 / IL15Rα comprises the amino acid sequence of SEQ ID NO: 24.

[0166]

Table 11

[0167] In yet other embodiments, the cytoplasmic domain of IL15Rα can be omitted without adversely affecting the autonomous properties of effector cells armed with IL15. In other embodiments, substantially all of IL15Rα is removed, except for a Sushi domain that is fused to IL15 at one end and a transmembrane domain at the other end (mb-Sushi), optionally with a linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of any membrane-bound protein. When only the desired trans-presentation of IL15 is retained, unwanted signaling via IL15Rα, including cis-presentation, is eliminated. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 25. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 25. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises the amino acid sequence of SEQ ID NO: 25.

[0168]

Table 12

[0169] In other embodiments, native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to enable constitutive signaling and maintain IL15 membrane binding and trans-presentation. In other embodiments, native or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling of the cytokine and membrane-bound trans-presentation. The common receptor γC is also referred to as the common gamma chain or CD132 and is also known as interleukin-2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukins, including but not limited to the IL2, IL4, IL7, IL9, IL15, and IL21 receptors. In other embodiments, engineered IL15Rβ that forms homodimers in the absence of IL15 is useful for generating constitutive signaling of the cytokine.

[0170] In various other embodiments, the cytokine signaling complex comprises an IL7 receptor fusion (IL7RF) comprising the full-length or partial-length of IL7 and the full-length or partial-length of the IL7 receptor (IL7R). The transmembrane (TM) domain can be native to the IL7 receptor or can be modified or replaced with the transmembrane domain of any other membrane-bound protein. In one embodiment, native (or wild-type) or modified IL7R is fused to IL7 at the C-terminus via a linker to enable constitutive signaling and maintain membrane-bound IL7. In some embodiments, such constructs comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 26, and the transmembrane domain, signal peptide, and linker are flexible and vary in length and / or sequence. In some embodiments, the sequence identity is at least 80%. In some embodiments, the sequence identity is at least 90%. In some embodiments, the sequence identity is at least 95%. In some embodiments, the sequence identity is 100%.

[0171]

Table 13

[0172] In one embodiment, natural or modified common receptor γC is fused to IL7 at the C-terminus via a linker for the constitutive and membrane-bound cytokine signaling complex. Common receptor γC, also called common gamma chain or CD132, is also known as interleukin-2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukins, including but not limited to the IL2, IL4, IL7, IL9, and IL21 receptors. Furthermore, engineered IL7R that forms homodimers in the absence of IL7 is also useful for generating constitutive signaling of the cytokine.

[0173] Those skilled in the art will understand that the signal peptides and linker sequences described above are exemplary and in no way limit those variations suitable for use as signal peptides or linkers. There are many suitable signal peptides or linker sequences known and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence can be replaced with another sequence without changing the activity of the functional peptide directed by the signal peptide or linked by the linker. Thus, iPSCs and derivative cells derived therefrom can autonomously maintain or improve cell proliferation, expansion, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo.

[0174] In iPSCs and derivative cells therefrom that contain both a CAR and an exogenous cytokine signaling complex (the "IL" in Table 1), the CAR and the IL can be expressed in separate constructs or co-expressed in a bicistronic construct that contains both the CAR and the IL. In some embodiments, the cytokine signaling complex can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence. Thus, the cytokine signaling complex (e.g., the IL7 signaling complex) and the CAR can be within a single open reading frame (ORF). In one embodiment, the cytokine signaling complex is contained 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 enables the expressed CAR and IL to dissociate, and then the dissociated IL can be presented on the cell surface with its transmembrane domain fixed to the cell membrane. The bicistronic construct design of CAR-2A-IL or IL-2A-CAR enables coordinated expression of the CAR and the IL under the same control mechanism that can be selected for incorporation, such as an inducible promoter or a promoter with temporal or spatial specificity for expression of a single ORF, in terms of both timing and quantity. Self-cleaving peptides are found in members of the Picornaviridae family, including the Aphthovirus genus such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine 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)), as well as the Cardiovirus genus such as Theiler's murine encephalomyelitis virus and encephalomyocarditis virus.The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV may also be referred to as "F2A", "E2A", "P2A", and "T2A", respectively.

[0175] The bicistronic CAR-2A-IL or IL-2A-CAR disclosed herein also contemplates the 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 the expression of one or more of IL2, IL4, IL7, IL9, IL15, IL21, and their cytokine signaling complexes.

[0176] Accordingly, in various embodiments, the cytokine signaling complexes provided herein may be introduced into iPSCs using one or more of the above construct designs, or may be introduced into their derivative cells upon iPSC differentiation. In addition to iPSCs, cloned iPSCs, cloned iPSC lines, or iPSC-derived effector cells comprising at least one engineered modality disclosed herein are provided. Thus, in some embodiments, the invention includes a Fas redirector, an exogenous cytokine signaling complex (the "IL" of Table 1) described herein, and optionally, a CAR, exogenous CD16 or a variant thereof, and CD38 - / -Provided are iPSCs and derivative cells therefrom that include one or more of the following, wherein the Fas redirector redirects Fas signaling upon binding to a Fas agonist, thereby providing improved apoptosis resistance and / or depletion resistance to cells including derivative T cells and derivative NK cells. In some embodiments, the iPSCs and their derivative cells include one or more additional genome edits provided in Table 1 without adversely affecting the differentiation ability of the iPSCs and the function of the derivative effector cells including derivative T cells and derivative NK cells. Also provided is a master cell bank including sorted single cells and expanded clonal engineered iPSCs having at least a Fas redirector and an exogenously introduced cytokine signaling complex as described in this section, the cell bank providing a platform for further iPSC engineering and a renewable source for manufacturing ready-to-use engineered homogeneous cell therapy products that can be mass-produced in a large scale in a manner that is clearly defined, homogeneous, and cost-effective.

[0177] 6. Deficiency of HLA-I and HLA-II To avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins need to match for the histocompatibility of allogeneic recipients. Provided herein are iPSC cell lines in which the expression of one or both of the HLA class I (“HLA-I”) and HLA class II (“HLA-II”) proteins is eliminated or substantially reduced. HLA class I deficiency can be achieved by a functional deletion of any region of the HLA class I locus (chromosome 6p21), or a deletion or reduced expression level of HLA class I-related 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 the cell surface expression of all HLA class I heterodimers. B2M-negative cells are HLA-I deficient.

[0178] HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II related genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator 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. However, the lack of HLA class I expression increases susceptibility to lysis by NK cells. Thus, the present application provides iPSCs and derivative cells therefrom that include HLA-I and / or HLA-II deficiencies, for example, by lack of B2M and / or CIITA expression, and the derived effector cells obtained enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoiding recognition and killing by host (allogeneic) T cells.

[0179] Furthermore, the lack of HLA class I expression triggers lysis by host NK cells. Thus, in addition to the aforementioned approach of CD38 conditioning to remove activated CD38-expressing host NK cells, HLA-G can be optionally knocked-in to avoid killing of HLA-I-deficient effector cells derived from iPSCs recognized and engineered by NK cells in order to overcome this "self-loss" response. In one embodiment, the provided HLA-I-deficient iPSCs and their derivative cells further comprise an HLA-G knock-in. Alternatively or in addition, the provided HLA-I-deficient iPSCs and their derivative cells further comprise one or both of a CD58 knockout and a CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and promote cell migration, including immune cells. It has previously been shown that disruption of CD58 and / or CD54 effectively reduces the sensitivity of HLA-I-deficient iPSC-derived effector cells to allogeneic NK cell killing. CD58 knockout results in higher efficiency in reducing allogeneic NK cell activation than CD54 knockout, and double knockout of both CD58 and CD54 has been shown to result in the most enhanced reduction of NK cell activation. In some observations, the CD58 and CD54 double knockout is even more effective than HLA-G overexpression on HLA-I-deficient cells in overcoming the "self-loss" effect.

[0180] Accordingly, in some embodiments, the present invention provides a strategy to enhance the persistence and / or survival of effector cells by reducing or preventing allograft rejection, optionally by additional HLA-I protein modification, by causing HLA-I deficiency and / or HLA-II deficiency without adversely affecting the differentiation potential of iPSCs and the function of the derived effector cells (including derived T cells and derived NK cells).

[0181] In some embodiments, effector cells have increased in vivo persistence and / or survival rate in the presence of and / or after exposure to therapeutic agents. Thus, in some embodiments, iPSCs and their derived cells containing a Fas redirector are HLA-I deficient (e.g., B2M negative or B2M - / - ), and optionally further comprise one or more of a CAR, exogenous CD16 or a variant thereof, CD38 - / - , and an exogenously introduced cytokine signaling complex (IL). In some embodiments, iPSCs and their derived cells containing a Fas redirector are HLA-I deficient and HLA-II deficient (e.g., B2M - / - and CIITA negative or CIITA - / - ), and optionally further comprise one or more of a CAR, exogenous CD16 or a variant thereof, CD38 - / - , and IL. In some embodiments, iPSCs and their derived cells containing a Fas redirector are HLA-I and / or HLA-II deficient, and further comprise an exogenous polynucleotide encoding HLA-G or HLA-E, and optionally one or more of a CAR, exogenous CD16 or a variant thereof, CD38 - / - , and IL. In some embodiments, iPSCs and their derived cells containing a Fas redirector are HLA-I and / or HLA-II deficient, are CD58 negative, and optionally further comprise one or more of a CAR, exogenous CD16 or a variant thereof, CD38 - / - , and IL. In some embodiments, iPSCs and their derived cells containing a Fas redirector are HLA-I and / or HLA-II deficient, are CD54 negative, and optionally further comprise one or more of a CAR, exogenous CD16 or a variant thereof, CD38 - / - , and IL.

[0182] In some embodiments, iPSCs and their derived cells containing a Fas redirector are HLA-I and / or HLA-II deficient, are both CD58 negative and CD54 negative, and optionally further comprise one or more of a CAR, exogenous CD16 or a variant thereof, CD38 - / -and further comprises one or more of IL. In some embodiments, a Fas redirector, and HLA-I and / or HLA-II deficiency, and optionally a CAR, exogenous CD16 or a variant thereof, CD38 - / - Effector cells comprising one or more of and IL are NK cells derived from iPSCs. In some embodiments, a Fas redirector, and HLA-I and / or HLA-II deficiency, and optionally a CAR, exogenous CD16 or a variant thereof, CD38 - / - Effector cells comprising one or more of and IL are T cells derived from iPSCs.

[0183] In some embodiments, iPSCs and their derivative cells have a Fas redirector and HLA deficiency or modification (e.g., HLA-I knockout with or without HLA-II knockout, and optionally HLA-E or HLA-G knock-in or overexpression, CD54 knockout and CD58 knockout, one or more of which are included in HLA-I and / or HLA-II modifications represented by "HLA" in Table 1), and optionally a CAR, exogenous CD16 or a variant thereof, CD38 - / - and one or more of IL, and the cells further comprise one or more additional genome edits provided in Table 1 without adversely affecting the differentiation ability of the iPSCs and the functions of the derived effector cells including the derived T cells and derived NK cells.

[0184] Also provided is a master cell bank comprising sorted single cells and expanded clonal engineered iPSCs having at least a Fas redirector and HLA deficiency as described in this section, the cell bank providing a platform for further iPSC engineering and a renewable source for manufacturing ready-to-use engineered homogeneous cell therapy products that can be mass-produced on a large scale in a clear, uniform, and cost-effective manner.

[0185] 7. Genetically engineered iPSC lines and derived cells provided herein In light of the above, the present application provides iPSCs, cloned iPS cells, iPS cell line cells, or derived cells therefrom, each cell comprising at least one Fas redirector (''Fas'' in Table 1) described herein. In some embodiments, the cell comprises (a) an extracellular Fas binding domain comprising an extracellular domain (ECD) of the Fas receptor (FAS) or a partial or complete peptide of a variant or allele thereof, and a cytoplasmic signaling domain comprising a partial or complete peptide of an intracellular domain (ICD) of one or more co-stimulatory molecules, an exogenous polynucleotide encoding a signal redirecting receptor, the signal redirecting receptor being a Fas redirector that redirects Fas signaling upon binding of a Fas agonist, thereby providing improved apoptosis resistance and / or exhaustion resistance to the cell or derived cells. In some embodiments, the derived cells are hematopoietic lineage cells, including but not limited to mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutrophils, dendritic cells, and macrophages. In some embodiments, the functional derived hematopoietic cells include effector cells having one or more functional traits not present in the corresponding primary T cells, NK cells, NKT cells, and / or B cells.

[0186] In some embodiments, the derived cells include NK or T lineage cells. As shown in Table 1, the Fas redirector and, optionally, CAR, exogenous CD16, CD38 - / - ,, iPSC-derived NK or T lineage cells comprising one or more of cytokine signaling complexes, HLA deficiency, and any other modality are useful for overcoming or reducing Fas-induced apoptosis or fratricide and / or tumor microenvironment suppression associated with solid tumors. As described above, derived CAR-T cells expressing hnCD16 have acquired ADCC and provide an additional mechanism for killing tumors in addition to CAR targeting. In some embodiments, the derived cells include NK lineage cells. Fas redirectors, optionally CAR, exogenous CD16, CD38, as shown in Table 1 - / - 、 iPSC-derived NK cells comprising cytokine signaling complexes, HLA deficiency, and one or more of any other modality have enhanced cytotoxicity and are effective in mobilizing bystander cells including T cells to infiltrate and kill tumor cells.

[0187] Fas redirectors, optionally CAR, exogenous CD16, CD38 - / - 、 In some embodiments of effector cells derived from engineered iPSCs as shown in Table 1, comprising cytokine signaling complexes, HLA deficiency, and one or more of any other modality, the cells are intact in HLA-II and still withstand allogeneic rejection by activated recipient T cells, activated recipient B cells, and activated recipient NK cells. In some embodiments, the iPSCs and their derived effector cells are intact in HLA-II and have synergistically increased persistence and / or survival rates in the presence of activated recipient T cells, recipient B cells, and recipient NK cells. In some embodiments, the iPSCs and their derived effector cells are useful for overcoming or reducing tumor microenvironment suppression associated with solid tumors. When anti-CD38 antibodies are used in combination therapy with the above-derived effector cells, the cells have synergistically increased persistence, survival rate, and effector function.

[0188] Also, a Fas redirector and, optionally, a CAR, exogenous CD16, CD38 - / - 、 An iPSC comprising one or more of a cytokine signaling complex (``IL'' in Table 1), HLA deficiency, and any other modality shown in Table 1, which can optionally use HLA-E or HLA-G knock-in and knockout of one or both of CD54 and CD58 to produce functionally derived hematopoietic cells and be directionally differentiated to overcome alloreactive NK cells is also provided.

[0189] Accordingly, the present application provides iPSCs and their functionally derived hematopoietic cells comprising any one of the following genotypes in Table 1. In some embodiments, the iPSCs and their functionally derived hematopoietic cells have a genotype comprising at least a Fas redirector (``Fas'' in Table 1). Additionally, ``HLA'' in Table 1 represents HLA-I and / or HLA-II modifications with or without HLA-II knockout and HLA-I knockout, and optionally one or more of HLA-E or HLA-G knock-in or overexpression, CD54 knockout, and CD58 knockout. In some embodiments, the iPSCs and their functionally derived hematopoietic cells have a genotype comprising at least a Fas redirector and, optionally, a checkpoint inhibitor (``CI + ''), an engager (``En + ''), or an antibody (``Ab + ''). Checkpoint inhibitors, engagers, and antibodies are discussed in more detail below.

[0190]

Table 14-1

[0191]

Table 14-2

[0192]

Table 14-3

[0193]

Table 14-4

[0194] 8. Additional Modifications In some embodiments, the genetically modified iPSCs and their derivative cells comprise the genotypes listed in Table 1, and the cells comprise one or more of the following genetic modification modalities: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or survival rate of iPSCs or their derivative cells. In some embodiments, iPSCs and their derivative effector cells comprising any one of the genotypes of Table 1 have at least one deletion or disruption of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and any gene within the chromosomal region 6p21; or HLA-E, HLA-G, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, TCR, Fc receptor, antibody, engager, and introduction of at least one of surface trigger receptors for coupling with bispecific, multispecific, or universal engagers may be further included.

[0195] 8.1 Engager An engager is a fusion protein consisting of two or more single-chain variable fragments (scFvs) of different antibodies or fragments thereof, or other functional variants, and having at least one scFv that binds to an effector cell surface molecule or surface trigger receptor, and at least another one that binds to a target cell via a target cell-specific surface molecule. The engager 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. 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.

[0196] In some embodiments, the engager is used in combination with a population of effector cells described herein by simultaneous or sequential administration, and the effector cells include surface molecules or surface trigger receptors recognized by the engager. In some other embodiments, the engager (the "En + " in Table 1) is a bispecific antibody expressed by derived effector cells through genetic engineering of iPSCs and directed differentiation of the engineered iPSCs as described herein. Exemplary effector cell surface molecules or surface trigger receptors that can be used for bispecific or multispecific engager recognition or their coupling include, but are not limited to, CD3, CD5, CD16, CD28, CD32, CD33, CD64, CD89, NKG2C, NKG2D, and the chimeric Fc receptors disclosed herein.

[0197] 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, CD52, 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, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROR1, and VEGF-R2. In various embodiments, the bispecific engager is a bispecific antibody. Exemplary bispecific antibodies include, but are not limited to, CD3-CD19 (specific for CD3 and CD19), CD16-CD30, CD64-CD30, CD16-BCMA, CD64-BCMA, and CD3-CD33.

[0198] 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 that promote cell proliferation (in some publications, called TriKE, or trispecific killer engager). Exemplary TriKEs include, but are not limited to, CD16-IL15-EPCAM, CD64-IL15-EPCAM, CD16-IL15-CD33, CD64-IL15-CD33, and NKG2C-IL15-CD33. In various embodiments, the IL15 in TriKE can also be derived from other cytokines including, but not limited to, IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21.

[0199] In some embodiments, surface trigger receptors for bispecific or multispecific engagers can sometimes be endogenous to effector cells, depending on the cell type. In some other embodiments, the methods and compositions provided herein are used to further engineer, for example, iPSCs comprising the genotypes listed in Table 1, to induce differentiation of the iPSCs into T cells, NK cells, or other effector cells that have the same genotype as the source iPSCs and surface trigger receptors, and one or more exogenous surface trigger receptors can be introduced into the effector cells.

[0200] 8.2 Antibodies for Immunotherapy In some embodiments, in addition to the genome-engineered effector cells provided herein, additional therapeutic agents comprising antibodies or antibody fragments that target antigens associated with a condition, disease, or indicator can be used in combination therapy with these effector cells. In some embodiments, the 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 (the "Ab" in Table 1) +") can be expressed by effector cells by genetically engineering iPSCs using an exogenous polynucleotide sequence encoding an antibody or a fragment thereof, and inducing differentiation of the engineered iPSCs. In some embodiments, the effector cells additionally express an exogenous CD16 variant, and the cytotoxicity of the effector cells is enhanced by an antibody via ADCC. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, as an additional therapeutic agent for the administered iPSC-derived effector cells, antibodies suitable for combination therapy include anti-CD20 (rituximab, belimumab, ofatumumab, ublituximab, ocrelizumab, obinutuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (cetuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars, but are not limited thereto.

[0201] In some embodiments, a Fas redirector and, optionally, a CAR, exogenous CD16, CD38 - / -, iPSC-derived effector cells containing iPSC-derived NK or T cells, including one or more of cytokine signal transduction complexes and HLA deficiencies, are used in combination therapies that include the effector cells and anti-CD38 antibodies such as daratumumab, isatuximab, and MOR202. In some embodiments of combinations useful for treating liquid or solid tumors, the combination includes a Fas redirector, optionally a CAR, exogenous CD16, CD38 - / - , iPSC-derived NK or T cells containing one or more of cytokine signal transduction complexes and HLA deficiencies, and daratumumab. In some further embodiments, the iPSC-derived NK cells included in the combination with one of the anti-CD38 antibodies are Fas redirectors, CARs, exogenous CD16, CD38 - / - , including HLA deficiency and IL15 or a cytokine signal transduction complex, and IL15 or its signal transduction complex is co-expressed with or separately expressed from the CAR. In some further embodiments, the iPSC-derived T cells included in the combination with one of the anti-CD38 antibodies are Fas redirectors, CARs, exogenous CD16, CD38 - / - , including HLA deficiency and IL7 or its cytokine signal transduction complex. In some specific embodiments, IL7 or its signal transduction complex is co-expressed with or separately expressed from the CAR.

[0202] In some embodiments, iPSC-derived effector cells containing iPSC-derived NK or T cells include a Fas redirector and, optionally, a CAR, exogenous CD16, CD38 - / - , one or more of cytokine signal transduction complexes, HLA deficiencies, and Ab + where "Ab + " refers to an exogenous polynucleotide sequence encoding an antibody or a fragment thereof, and the antibody or a fragment thereof is expressed by the effector cells. In various embodiments, Ab + includes any one or more of the above therapeutic antibodies or functional fragments thereof.

[0203] 8.3 Checkpoint Inhibitors Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate the immune response when not inhibited. It has become clear that tumors utilize specific immune checkpoint pathways as a major mechanism of immune tolerance to T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can block inhibitory checkpoints and restore immune system function by reducing the expression of checkpoint genes or gene products or by decreasing the activity of checkpoint molecules. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has changed the landscape of oncology, and these agents have produced long-term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a major concern.

[0204] Accordingly, in some embodiments, the present application provides a therapeutic approach for overcoming CI resistance by including genomically engineered functional derivative cells as provided herein in combination therapy with a CI. In some embodiments, the checkpoint inhibitor is used in combination with a population of effector cells described herein by its simultaneous or sequential administration to a subject. Some embodiments of the combination therapy with the effector cells described herein include at least one checkpoint inhibitor for targeting at least one checkpoint molecule, and the derivative cells have the genotypes listed in Table 1. In other embodiments, the checkpoint inhibitor (the "CI" in Table 1) is expressed by effector cells by genetically engineering iPSCs using an exogenous polynucleotide sequence encoding the checkpoint inhibitor or a fragment or variant thereof and inducing the differentiation of the engineered iPSCs. + ” is expressed by effector cells by genetically engineering iPSCs using an exogenous polynucleotide sequence encoding the checkpoint inhibitor or a fragment or variant thereof and inducing the differentiation of the engineered iPSCs.

[0205] In some embodiments, the exogenous polynucleotide sequence encoding a checkpoint inhibitor or a fragment thereof is co-expressed with the CAR, either within a separate construct or within a bicistronic construct. In some further embodiments, the sequence encoding the checkpoint inhibitor or a fragment thereof can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence exemplified, for example, as CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences 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 the derived effector cells that can infiltrate the tumor microenvironment (TME), it neutralizes inhibitory checkpoint molecules when binding to the TME, activates modalities such as the CAR, or activates the effector cells by activating receptors. In one embodiment of the combination therapy, the derived effector cells comprising the genotypes listed in Table 1 are NK lineage cells. In another embodiment of the combination therapy, the derived effector cells comprising the genotypes listed in Table 1 are T lineage cells.

[0206] Checkpoint inhibitors suitable for combination therapy with derived effector cells and / or expression by derived effector cells provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A 2AAntagonists of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2) are included, but not limited thereto.

[0207] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab′)2, F(ab′)3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which can be more cost-effective to manufacture, easier to use, or more sensitive than the whole antibody. In some embodiments, the checkpoint inhibitor includes at least one of atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR), IPH43 (anti-MICA), IPH33 (anti-TLR3), lirilumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0208] In some embodiments, since many miRNAs are found as regulators that control the expression of immune checkpoints, antagonists that inhibit any of the checkpoint molecules described above are miRNA-based (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint antagonist miRNAs include, but are not limited to, miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197, miR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c.

[0209] In some embodiments, the checkpoint inhibitor (the "CI" in Table 1) + is co-expressed with the CAR and the following checkpoint molecules: PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AInhibit at least one of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR. In some embodiments, the checkpoint inhibitor co-expressed with CAR in derived cells having the genotypes listed in Table 1 is selected from the group consisting of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and their humanized, or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is atezolizumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is nivolumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is pembrolizumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar.

[0210] In some other embodiments of the combination therapies provided herein that include the derived effector cells and at least one antibody that inhibits checkpoint molecules, the antibody is not produced by or within the derived cells and is further administered before, at the same time as, or after the administration of the derived cells as provided herein. In some embodiments, the administration of one, two, three, or more checkpoint inhibitors in combination therapy with the provided derived NK lineage cells or derived T lineage cells is simultaneous or sequential. In one embodiment of the combination treatment, the checkpoint inhibitors included in the treatment are atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments of the combination therapy, the checkpoint inhibitor included in the treatment is atezolizumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof. In some embodiments of the combination therapy, 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 the combination therapy, the checkpoint inhibitor included in the treatment is pembrolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0211] In some embodiments, the iPSC-derived effector cells, including iPSC-derived NK or T cells, include a Fas redirector and, optionally, a CAR, exogenous CD16, CD38 - / - , a cytokine signaling complex, HLA deficiency, and CI + , where "CI + " refers to an exogenous polynucleotide sequence encoding a checkpoint inhibitor or a fragment thereof, and the checkpoint inhibitor or a fragment thereof is expressed by the effector cells. In various embodiments, CI +It contains any one or more of the above therapeutic antibodies or functional fragments thereof.

[0212] II. Methods for Targeted Genome Editing at Selected Loci of iPSCs Genome editing, genomic editing, or gene editing, which are used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and / or substituted in the genome of a target cell. Targeted genome editing (interchangeable with "targeted genome editing" or "targeted gene editing") enables insertion, deletion, and substitution at a preselected site within the genome. When the endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down due to the sequence deletion. Therefore, targeted editing can also be used to accurately disrupt endogenous gene expression. The term "targeted integration" as used herein refers to a process that includes the insertion of one or more exogenous sequences with or without deletion of the endogenous sequence at the insertion site. In contrast, randomly integrated genes are susceptible to the effects of position effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomeric regions are particularly prone to transgene silencing. Newly integrated genes can affect the surrounding endogenous genes and chromatin, change cell behavior, or promote cell transformation. Therefore, inserting exogenous DNA into a preselected locus such as a safe harbor locus or genomic safe harbor (GSH) is important for safe, efficient, copy number control, and reliable gene response control.

[0213] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences adjacent to the exogenous polynucleotide to be inserted through the enzymatic machinery of the host cell.

[0214] Alternatively, by specifically introducing double strand breaks (DSBs) with a specific rare-cut endonuclease, targeted editing can be achieved at a higher frequency. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non-homologous end joining (NHEJ) that occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often causes random insertions or deletions (indels) of a few endogenous nucleotides. In contrast, when a donor vector containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in "targeted integration". 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 a single editing step.

[0215] One or more transgenes can be inserted at selected positions in a gene locus of interest (GOI), achieving simultaneous gene knockout. Gene loci suitable for simultaneous knock-in and knockout (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, the TCRα or TCRβ 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 position-selective insertion enables the transgene to be expressed either under the endogenous promoter of the site or under an exogenous promoter contained in the construct. When two or more transgenes are inserted at selected positions in the CD38 gene locus, a linker sequence, such as a 2A linker or an IRES, is placed between any two transgenes. The 2A linker encodes a self-cleaving peptide (referred to as "F2A", "E2A", "P2A", and "T2A", respectively) derived from, for example, FMDV, ERAV, PTV-I, or TaV, allowing separate proteins to be produced from a single translation. In some embodiments, the construct contains insulators to reduce the risk of transgene and / or exogenous promoter silencing. In various embodiments, the exogenous promoter can be CAG, or other constitutive, inducible, temporally specific, tissue-specific, or cell-type specific promoters including, but not limited to, CMV, EF1α, PGK, and UBC.

[0216] 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 Regular Interspaced Short Palindromic Repeats) systems. Furthermore, the DICE (Dual Integrase Cassette Exchange) system that utilizes phiC31 and Bxb1 integrases is also a promising tool for targeted integration.

[0217] ZFN is a targeted nuclease that contains a nuclease fused to a zinc finger DNA binding domain. The "zinc finger DNA binding domain" or "ZFBD" means 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 a zinc finger binding domain whose structure is stabilized by coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A "designed" zinc finger domain is a domain that does not occur in nature, and its design / construction results primarily from the application of rational criteria, such as substitution rules and computerized algorithms for processing information in databases storing information on existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261. Also see International Publication Nos. 98 / 53058, 98 / 53059, 98 / 53060, 02 / 016536, and 03 / 016496 (the complete disclosures of which are incorporated herein by reference). A "selected" zinc finger domain is a domain that does not occur in nature and whose production results primarily from empirical processes such as phage display, interaction trap, or hybrid selection. ZFNs are described in detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is a fusion of the FokI nuclease and a zinc finger DNA binding domain.

[0218] TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. The "transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" means the polypeptide domain of the TAL effector protein that causes binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA binding domain depends on the effector variable number of imperfect 34-amino acid repeats containing 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 the FokI nuclease to a TAL effector DNA binding domain.

[0219] Another example of a targeted nuclease used in the methods of the subject invention is a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA binding domain having specificity for a DNA sequence of interest, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, and the like.

[0220] 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.

[0221] Other non-limiting examples of targeted nucleases include naturally occurring nucleases 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.

[0222] As an example using Cas9, CRISPR / Cas9 requires two main components, namely, (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to a target DNA sequence containing a PAM and a PAM-proximal seeding region. Combining the crRNA and tracrRNA to form a chimeric guide RNA (gRNA) can guide Cas9 to target a selected sequence. These two components can then be delivered into mammalian cells via transfection or transduction.

[0223] Insertion via DICE provides for unidirectional integration of exogenous DNA, which is tightly restricted to the small attB and attP recognition sites of each enzyme itself, using a pair of recombinases such as phiC31 and Bxb1, for example. Since these targeted att sites do not naturally occur in the mammalian genome, they need to be first introduced into the genome at the desired integration site. See, for example, U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0224] 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 for a desired integration site, and the method of targeted integration comprises introducing the construct into a cell to enable site-specific homologous recombination by the cell host enzymatic machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site into the cell to enable insertion via ZFN. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site into the cell to enable insertion via TALEN. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a gRNA comprising a Cas9 expression cassette and a guide sequence specific for a desired integration site into the cell to enable insertion via Cas9. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases into a desired integration site within the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable targeted integration via DICE.

[0225] Promising sites for targeted integration are regions within or outside genes in the human genome, and theoretically include, but are not limited to, safe harbor loci, or genomic safe harbors (GSH), that can accommodate predictable expression of newly integrated DNA without adversely affecting the host cell or organism. A useful safe harbor needs to allow sufficient transgene expression to produce desirable levels of the protein or non-coding RNA encoded by the vector. A safe harbor also must not predispose cells to malignant transformation or alter cellular functions. For an integration site to be a potential safe harbor locus, it ideally needs to meet criteria including, but not limited to: the absence of disruption of regulatory elements or genes as determined 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 potential for long-range interactions between a transcriptional activator encoded by the vector and the promoters of adjacent genes, particularly cancer-related genes and microRNA genes, and having clearly ubiquitous transcriptional activity as reflected by a broad spatial and temporal expressed sequence tag (EST) expression pattern indicative of ubiquitous transcriptional activity. This latter property is particularly important in stem cells, where chromatin remodeling during differentiation usually results in the potential for silencing of some loci and activation of other loci. Within regions suitable for exogenous insertion, the exact locus selected for insertion should lack repetitive elements and conserved sequences and should allow for easy design of primers for amplification of homology arms.

[0226] Sites suitable for human genome editing, or specifically targeted integration, include, but are not limited to, adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the human ortholog of the mouse ROSA26 locus. In addition, the human ortholog of the mouse H11 locus can also be a suitable site for insertion using the compositions and methods of targeted integration disclosed herein. Furthermore, the collagen and HTRP loci can also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be particularly necessary in stem cells for a particular integration event, and optimization of insertion strategies such as promoter selection, the sequence and placement of exogenous genes, and construct design is often required.

[0227] For targeted indels, the editing site is often contained within an endogenous gene whose expression and / or function is intended to be disrupted. In some embodiments, the endogenous gene containing the targeted indel is related to the regulation and modification of the immune response. In some other embodiments, the endogenous gene containing the targeted indel is related to a targeted modality, receptor, signaling molecule, transcription factor, drug target candidate, immune response regulation and modification, or a protein that suppresses engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells, and cells derived therefrom.

[0228] Accordingly, one aspect of the present invention provides a method of targeted integration at a selected locus comprising a genomic safe harbor, or, in the case of T cells, at a preselected locus that has been found or proven to be safely and adequately regulated for constitutive or transient gene expression, such as the constant region of the T cell receptor (TCR). In one embodiment, the genomic safe harbor for the method of targeted integration comprises one or more desirable integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of a genomic safe harbor. In one embodiment, the method of targeted integration into a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell a construct comprising a pair of homologous arms specific for the desired integration site and one or more exogenous sequences to enable site-specific homologous recombination by the cell host enzymatic machinery, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of a genomic safe harbor. Further integration sites include endogenous loci intended for disruption, e.g., reduction or knockout, which include B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or TCRβ constant regions (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.

[0229] In another embodiment, a method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site to enable ZFN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, the TCRα or TCRβ constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD54, CD56, CD58, 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 into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site to enable TALEN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, the TCRα or TCRβ 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 for targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing into the cell a Cas9 expression cassette specific for a desired integration site and a gRNA comprising a guide sequence to enable Cas9-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, the TCRα or TCRβ 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 for targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette of DICE recombinase to enable DICE-mediated targeted integration, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, the TCRα or TCRβ 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.

[0230] Furthermore, as provided herein, the above-described method for targeted integration at a safe harbor is used to insert a polynucleotide of interest, such as a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, and a polynucleotide encoding a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some other embodiments, a construct comprising one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the 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 by the B cell molecule CD20 can eliminate them upon administration of the mAb rituximab. Furthermore, a modified EGFR comprising an epitope recognized by cetuximab can be used to deplete genetically engineered cells when the cells are exposed to cetuximab. Accordingly, one aspect of the invention provides a method for targeted integration of one or more suicide genes encoding a safety switch protein selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0231] In some embodiments, one or more exogenous polynucleotides incorporated by the methods described herein are driven by an exogenous promoter operably linked that is included in a construct for targeted integration. The promoter can be inducible or constitutive, and can be temporally specific, tissue specific or cell-type specific. Constitutive promoters suitable for the methods of the invention include, but are not limited to, the cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0232] An exogenous polynucleotide incorporated by the method described herein can be driven by an endogenous promoter in the host genome at the integration site. In one embodiment, the method described herein is used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the method described herein is used for targeted integration at the ROSA26 locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the method described herein is used for targeted integration at the H11 locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the method described herein is used for targeted integration at the collagen locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method described herein is used for targeted integration at the HTRP locus in the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous HTRP promoter. In theory, only correct insertion at the desired location will allow gene expression of the exogenous gene driven by the endogenous promoter.

[0233] In some embodiments, one or more exogenous polynucleotides included in a construct for a method of targeted integration are driven by one promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter, enhancing the physical separation between the parts and maximizing access to the enzymatic machinery. The linker peptide of the linker sequence can consist of amino acids selected to make the physical separation between the parts (exogenous polynucleotides and / or the proteins or peptides encoded therefrom) more flexible or more rigid, depending on the relevant function. The linker sequence can be cleavable by a protease or chemically cleavable to yield separate parts. Examples of enzymatic cleavage sites in the linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is naturally produced by the host or is introduced exogenously. Alternatively, the cleavage site in the linker can be a site cleavable by a selected chemical, such as cyanogen bromide, hydroxylamine, or exposure to low pH. The linker sequence as required can serve purposes other than providing a cleavage site. The linker sequence should enable the effective placement of a part with respect to another adjacent part for the parts to function properly. The linker can also be a simple amino acid sequence of sufficient length to prevent any steric hindrance between the parts. In addition, the linker sequence can provide post-translational modifications including, but not limited to, phosphorylation sites, biotinylation sites, sulfation sites, γ-carboxylation sites, etc. In some embodiments, the linker sequence is flexible so as not to hold a biologically active peptide in a single undesirable conformation. The linker can mainly consist of amino acids with small side chains such as glycine, alanine, and serine to provide flexibility. In some embodiments, about 80 to 90 percent or more of the linker sequence contains glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, the G4S linker peptide separates the fusion protein terminal processing and the endonuclease domain. In other embodiments, the 2A linker sequence enables two separate proteins to be produced from a single translation. Suitable linker sequences can be readily identified empirically. Additionally, 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 sequence (IRES) within the sequence. In some embodiments, any two consecutive linker sequences are different.

[0234] A method of introducing a construct containing an exogenous polynucleotide for targeted integration into a cell can be achieved using methods of gene transfer into cells known per se. In one embodiment, the construct comprises the backbone of a viral vector such as an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a Sendai viral vector. In some embodiments, plasmid vectors are used to deliver and / or express exogenous polynucleotides into target cells (such as pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo). In some other embodiments, episomal vectors are used to deliver exogenous polynucleotides into target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used in genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV does not integrate into the host genome. In addition, episomal rAAV vectors mediate homologous-directed gene targeting at a much higher rate compared to transfection of conventional targeting plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the genome of iPSCs. In some embodiments, the genome-modified iPSCs and their derivative cells obtained using the methods and compositions described herein comprise at least one genotype listed in Table 1.

[0235] III. Methods for obtaining and maintaining genome-engineered iPSCs In various embodiments, the present invention provides a method for obtaining and maintaining genome-edited iPSCs that include one or more targeted edits at one or more desired sites, where the one or more targeted edits continue to be intact and functional at their respective selected edit sites in the expanded genome-edited iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions (i.e., targeted integration and / or indels at the selected sites) into the genomes of the iPSCs and their derivative cells. Among the many advantages of obtaining genome-edited derivative cells derived through the editing and differentiation of iPSCs provided herein, as compared to the direct manipulation of patient-derived primary effector cells from peripheral blood, include, but are not limited to, the unlimited source of the manipulated effector cells, the lack of a need for repeated manipulation of the effector cells, particularly when multiple engineered modalities are included, the resulting effector cells having elongated telomeres and being rejuvenated due to less senescence, the effector cell population being homogeneous with respect to the edit site, copy number, and the absence of allelic mutations, random mutations, and variegation, which is mainly due to the possibility of clone selection in the engineered iPSCs provided herein.

[0236] In certain embodiments, genome-edited iPSCs that include one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells for extended periods in the cell culture medium shown in Table 2 as the Fate Maintenance Medium (FMM), where the iPSCs retain the targeted edits and functional modifications at the selected sites. The components of the medium may be present in the medium in amounts within the optimal ranges shown in Table 2. iPSCs cultured in FMM continue to maintain their undifferentiated, basal, or naive profile; provide genomic stability that does not require culture washes or selections, readily give rise to all three somatic cell lineages by in vitro differentiation through embryoid bodies or monolayers (that do not form embryoid bodies), and readily give rise to in vivo differentiation by teratoma formation. See, for example, International Publication No. WO 2015 / 134652, the disclosure of which is incorporated herein by reference.

[0237]

Table 15

[0238] In some embodiments, genome-engineered iPSCs comprising one or more targeted integrations and / or indels are maintained, passaged, and expanded in a medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and do not contain, or are essentially free of, a TGFβ receptor / ALK5 inhibitor, and the iPSCs retain an intact and functional targeted edit at a selected site.

[0239] Another aspect of the invention provides a method of generating genome-engineered iPSCs through targeted editing of iPSCs or, alternatively, first generating genome-engineered non-pluripotent cells by targeted editing, and then reprogramming the selected / isolated genome-engineered non-pluripotent cells to obtain iPSCs comprising the same targeted edit as the non-pluripotent cells. A further aspect of the invention provides genome-engineered non-pluripotent cells that are undergoing reprogramming simultaneously by introducing a targeted integration and / or a targeted indel into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and the conditions for reprogramming include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genome engineering and reprogramming, the targeted integration and / or the targeted indel can be introduced into the non-pluripotent cells before, or essentially simultaneously with, initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally one or more small molecules.

[0240] In some embodiments, for simultaneous genome engineering and reprogramming of non-pluripotent cells, targeted integration and / or indels can also be introduced into non-pluripotent cells after the multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes including, but not limited to, SSEA4, Tra181, and CD30, a vector carrying the construct is introduced.

[0241] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 2). It is further maintained and expanded using (Table 2). In some embodiments, genomically engineered iPSCs produced by any of the methods described above are further maintained and expanded using a mixture (FMM; Table 2) comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor.

[0242] In some embodiments of methods for generating genome-edited iPSCs, the method comprises introducing one or more targeted integrations and / or indels into the iPSCs to genome-edit the iPSCs and obtaining genome-edited iPSCs having at least one genotype listed in Table 1. Alternatively, a method for generating genome-edited iPSCs comprises: (a) introducing one or more targeted edits into non-pluripotent cells to obtain genome-edited non-pluripotent cells comprising targeted integration and / or indels at a selected site; and (b) contacting the genome-edited 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 obtain genome-edited iPSCs comprising targeted integration and / or indels at a selected site. Alternatively, a method for generating genome-edited iPSCs comprises: (a) contacting 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; (b) introducing one or more targeted integrations and / or indels into the reprogramming non-pluripotent cells for genome editing; and (c) obtaining clonal genome-edited iPSCs comprising targeted integration and / or indels at a selected site. Any of the above methods may further comprise single cell sorting of the genome-edited iPSCs to obtain clonal iPSCs. Through clonal expansion of the genome-edited iPSCs, a master cell bank is generated to comprise single cell sorting and expanded clonal engineered iPSCs having at least one phenotype as provided herein. The master cell bank is subsequently cryopreserved and provides a platform for further iPSC engineering and a renewable source for manufacturing off-the-shelf engineered homogeneous cell therapy products that are compositionally defined, uniform, and can be mass-produced cost-effectively at a significant scale.

[0243] The reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in International Publication Nos. 2015 / 134652 and 2017 / 066634, the disclosures of which are incorporated herein by reference. One or more reprogramming factors can be in the form of a polypeptide. The reprogramming factors can also be in the form of a polynucleotide encoding a reprogramming factor and can thus be introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai viral vector. In some embodiments, one or more polynucleotides are introduced by a combination of plasmids. See, for example, International Publication No. 2019 / 075057(A1), the disclosure of which is incorporated herein by reference.

[0244] In some embodiments, non-pluripotent cells are transfected with multiple constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides can include suicide genes, or genes encoding targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derivative cells. In some embodiments, the exogenous polynucleotides encode RNAs including, but not limited to, siRNA, shRNA, miRNA, and antisense nucleic acids. These exogenous polynucleotides can be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, temporally specific promoters, and tissue-specific or cell-type specific promoters. Thus, the polynucleotide is 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 polynucleotide is expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, one or more suicide genes are driven by a constitutive promoter, for example, caspase-9 driven by CAG. These constructs containing different exogenous polynucleotides and / or different promoters can be transfected into non-pluripotent cells simultaneously or sequentially. The non-pluripotent cells subjected to targeted integration of multiple constructs can be simultaneously contacted with one or more reprogramming factors to initiate reprogramming simultaneously with genome manipulation, thereby obtaining genome-manipulated iPSCs containing multiple targeted integrations in the same cell pool. Thus, by this robust method, clonal genome-manipulated iPSCs with multiple modalities integrated within one or more selected target sites can be induced by simultaneous reprogramming and engineering strategies. In some embodiments, the genome-modified iPSCs and their derivative cells obtained using the methods and compositions herein include at least one genotype listed in Table 1.

[0245] IV. Method for obtaining genetically engineered effector cells by differentiating genomically engineered iPSCs A further aspect of the present invention provides a method for in vivo differentiation of genome-edited iPSCs by teratoma formation, wherein the differentiated cells induced in vivo from the genome-edited iPSCs retain intact and functional targeted editing including targeted integration and / or indels at a desired site. In some embodiments, the differentiated cells induced in vivo from genome-edited iPSCs via teratoma formation comprise 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 meeting the criteria of genomic safe harbors. In some other embodiments, the differentiated cells induced in vivo from genome-edited iPSCs via teratoma formation comprise a polynucleotide encoding a targeting modality, or a polynucleotide encoding a protein that promotes the transport, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells. In some embodiments, the differentiated cells induced in vivo from genome-edited iPSCs via teratoma formation comprising one or more inducible suicide genes further comprise one or more indels of endogenous genes associated with the regulation and mediation of the immune response. In some embodiments, the indels are contained in one or more endogenous checkpoint genes. In some embodiments, the indels are contained in one or more endogenous T cell receptor genes. In some embodiments, the indels are contained in one or more endogenous MHC class I suppressor genes. In some embodiments, the indels are contained in one or more endogenous genes associated with the major histocompatibility complex.In some embodiments, the indel is included 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, the TCRα or TCRβ 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. In one embodiment, the genome-engineered iPSC comprising one or more exogenous polynucleotides at a selected site further comprises a targeted edit in the gene encoding B2M (beta-2-microglobulin).

[0246] In certain embodiments, the genome-engineered iPSC comprising one or more genetic modifications provided herein is used to induce hematopoietic cell lineages or other specific cell types in vitro, and the induced non-pluripotent cells retain the functional genetic modification including the targeted edit at the selected site. In one embodiment, the genome-engineered iPSC-derived cells include, but are not limited to, mesodermal cells with definitive hematopoietic endothelium (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem progenitor cells, hematopoietic multipotent progenitor bodies (MPP), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, where the cells derived from the genome-engineered iPSC retain the functional genetic modification including the targeted edit at the desired site.

[0247] Applicable differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lineages include, for example, those disclosed in International Publication No. WO 2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, the methods and compositions for generating hematopoietic cell lineages are via definitive hematopoietic endothelium (HE) derived from pluripotent stem cells in a culture platform that is serum-free, feeder-free, and / or stroma-free, scalable, and does not require monolayer EB formation. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated cells and transdifferentiated cells, and to various lineages of cells that have directly transitioned to a hematopoietic fate without passing through pluripotent intermediates. Similarly, cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells and to all intervening hematopoietic cell lineages.

[0248] A method for differentiating and expanding cells of the hematopoietic lineage from pluripotent stem cells in monolayer culture involves 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 proliferating mesodermal cells with definitive hematopoietic endothelium (HE) ability without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF and optionally a ROCK inhibitor and / or a WNT pathway activator causes the mesodermal cells with definitive HE ability to differentiate into definitive HE cells that also proliferate during differentiation.

[0249] The method for obtaining cells of the hematopoietic lineage provided herein is superior to pluripotent stem cell differentiation via EBs because EB formation results in minimal cell proliferation from modest proliferation, does not allow for important monolayer culture and uniform differentiation of cells within the population, which is crucial for many applications that require uniform proliferation, and is brittle and inefficient.

[0250] The provided monolayer differentiation platform promotes definitive hematopoietic endothelial differentiation that leads to the derivation of differentiated progeny such as hematopoietic stem cells and T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion, enabling the delivery of a therapeutically relevant number of pluripotent stem cell-derived hematopoietic cells for various therapeutic applications. Furthermore, monolayer culture using the methods provided herein yields functional hematopoietic lineage cells that enable the full range of in vitro differentiation, in vivo engraftment, and long-term hematopoietic self-renewal, reconstitution, and engraftment in vivo. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, definitive hematopoietic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem and progenitor cells, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0251] Accordingly, in various embodiments, a method for directing the differentiation of pluripotent stem cells into cells of the definitive hematopoietic lineage comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP agonist and, optionally, bFGF to initiate the differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a BMP agonist, bFGF, and a GSK3 inhibitor to initiate the differentiation and expansion of mesodermal cells with the potential for definitive HE from the mesodermal cells, wherein the composition optionally does not contain a TGFβ receptor / ALK inhibitor; and (iii) contacting the mesodermal cells with the potential for definitive HE with a composition comprising a ROCK inhibitor and 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 the differentiation and expansion of definitive hematopoietic endothelium from the pluripotent stem cell-derived mesodermal cells with definitive hematopoietic endothelial potential, wherein the composition optionally does not contain a TGFβ receptor / ALK inhibitor.

[0252] In some embodiments, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor and not comprising a TGFβ receptor / ALK inhibitor to seed and 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 iPSCs are retained in the hematopoietic cells differentiated therefrom. In some embodiments of the method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of the hematopoietic lineage is in a monolayer culture format without embryoid body formation.

[0253] In some embodiments of the method described above, the determined hematopoietic endothelial cells derived from the obtained pluripotent stem cells are CD34 + positive. In some embodiments, the obtained determined hematopoietic endothelial cells are CD34 + CD43 - positive. In some embodiments, the determined hematopoietic endothelial cells are CD34 + CD43 - CXCR4 - CD73 - positive. In some embodiments, the determined hematopoietic endothelial cells are CD34 + CXCR4 - CD73 - positive. In some embodiments, the determined hematopoietic endothelial cells are CD34 + CD43 - CD93 - positive. In some embodiments, the determined hematopoietic endothelial cells are CD34 + CD93 - positive.

[0254] In some embodiments of the above method, the method comprises: (i) contacting definitive hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, and optionally a BMP activator, to initiate the differentiation of definitive hematopoietic endothelium into pre-T cell precursors; and optionally, (ii) contacting the pre-T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate the differentiation of the pre-T cell precursors into T cell precursors or T cells. In some embodiments of this method, the pluripotent stem cell-derived T cell precursors are CD34 + CD45 + CD7 + positive. In some embodiments of this method, the pluripotent stem cell-derived T cell precursors are CD45 + CD7 + positive.

[0255] In still further embodiments of the above method for directing the differentiation of pluripotent stem cells into hematopoietic lineage cells, the method comprises: (i) contacting definitive hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator, to initiate the differentiation of the definitive hematopoietic endothelium into pre-NK cell precursors; and optionally, (ii) contacting the pre-NK cell precursors derived from pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, to initiate the 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 activator, and ROCK inhibitor. In some embodiments, the pluripotent stem cell-derived NK precursors are CD3 - CD45 + CD56+ CD7 + is. In some embodiments, the pluripotent stem cell-derived NK cells are CD3 - CD45 + CD56 + and, optionally, NKp46 + , CD57 + and CD16 + is further defined by.

[0256] Thus, using the above differentiation method, one or more populations of the following iPSC-derived hematopoietic cells can be obtained: (i) CD34 using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2 + HE cells (iCD34), (ii) definitive hematopoietic endothelium (iHE) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2, (iii) definitive HSC using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2, (iv) pluripotent progenitor cells (iMPP) using iMPP-A, (v) T cell precursors (ipro-T) using one or more culture media selected from iTC-A2 and iTC-B2, (vi) T cells (iTC) using iTC-B2, (vii) NK cell precursors (ipro-NK) using one or more culture media selected from iNK-A2 and iNK-B2, and / or (viii) NK cells (iNK) and iNK-B2. In some embodiments, the medium is as follows: a. iCD34-C contains a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator, and does not contain a TGFβ receptor / ALK inhibitor. b. iMPP-A contains a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. c. iTC-A2 comprises a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7, and optionally a BMP activator. d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. e. iNK-A2 comprises a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator. f. iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0257] In some embodiments, the genome-edited iPSC-derived cells obtained from the above method 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, the constant region of TCRα or TCRβ, 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 of genomic safe harbors. In some other embodiments, the genome-edited iPSC-derived cells comprise a polynucleotide encoding a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes the trafficking, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells. In some embodiments, the genome-edited 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.

[0258] In addition, applicable dedifferentiation methods and compositions for obtaining genomically engineered hematopoietic cells of a second fate from genomically engineered hematopoietic cells of a first fate include, for example, those shown in International Publication No. WO 2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided therein enable the partial reprogramming of starting non-pluripotent cells into non-pluripotent intermediate cells by restricting the expression of the endogenous Nanog gene during reprogramming, and subjecting the non-pluripotent intermediate cells to conditions for differentiating the intermediate cells into the desired cell type. In some embodiments, the genomically modified iPSCs and their derivative cells obtained using the methods and compositions described herein include at least one genotype listed in Table 1.

[0259] V. Therapeutic use of derivative immune cells with functional modalities differentiated from genetically engineered iPSCs In some embodiments, the present invention provides a composition comprising an isolated population or subpopulation of functionally enhanced derivative immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSCs comprise one or more targeted gene edits that can be retained in iPSC-derived effector cells, and the genetically engineered iPSCs and their derivative cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells comprises iPSC-derived CD34 cells. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells comprises iPSC-derived pro-T cells or T cells. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells comprises iPSC-derived pro-NK cells or NK cells. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells comprises iPSC-derived immunomodulatory cells or myeloid-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived genetically engineered effector cells are further engineered ex vivo for improved therapeutic potential. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells derived from iPSCs comprises an increase in the number or ratio of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells derived from iPSCs comprises an increase in the number or ratio of type I NKT cells. In another embodiment, the isolated population or subpopulation of genetically engineered effector cells derived from iPSCs comprises an increase in the number or ratio of adaptive NK cells. In some embodiments, the isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, or myeloid-derived suppressor cells derived from iPSCs are allogeneic. In some other embodiments, the isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, or MDSCs derived from iPSCs are autologous.

[0260] In some embodiments, the iPSCs for differentiation contain a genetic imprint selected to confer desirable therapeutic attributes in the derived effector cells, on the condition that the developmental biology of the cells during differentiation is not disrupted, the genetic imprint is retained and functional in the differentiated hematopoietic cells derived from the iPSCs.

[0261] In some embodiments, the genetic imprint of the pluripotent stem cells comprises (i) one or more gene recombination modalities obtained through genomic insertion, deletion, or substitution in the genome of the pluripotent cells during or after reprogramming non-pluripotent cells into iPSCs, or (ii) one or more retainable therapeutic properties of the source-specific immune cells that are specific to the donor, disease, or treatment response, the pluripotent cells are reprogrammed from source-specific immune cells, the iPSCs retain the source therapeutic properties, and the source therapeutic properties are also included in the hematopoietic lineage cells derived from the iPSCs.

[0262] In some embodiments, the gene recombination modality includes one or more of a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or survival rate of iPSCs or their derived cells. In some embodiments, the genetically modified iPSCs and their derived cells include the genotypes listed in Table 1. In some other embodiments, the genetically modified iPSCs and their derived cells that include the genotypes listed in Table 1 are (1) one or more of the deletion or disruption of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or TCRβ 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) HLA-E, HLA-G, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptor, or surface trigger receptor, and further includes an additional gene recombination modality involving the introduction of

[0263] In still some other embodiments, iPSC-derived hematopoietic lineage cells include the therapeutic attributes of source-specific immune cells regarding at least two of the following combinations: (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) resistance to the tumor microenvironment, (iv) recruitment of bystander immune cells and immune modification, (v) improved on-target specificity with reduced off-tumor effects, (vi) overcoming or reducing tumor microenvironment suppression associated with solid tumors, (vii) improved homing, persistence, cytotoxicity, or antigen escape rescue, and (viii) improved apoptosis resistance and / or exhaustion resistance.

[0264] In some embodiments, the iPSC-derived hematopoietic cells comprise the genotype listed in Table 1, the cells express a Fas redirector (''Fas'' in Table 1), and the Fas redirector comprises (a) an extracellular Fas binding domain comprising the extracellular domain (ECD) of the Fas receptor (FAS) or a partial or complete peptide of a variant or allele thereof, and (b) a cytoplasmic signaling domain comprising a partial or complete peptide of the intracellular domain (ICD) of one or more costimulatory molecules, and the Fas redirector redirects Fas signaling upon binding to a Fas agonist, thereby providing the cells with improved apoptosis resistance and / or exhaustion resistance. In various embodiments, the Fas redirector further comprises a transmembrane region comprising the transmembrane domain of a transmembrane protein or a portion thereof. In some embodiments, the one or more costimulatory molecules comprise CD27, CD28, CD40, MyD88, OX40, IL12Rβb2, IL18R1, IL21R, or a combination thereof. In some embodiments, the one or more costimulatory molecules do not include 41BB. In some embodiments, the Fas agonist comprises Fas ligand (FasL). In some embodiments, the cells comprising the Fas redirector further comprise one or more of (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), (b) an exogenous polynucleotide encoding CD16 or a variant thereof, (c) a CD38 knockout, and (d) an exogenous polynucleotide encoding a cytokine signaling complex comprising a partial or complete peptide of an extracellular cytokine and / or its receptor expressed on the cell surface.

[0265] Specifically, the present application provides a method for improving the efficacy of adoptive cell therapy provided to a subject in need of adoptive cell therapy, the method comprising administering the derived effector cells or a population thereof described herein. In various embodiments, the derived effector cells comprise a Fas redirector, the Fas redirector comprising (a) an extracellular Fas binding domain comprising the extracellular domain (ECD) of the Fas receptor (FAS) or a partial or complete peptide of a variant or allele thereof, and (b) a cytoplasmic signaling domain comprising a partial or complete peptide of the intracellular domain (ICD) of one or more costimulatory molecules, the Fas redirector redirecting Fas signaling upon binding of a Fas agonist, thereby providing the cell with improved apoptosis resistance and / or exhaustion resistance. In various embodiments, the induced effector cells optionally comprise, as shown in Table 1, CAR, exogenous CD16, CD38 - / -、further comprises one or more of a cytokine signaling complex, HLA deficiency / modification, an antibody, a checkpoint inhibitor, an engager, and any other modality. In various embodiments, the transmembrane region of the Fas redirector is (i) the full length or at least a portion of the native or modified transmembrane region of FAS, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, 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, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide, or (ii) the full length or partial length of the transmembrane domain of FAS. In certain embodiments, (i) the extracellular binding domain of the Fas redirector comprises a sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 1, (ii) the cytoplasmic signaling domain of the Fas redirector is (a) the full length or partial length of the intracellular domain (ICD) of MyD88 and CD40 represented by SEQ ID NO: 2, or (b) the full length or partial length of the intracellular domain (ICD) of CD27 represented by SEQ ID NO: 3, or (c) the full length or partial length of the intracellular domain (ICD) of CD28 represented by SEQ ID NO: 4, or (d) the full length or partial length of the intracellular domain (ICD) of OX40 represented by SEQ ID NO: 5, or (e) the full length or partial length of the intracellular domain (ICD) of IL12Rβ2 represented by SEQ ID NO: 6, or (f) the full length or partial length of the intracellular domain (ICD) of IL18R1 represented by SEQ ID NO: 7, or (g) the full length or partial length of the intracellular domain (ICD) of IL21R represented by SEQ ID NO: 8, or (iii) the Fas redirector comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 9-15.

[0266] In various embodiments of methods for improving the efficacy of adoptive cell therapy, effector cells are administered to a subject as combination therapy with a therapeutic agent. In some embodiments, the therapeutic agent of the combination therapy is an anti-CD38 antibody or a fragment thereof. In some embodiments, the anti-CD38 antibody is daratumumab, isatuximab, or MOR202. In some embodiments, the anti-CD38 therapeutic agent is administered simultaneously with, prior to, or after the administration of the derived effector cells. Thus, in some embodiments, the antibody is used in combination with the population of effector cells described herein by simultaneous or sequential administration to the subject. In other embodiments, such an antibody or a fragment thereof can be expressed by effector cells by genetically engineering iPSCs using an exogenous polynucleotide sequence encoding the antibody or a fragment thereof and inducing the differentiation of the engineered iPSCs as described herein. In some embodiments of the method, the effector cells are iPSC-derived hematopoietic cells. In some embodiments of the method, the effector cells are iPSC-derived T cells, NK cells, or NKT cells.

[0267] In a further embodiment of a method of improving the effectiveness of adoptive cell therapy provided to a subject in need thereof, the method further comprises administering an antibody specific for an upregulated surface protein that is the same as or different from that targeted by the CAR, and / or one or more additional therapeutic agents. In some embodiments of the method, the antibody comprises at least one of anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, anti-CD38 antibody, or a humanized or Fc-modified variant or fragment, functional equivalent, and biosimilar thereof. In some embodiments of the therapeutic agent used in the method, the therapeutic agent comprises a peptide, cytokine, checkpoint inhibitor, mitogen, growth factor, small molecule RNA, dsRNA (double-stranded RNA), mononuclear cells, feeder cells, feeder cell components or replenishing factors thereof, a vector comprising one or more polynucleotides of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

[0268] By introducing the derived effector cells of the present invention into a subject suitable for adoptive cell therapy, various diseases can be improved. In some embodiments, the iPSC-derived hematopoietic cells provided herein are for allogeneic adoptive cell therapy. In other embodiments, the iPSC-derived hematopoietic cells provided herein are for preventing or reducing tumor microenvironment suppression associated with solid tumors. In other embodiments, the iPSC-derived hematopoietic cells provided herein are for improving the effectiveness of adoptive cell therapy. Additionally, the present invention, in some embodiments, is the therapeutic use of the above therapeutic composition and / or combination therapy by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, hematological malignancy, solid tumor, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0269] Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML)), lymphomas, non-Hodgkin lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes. Examples of solid cancers include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpesvirus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella-zoster virus), adenovirus-, lentivirus-, BK polyomavirus-related diseases.

[0270] Treatment using the derived hematopoietic stem cells of the embodiments disclosed herein can be performed based on the presentation of symptoms or for relapse prevention. The terms "treating", "treatment", etc. are generally used herein to mean obtaining the desired pharmacological and / or physiological effect. The effect can be prophylactic with respect to completely or partially preventing a disease and / or can be therapeutic with respect to the partial or complete cure of a disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any intervention in a subject's disease and includes: preventing the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed as having it, inhibiting the disease, i.e., preventing its onset, or alleviating the disease, i.e., causing the disease to regress. A therapeutic agent or composition can be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease where the treatment stabilizes or reduces the subject's undesirable clinical symptoms is also particularly important. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury that can have at least one related symptom suppressed, remitted, and / or improved by cell therapy. Certain embodiments contemplate that the subject in need of cell therapy includes, but is not limited to, a candidate for bone marrow or stem cell transplantation, a subject who has received chemotherapy or radiation therapy, a subject who has or is at risk of having a proliferative disorder or cancer, such as a hematopoietic proliferative disorder or cancer, a subject who has or is at risk of developing a tumor, such as a solid tumor, and a subject who has or is at risk of having a viral infection or a disease associated with a viral infection.

[0271] When evaluating the responsiveness to a treatment comprising the derived hematopoietic lineage cells of the embodiments disclosed herein, the response can be measured by at least one of the clinical benefit rate, survival rate until death, pathological complete remission, quantitative measurement of the pathological response version, clinical complete remission, clinical partial remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Response Evaluation Criteria In Solid Tumors) criteria.

[0272] The therapeutic composition comprising iPSC-derived hematopoietic lineage cells disclosed herein can be administered to a subject before, during, and / or after another treatment. Accordingly, the method of combination therapy can include the administration or preparation of iPSC-derived effector cells before, during, and / or after the use of an additional therapeutic agent. As provided herein, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small interfering RNAs, dsRNAs (double-stranded RNAs), monocytes, feeder cells, feeder cell components or their replenishing factors, vectors containing one or more polynucleotides of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). Administration of iPSC-derived immune cells can be temporally separated from the administration of the additional therapeutic agent by hours, days, or weeks. Additionally or alternatively, the administration can be combined with other bioactive agents or modalities such as, but not limited to, anti-tumor agents, non-pharmacological therapies such as surgery, and the like.

[0273] In some embodiments of the combinatorial cell therapy, the therapeutic combination comprises the iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent that is an antibody or antibody fragment. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody may be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor- or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability. In some embodiments, as additional therapeutic agents for the administered iPSC-derived hematopoietic lineage cells, antibodies suitable for combinatorial therapy include anti-CD20 (e.g., rituximab, belimumab, ofatumumab, ublituximab, ocrelizumab, obinutuzumab), anti-HER2 (e.g., trastuzumab, pertuzumab), anti-CD52 (e.g., alemtuzumab), anti-EGFR (e.g., cetuximab), anti-GD2 (e.g., dinutuximab), anti-PDL1 (e.g., avelumab), anti-CD38 (e.g., daratumumab, isatuximab, MOR202), anti-CD123 (e.g., 7G3, CSL362), anti-SLAMF7 (elotuzumab), and their humanized or Fc-modified variants or fragments, or functional equivalents or biosimilars thereof, but are not limited thereto.

[0274] In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. A checkpoint refers to a cellular molecule, often a cell surface molecule, that can suppress or downregulate an immune response when not inhibited. A checkpoint inhibitor is an antagonist that can reduce the gene expression or gene product of a checkpoint or decrease the activity of a checkpoint molecule. Checkpoint inhibitors suitable for combination therapy with derived effector cells, including NK cells or T cells, are provided above.

[0275] Some embodiments of the combination therapy comprising the provided derived effector cells further comprise at least one inhibitor that targets a checkpoint molecule. Some other embodiments of the combination therapy with the provided derived effector cells comprise two, three, or more inhibitors such that two, three, or more checkpoint molecules are targeted. In some embodiments, the effector cells for the combination therapy described herein are derived NK cells as provided. In some embodiments, the effector cells for the combination therapy described herein are derived T cells. In some embodiments, the derived NK cells or T cells for the combination therapy are functionally enhanced as provided herein. In some embodiments, two, three, or more checkpoint inhibitors can be administered in the combination therapy simultaneously with, prior to, or after the administration of the derived effector cells. In some embodiments, two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

[0276] In some embodiments, the antagonist that inhibits any of the above-described checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab′)2, F(ab′)3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which can be more cost-effective to manufacture, easier to use, or more sensitive than the whole antibody. In some embodiments, one or two or three or more checkpoint inhibitors include at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0277] A combination therapy comprising derived effector cells and one or more checkpoint inhibitors is useful for cutaneous T-cell lymphoma, non-Hodgkin lymphoma (NHL), mycosis fungoides, pagetoid reticulosis, Sézary syndrome, granulomatous slack skin, lymphomatoid papulosis, pityriasis lichenoides chronica, pityriasis lichenoides et varioliformis acuta, CD30 + cutaneous T-cell lymphoma, secondary cutaneous CD30 +It is applicable to the treatment of liquid and solid cancers, including, but not limited to, large cell lymphoma, mycosis fungoides, CD30 cutaneous large T cell lymphoma, polymorphic T cell lymphoma, Lennert lymphoma, subcutaneous T cell lymphoma, angiocentric lymphoma, blastic NK cell lymphoma, B cell lymphoma, Hodgkin lymphoma (HL), head and neck tumors: squamous cell carcinoma, rhabdomyosarcoma, Lewis lung carcinoma (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostatic small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, liver cancer, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid cancer, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal carcinoma.

[0278] In some embodiments other than the derived effector cells as provided herein, the combination for therapeutic use comprises one or more additional therapeutic agents comprising a chemotherapeutic agent or a radioactive moiety. A chemotherapeutic agent refers to a cytotoxic anti-tumor agent, i.e., a chemical agent that has been found to preferentially kill tumor cells, or disrupt the cell cycle of rapidly proliferating cells, or eradicate cancer stem cells, and is used therapeutically to prevent or reduce the growth of neoplastic cells. Chemotherapeutic agents are also sometimes referred to as anti-tumor or cytotoxic drugs or agents and are well known in the art.

[0279] In some embodiments, the chemotherapeutic agent includes anthracycline, alkylating agent, alkyl sulfonate, aziridine, ethyleneimine, methylmelamine, nitrogen mustard, nitrosourea, antibiotic, antimetabolite, folic acid analog, purine analog, pyrimidine analog, enzyme, podophyllotoxin, platinum-containing agent, interferon, and interleukin. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, mechlorethamine, melphalan, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisantrene, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine.Additional agents include aminoglutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzumab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, celecoxib, cetuximab, cladribine, clofarabine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, drostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegademase, pegaspargase, pentostatin, pipobroman, plicamycin, polyhepcosan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those approved as chemotherapeutic or radiation therapy agents and known in the art.Such agents can be referenced from either many standard physician and oncologist references (e.g., Goodman & Gilman’s The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, N.Y., 1995) or the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated as needed.

[0280] Immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, stimulate both NK cells and T cells. As provided herein, IMiDs can be used with iPSC-derived therapeutic immune cells for cancer treatment.

[0281] In addition to the isolated population of iPSC-derived hematopoietic lineage cells contained in the therapeutic composition, a composition suitable for administration to a patient can further comprise one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., a pharmaceutically acceptable medium, such as a cell culture medium), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the particular composition being administered and by the particular method used to administer the therapeutic composition. Accordingly, there are a variety of suitable formulations of the therapeutic compositions of the embodiments of the present invention (see, e.g., Remington’s Pharmaceutical Sciences, 17 th ed. 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0282] In one embodiment, the therapeutic composition comprises pluripotent cell-derived T cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived NK cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived CD34 + HE cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived HSCs produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived MDSCs produced by the methods and compositions disclosed herein. A therapeutic composition comprising a population of iPSC-derived hematopoietic lineage cells disclosed herein can be administered individually or in combination with other suitable compounds by intravenous, intraperitoneal, enteral, or tracheal administration methods to affect a desired therapeutic goal.

[0283] These pharmaceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition from about 3 to about 10. Thus, the buffer can be as much as about 5% by weight of the total composition. Electrolytes, such as, but not limited to, sodium chloride and potassium chloride, can also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition ranges from about 4 to about 10. Alternatively, the pH of the therapeutic composition ranges from about 5 to about 9, from about 6 to about 9, or from about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH within one of these pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH ranging from about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0284] The present invention also provides, in part, the use of a pharmaceutically acceptable cell culture medium in certain compositions and / or cultures of embodiments of the present invention. Such compositions are suitable for administration to a human subject. Generally speaking, any medium that supports the maintenance, proliferation, and / or health of iPSC-derived effector cells according to embodiments of the present invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, the serum-free medium contains no animal components and may optionally be protein-free. Optionally, the medium may contain a biopharmaceutically acceptable recombinant protein. A medium that contains no animal components refers to a medium whose components are derived from non-animal sources. The recombinant protein replaces the natural animal protein in a medium that does not contain animals, and the nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a protein-free medium is defined as substantially free of protein. Those skilled in the art will understand that the examples of media described above are illustrative and in no way limit the formulation of media suitable for use in the present invention, and that there are many suitable media known and available to those skilled in the art.

[0285] iPSC-derived hematopoietic lineage cells can have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34 + HE cells, HSCs, B cells, myeloid-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, the iPSC-derived hematopoietic lineage cells have about 95% to about 100% T cells, NK cells, pro-T cells, pro-NK cells, CD34 + HE cells, or myeloid-derived suppressor cells (MDSCs). In some embodiments, the present invention provides a therapeutic composition having purified T cells or NK cells, such as a composition having an isolated population of about 95% T cells, NK cells, pro-T cells, pro-NK cells, CD34 + HE cells, or myeloid-derived suppressor cells (MDSCs) for treating a subject in need of cell therapy.

[0286] As will be understood by those skilled in the art, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs based on the methods and compositions of this specification can be used in cell therapies such as those described above. In the case of autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA-compatible with the patient. In another embodiment, the derived hematopoietic lineage cells are not HLA-matched to the subject, and the derived hematopoietic lineage cells are NK cells or T cells containing a Fas redirector, and the Fas redirector is an extracellular Fas-binding domain containing the extracellular domain (ECD) of the Fas receptor (FAS) or a partial or complete peptide of its variant or allele, and a cytoplasmic signaling domain containing a partial or complete peptide of the intracellular domain (ICD) of one or more co-stimulatory molecules. The Fas redirector redirects Fas signaling upon binding to a Fas agonist, and the NK cells or T cells optionally contain one or more modalities listed in Table 1.

[0287] In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is at least 0.1×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 5×10 6 cells, at least 1×10 7 cells, at least 5×10 7 cells, at least 1×10 8 cells, at least 5×10 8 cells, at least 1×10 9 cells, or at least 5×10 9 cells. In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is from about 0.1×10 5 cells to about 1×10 6 cells, from about 0.5×10 6 cells to about 1×10 7 cells, from about 0.5×10 7 cells to about 1×10 8 cells, from about 0.5×108 Cells ~ about 1×10 9 Cells, per dose, about 1×10 9 Cells ~ about 5×10 9 Cells, per dose, about 0.5×10 9 Cells ~ about 8×10 9 Cells, per dose, about 3×10 9 Cells ~ about 3×10 10 Cells, or any range therebetween. Generally, for a 60 kg patient / subject, 1×10 8 Cells / dose is converted to 1.67×10 6 Cells / kg.

[0288] In one embodiment, the number of hematopoietic lineage-derived cells in the therapeutic composition is the number of immune cells in partial or single umbilical cord blood, or at least 0.1×10 5 Cells / kg body weight, at least 0.5×10 5 Cells / kg body weight, at least 1×10 5 Cells / kg body weight, at least 5×10 5 Cells / kg body weight, at least 10×10 5 Cells / kg body weight, at least 0.75×10 6 Cells / kg body weight, at least 1.25×10 6 Cells / kg body weight, at least 1.5×10 6 Cells / kg body weight, at least 1.75×10 6 Cells / kg body weight, at least 2×10 6 Cells / kg body weight, at least 2.5×10 6 Cells / kg body weight, at least 3×10 6 Cells / kg body weight, at least 4×10 6 Cells / kg body weight, at least 5×10 6 Cells / kg body weight, at least 10×10 6 Cells / kg body weight, at least 15×10 6 Cells / kg body weight, at least 20×10 6 Cells / kg body weight, at least 25×10 6 Cells / kg body weight, at least 30×10 6 Cells / kg body weight, 1×10 8 Cells / kg body weight, 5×10 8cells / kg body weight, or 1×10 9 cells / kg body weight.

[0289] In one embodiment, a certain dosage of hematopoietic lineage cells is delivered to a subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2×10 6 cells / kg, at least 3×10 6 cells / kg, at least 4×10 6 cells / kg, at least 5×10 6 cells / kg, at least 6×10 6 cells / kg, at least 7×10 6 cells / kg, at least 8×10 6 cells / kg, at least 9×10 6 cells / kg, or at least 10×10 6 cells / kg, or more cells / kg, including all intervening cell dosages.

[0290] In another exemplary embodiment, the effective amount of cells provided to the subject is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, or about 10×10 6 cells / kg, or more cells / kg, including all intervening cell dosages.

[0291] In another exemplary embodiment, the effective amount of cells provided to the subject is about 2×10 6 cells / kg to about 10×10 6 cells / kg, about 3×10 6 cells / kg to about 10×10 6 cells / kg, about 4×10 6 cells / kg to about 10×10 6 cells / kg, about 5×10 6 cells / kg to about 10×10 6cells / kg, 2×10 6 cells / kg to approximately 6×10 6 cells / kg, 2×10 6 cells / kg to approximately 7×10 6 cells / kg, 2×10 6 cells / kg to approximately 8×10 6 cells / kg, 3×10 6 cells / kg to approximately 6×10 6 cells / kg, 3×10 6 cells / kg to approximately 7×10 6 cells / kg, 3×10 6 cells / kg to approximately 8×10 6 cells / kg, 4×10 6 cells / kg to approximately 6×10 6 cells / kg, 4×10 6 cells / kg to approximately 7×10 6 cells / kg, 4×10 6 cells / kg to approximately 8×10 6 cells / kg, 5×10 6 cells / kg to approximately 6×10 6 cells / kg, 5×10 6 cells / kg to approximately 7×10 6 cells / kg, 5×10 6 cells / kg to approximately 8×10 6 cells / kg, or 6×10 6 cells / kg to approximately 8×10 6 cells / kg and includes all intervening cell doses.

[0292] In some embodiments, the therapeutic use of hematopoietic system-derived cells is a single-dose treatment. In some embodiments, the therapeutic use of hematopoietic system-derived cells is a multiple-dose treatment. In some embodiments, the multiple-dose treatment is one administration per day, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days, every 45 days, every 50 days, or any number of administrations for any number of days in between.

[0293] Compositions containing a population of derived hematopoietic cells of the present invention can be sterile, suitable for administration to a human patient / subject, and can be administered immediately (i.e., can be administered without further treatment). A cell-based composition that is ready for administration means that the composition does not require further processing or manipulation prior to transplantation or administration to a subject. In other embodiments, the present invention provides an isolated population of derived hematopoietic cells that are expanded and / or conditioned prior to administration of one or more agents comprising small chemical molecules. Compositions and methods for modulating immune cells comprising iPSC-derived effector cells are described in detail, for example, in International Publication No. WO 2017 / 127755, the relevant disclosure of which is incorporated herein by reference. For derived hematopoietic cells genetically engineered to express a recombinant TCR or CAR, the cells can be activated and expanded using, for example, the methods described in U.S. Patent No. 6,352,694.

[0294] In certain embodiments, the primary stimulation signal and the co-stimulation signal for the derived hematopoietic cells can be provided by different protocols. For example, the agents providing each signal can be in solution or can be bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other agent can be present in solution. In one embodiment, the agent providing the co-stimulation signal can be bound to the cell surface, and the agent providing the primary activation signal can be in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agent is in a soluble form and is then cross-linked to a surface such as a cell expressing an Fc receptor, an antibody, or other binding agent that binds to the agent, as disclosed in U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 (the disclosures of which are incorporated by reference), which are contemplated for use in the activation and expansion of T lymphocytes in embodiments of the present invention.

[0295] Some variation in dosage, frequency, and protocol will necessarily occur depending on the condition of the subject being treated. In any case, the person responsible for administration shall determine the appropriate dosage, frequency, and protocol for each individual subject.

Example

[0296] The following examples are provided for illustration and not for limitation.

[0297] Example 1 - Materials and Methods The hiPSC platform was used for single cell passage and high-throughput 96-well plate-based flow cytometry sorting to enable the derivation of clonal hiPSCs with single or multiple gene regulations.

[0298] Maintenance of hiPSCs in small molecule culture: hiPSCs were routinely passaged as single cells when the confluence of the culture reached 75% - 90%. For single cell dissociation, hiPSCs were washed once with PBS (Mediatech), treated with Accutase (Millipore) at 37 °C for 3 - 5 minutes, and then pipetted to ensure single cell dissociation. The single cell suspension was then mixed with an equal volume of conventional medium, centrifuged at 225×g for 4 minutes, resuspended in FMM, and seeded onto a surface coated with Matrigel. Passage was usually at 1:6 - 1:8, transferred to a tissue culture plate pre-coated with Matrigel at 37 °C for 2 - 4 hours, and FMM was supplied every 2 - 3 days. Cell culture was maintained in a humidified incubator set at 37 °C and 5% CO2.

[0299] Human iPSC manipulation by ZFN, using CRISPR:ROSA26 targeted insertion for targeted editing of the modality of interest as an example, in genome editing via ZFN, 2 million iPSCs were transfected with a mixture of 2.5 μg of ZFN-L (FTV893), 2.5 μg of ZFN-R (FTV894), and 5 μg of donor construct for AAVS1 targeted insertion. In genome editing via CRISPR, 2 million iPSCs were transfected with a mixture of 5 μg of ROSA26-gRNA / Cas9 (FTV922) and 5 μg of donor construct for ROSA26 targeted insertion. Transfection was performed using the Neon transfection system (Life Technologies) with parameters 1500V, 10 ms, 3 pulses. On the 2nd or 3rd day after transfection, when the plasmid contained an artificial promoter-driven GFP and / or RFP expression cassette, flow cytometry was used to measure the transfection efficiency. On the 4th day after transfection, puromycin was added to the medium at a concentration of 0.1 μg / mL for the first 7 days and at a concentration of 0.2 μg / mL 7 days later to select for target cells. During puromycin selection, the cells were passaged into freshly Matrigel-coated wells on the 10th day. After the 16th day of puromycin selection, the surviving cells were analyzed by flow cytometry for the percentage of GFP + iPS cells.

[0300] Bulk sorting and clonal sorting of genome-edited iPSCs: iPSCs containing genome-targeted editing using ZFN or CRISPR-Cas9 were GFP-sorted 20 days after puromycin selection. + SSEA4 + TRA181 +Bulk sorting and clone sorting of iPSCs were performed. A single-cell dissociation-targeted iPSC pool was newly prepared and resuspended in a chilled staining buffer containing Hank's balanced salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1× penicillin / streptomycin (Mediatech), and 10 mM Hepes (Mediatech) for optimal performance. Freshly made for optimal performance. Conjugated primary antibodies such as SSEA4-PE and TRA181-Alexa Fluor-647 (BD Biosciences) were added to the cell solution and incubated on ice for 15 minutes. All antibodies were used at 7 μL in 100 μL of staining buffer per million cells. The solution was washed once with staining buffer, spun down at 225 g for 4 minutes, resuspended in staining buffer containing 10 μM thiazobib, and maintained on ice for sorting by flow cytometry. Sorting by flow cytometry was performed using a FACS Aria II (BD Biosciences). For bulk sorting, GFP + SSEA4 + TRA181 +Cells were gated and sorted into 15 mL standard tubes filled with 7 mL of FMM. For clone sorting, a 100 μM nozzle was used to directly dispense the sorted cells into 96-well plates at a concentration of 3 events per well. Each well was pre-filled with 200 μL of FMM supplemented with 5 μg / mL fibronectin and 1× penicillin / streptomycin (Mediatech) and had been pre-coated with 5× Matrigel overnight. For 5× Matrigel pre-coating, one aliquot of Matrigel was added to 5 mL of DMEM / F12, incubated overnight at 4 °C to resusp...

Claims

1. A cell or a group thereof, (i) The cells are (a) immune cells, (b) induced pluripotent cells (iPSCs), cloned iPSCs, or iPS cell lines, or (c) derived cells obtained by differentiating the cells of (b), (ii) The cells mentioned above, (a) Extracellular Fas-binding domains comprising a partial or complete peptide of the extracellular domain (ECD) of the Fas receptor (FAS), or a variant or allele thereof, and (b) comprising an exogenous polynucleotide encoding a signaling redirector receptor, which includes a cytoplasmic signaling domain containing a partial or complete peptide of the intracellular domain (ICD) of one or more co-stimulatory molecules, A cell or population thereof in which the signal transduction redirector receptor is a Fas redirector that redirects Fas signaling upon binding to a FAS agonist, thereby providing the cell or derived cell with improved apoptosis resistance and / or attrition resistance.

2. (i) The Fas redirector further comprises a transmembrane region including the transmembrane domain of a transmembrane protein or a part thereof, (ii) The one or more co-stimulatory molecules include CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof. (iii) The one or more co-stimulatory molecules that do not contain 41BB, (iv) The FAS agonist comprises Fas ligand (FasL), or (v) The cells said to be (a) Exogenous polynucleotides encoding chimeric antigen receptors (CARs), (b) Exogenous polynucleotides or variants thereof that encode CD16, (c) CD38 knockout, and (d) one or more exogenous polynucleotides encoding a cytokine signaling complex comprising a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor, according to claim 1, the cell or population thereof.

3. The transmembrane region of the Fas redirector is (i) the full length or at least a portion of the native or modified transmembrane domain of FAS, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, 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, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or the full length or at least a portion of the native or modified transmembrane domain of a T cell receptor polypeptide, or (ii) The cell or population thereof according to claim 2, comprising the full length or partial length of the transmembrane domain of FAS.

4. (i) The extracellular binding domain of the Fas redirector contains a sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with respect to Sequence ID No. 1, (ii) The cytoplasmic signaling domain of the Fas redirector (a) The full length or partial length of the intracellular domain (ICD) of MyD88 and CD40 as represented by Sequence ID No. 2, or (b) The full length or partial length of the intracellular domain (ICD) of CD27, as represented by Sequence ID No. 3, or (c) The full length or partial length of the intracellular domain (ICD) of CD28, as represented by Sequence ID No. 4, or (d) The full length or partial length of the intracellular domain (ICD) of OX40 as represented by Sequence ID No. 5, or (e) The full length or partial length of the intracellular domain (ICD) of IL12Rβ2 as represented by Sequence ID No. 6, or (f) The full length or partial length of the intracellular domain (ICD) of IL18R1, as represented by Sequence ID No. 7, or (g) including the full length or partial length of the intracellular domain (ICD) of IL21R as represented by Sequence ID No. 8, or (iii) The cell or population thereof according to claim 1, wherein the Fas redirector comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with any one of sequence numbers 9 to 15.

5. The aforementioned cells, (i) At least one of the genotypes listed in Table 1, (ii) HLA-I deficiency and / or HLA-II deficiency, (iii) Introduction of HLA-G or non-cleaving HLA-G, or knockout of one or both CD58 and CD54. (iv) Deletion or destruction of at least one of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (v) HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ A cell or population thereof according to any one of claims 1 to 4, further comprising at least one of the following: R, introduction of at least one of an antigen-specific TCR, a chimeric fusion receptor (CFR), an Fc receptor, an antibody or a functional variant or fragment thereof, a checkpoint inhibitor, an engager, and a surface trigger receptor for coupling with a bispecific, multispecific, or universal engager.

6. The aforementioned derived cells, (a) Derivative CD34 ＋ Includes cells, derived hematopoietic stem progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional characteristics not present in the corresponding primary T cells, NK cells, NKT cells, and / or B cells, or (b) Allogeneic effector cells, wherein the effector cells are compared to their native corresponding cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue. (i) Improved persistence and / or survival rate, (ii) Increased resistance to activated recipient immune cells, (iii) Increased cytotoxicity, (iv) Improved tumor penetration, (v) Enhanced or acquired ADCC, (vi) Enhanced ability to migrate bystander immune cells to tumor sites and / or activate or mobilize them. (vii) Enhanced ability to reduce tumor immunosuppression, (viiii) Improved ability to rescue tumor antigen escapes, and (ix) Apoptosis and / or reduction of flatorides, The cell or population thereof according to claim 1, which is a derived NK cell or derived T cell having at least one of the features including the above.

7. The exogenous CD16 is, (a) High affinity non-cleavable CD16 (hnCD16) or its variant, (b) F176V and S197P in the external domain domain of CD16, (c) All or partial external domains derived from CD64, (d) Non-natural (or non-CD16) transmembrane domains, (e) Non-natural (or non-CD16) intracellular domains, (f) Non-natural (or non-CD16) signaling domains, (g) unnatural irritant domains, and (h) The cell or population thereof according to claim 2, comprising at least one of a transmembrane domain, a signaling domain, and a stimulating domain, which are not derived from CD16 and are derived from the same or a different polypeptide.

8. The cytokine signaling complex, (a) cell surface-expressed exogenous cytokines and / or partial or complete peptides of their receptors, comprising at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors, (b) (i) Co-expression of IL15 and IL15Rα with a self-cleaving peptide in between, (ii) Fusion protein of IL15 and IL15Rα (iii) IL15 / IL15Rα fusion protein (IL15Δ) in which the intracellular domain of IL15Rα is shortened. (iv) A fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) Fusion protein of IL15 and IL15Rβ, (vi) A fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either natural or modified, and (vii) At least one of the homodimers of IL15Rβ, (b) comprising at least one of (i) to (vii) which can be co-expressed with CAR in a separate construct or in a bicistronic construct, or (c) (i) A fusion protein of IL7 and IL7Rα, (ii) A fusion protein of IL7 and the common receptor γC, wherein the common receptor γC is either native or modified, and (iii) At least one of the homodimers of IL7Rβ, (c) Any one of (i) to (iii) is optionally co-expressed with CAR in a separate construct or in a bicistronic construct. And optionally, (d) The cells or population thereof according to claim 2, which are transiently expressed.

9. The cytoplasmic signaling domain of the Fas redirector (a) The full length or partial length of the intracellular domain (ICD) of MyD88 and CD40 as represented by Sequence ID No. 2, or (b) The full length or partial length of the intracellular domain (ICD) of CD27, as represented by Sequence ID No. 3, or (c) The cell or population thereof according to claim 1, comprising the full length or partial length of the intracellular domain (ICD) of OX40 as represented by Sequence ID No.

5.

10. The cells or population thereof according to claim 9, further comprising an exogenous polynucleotide encoding a fusion protein of a partial or complete peptide of IL7 and a partial or complete peptide of IL7Rα.

11. A composition comprising cells or a population thereof as described in any one of claims 1 to 4.

12. The composition according to claim 11, further comprising one or more therapeutic agents.

13. A composition according to claim 11 or 12 for use in adoptive cell therapy in a subject, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection.

14. A master cell bank (MCB) comprising cloned iPSCs according to any one of claims 1 to 4.

15. A method for producing a derived cell according to any one of claims 1 to 4, wherein the derived cell is an effector cell, and the method is Differentiating a genetically modified iPSC, wherein the genetically modified iPSC contains an exogenous polynucleotide that encodes a Fas redirector that redirects Fas signaling upon binding to a FAS agonist, A method that thereby provides effector cells with improved apoptosis resistance and / or attrition resistance.

16. The method further comprises genomically manipulating an iPSC to knock in (a) the polynucleotide encoding the signal transduction redirector receptor, optionally (b) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), and optionally (c) an exogenous polynucleotide encoding CD16 or a variant thereof, wherein optionally the iPSC is, (i) In order to knock out CD38, (ii) In order to knock out either or both B2M and CIITA, (iii) To knock out one or both of CD58 and CD54, and / or The method according to claim 15, further comprising genomically manipulating a signaling complex comprising (iv) HLA-G or uncleaved HLA-G, and / or a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor.

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