Enhancing effector cell persistence and efficacy in adoptive cell therapy

Abstract

The present invention provides methods and compositions for obtaining functionally enhanced derived effector cells obtained by directed differentiation of genome engineered iPSCs. Embodiments of the derived cells provided herein have stable and functional genome editing that delivers improved or enhanced therapeutic effects. Also provided are therapeutic compositions comprising functionally enhanced derived effector cells alone, or functionally enhanced derived effector cells treated in combination with an antibody or checkpoint inhibitor, and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 367,596, filed on July 1, 2022, which is hereby incorporated by reference in its entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically. The sequence listing entitled 184143-644601_SL.xml, created on June 28, 2023 and having a size of 30,164 bytes, is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates broadly to the field of off-the-shelf immune cell products. More specifically, the present disclosure relates to strategies for developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. Cell products developed according to the present disclosure address key limitations of patient-derived cell therapies. Background Art

[0006] The current focus of the adoptive cell therapy field is to use patient-derived cells and donor-derived cells, which makes it particularly difficult to realize the continuous manufacture of cancer immunotherapy and to deliver therapy to all patients who may benefit therefrom. It is also necessary to improve the efficacy and retention of adoptively transferred lymphocytes to promote good patient outcomes. Lymphocytes (such as T cells and natural killer (NK) cells) are effective anti-tumor effectors that play an important role in innate and adaptive immunity. However, it is still challenging to use these immune cells for adoptive cell therapy, and the demand for improvement has not yet been met. Therefore, in adoptive immunotherapy, there are still a lot of opportunities to utilize the full potential of T cells and NK cells or other lymphocytes. Summary of the invention

[0007] Functionally improved effector cells are needed to address issues ranging from response rate, cell exhaustion, transfused cell loss (survival and / or retention), tumor escape via target loss or lineage switching, tumor targeting precision, off-target toxicity, extra-tumor effects to efficacy against solid tumors, i.e., the tumor microenvironment and associated immunosuppression, recruitment, trafficking, and infiltration.

[0008] The purpose of the embodiments of the present invention is to generate a method and composition for the differentiation of a derived non-pluripotent cell derived from a single cell iPSC (induced pluripotent stem cell) clone, wherein the iPSC contains one or more genetic modifications in its genome. In some embodiments, the one or more genetic modifications include DNA insertion, deletion and substitution, and the modification is retained and remains functional in subsequent derived cells after differentiation, amplification, passage and / or transplantation.

[0009] The iPSC-derived non-pluripotent cells of the present application include but are not limited to CD34 cells, hemogenic endothelial cells, HSC (hematopoietic stem cells and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells and B cells.In some embodiments, the iPSC-derived non-pluripotent cells of the present application are differentiated by iPSCs comprising the same genetic modification and include one or more genetic modifications in their genomes.In some embodiments, the clone iPSC differentiation strategy for obtaining the engineered derivative cells of genetically engineered cells benefits from the developmental potential of iPSC in directed differentiation, which is not significantly affected by the adverse effects of the engineered mode in iPSC, and the engineered mode is also required to work in the derived cells as expected.In addition, this strategy overcomes the existing obstacles of engineered primary lymphocytes (such as T cells or NK cells obtained from peripheral blood), so cells are difficult to engineer, and such cells are generally lacking in reproducibility and uniformity, so that cells show the poor cell retention with high cell death and low cell amplification. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations that would otherwise be obtained using a starting heterogeneous primary cell source.

[0010] Therefore, 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 cell (iPSC), a cloned iPSC or an iPS cell line cell; or (c) a derived cell obtained by differentiating a cell of (b); (ii) the cell comprises an exogenous polynucleotide encoding a signaling redirection receptor, the signaling redirection receptor comprising (a) a partial or complete peptide of the extracellular domain (ECD) of the Fas receptor (FAS), or an extracellular Fas binding domain of a variant or allele thereof; and (b) a cytoplasmic signaling domain, the cytoplasmic signaling domain comprising a partial or complete peptide of the intracellular domain (ICD) of one or more co-stimulatory molecules, wherein the signaling redirection receptor is a Fas redirector, which redirects Fas signaling when bound to a FAS agonist, thereby providing the cell or the derivative cell with improved apoptosis resistance and / or exhaustion resistance. In various embodiments, (i) the Fas redirector further comprises a transmembrane region comprising a transmembrane domain or a portion thereof of a transmembrane protein; (ii) the one or more co-stimulatory molecules comprise CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof; (iii) the one or more co-stimulatory molecules do not include 41BB; (iv) the FAS agonist comprises Fas ligand (FasL); or (v) the cell further comprises one or more of the following: (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (b) an exogenous polynucleotide encoding CD16 or a variant thereof; (c) CD38 knockout; and (d) an exogenous polynucleotide encoding a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface. In some embodiments, the transmembrane region of the Fas redirector comprises (i) all or at least a portion 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) all or a portion of the transmembrane domain of FAS.

[0011] In some embodiments of the 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 OX40 represented by SEQ ID NO: 6. NO:6; or (f) the full length or a portion of the intracellular domain (ICD) of IL18R1 represented by SEQ ID NO:7; or (g) the full length or a portion of the intracellular domain (ICD) of IL21R represented by SEQ ID NO:8; or (iii) the Fas reorientor comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any one of SEQ ID NOs:9-15.

[0012] In various embodiments of the cell or its population, the cell further comprises one or more of the following: (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-cleavable HLA-G, or knockout of one or both of CD58 and CD54; (iv) B2M, CIITA, TAP1, TAP2, TAP-associated protein, NLRC5, RFXANK , RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (v) deletion or disruption of at least one of HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AR, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or its functional variant or fragment, checkpoint inhibitor, adapter and for the introduction of at least one of the surface triggering receptors coupled with bispecific or multispecific or universal adapter. In various embodiments of a cell or its population, the cell comprises HLA-I deficiency and / or HLA-II deficiency; and optionally, wherein the cell comprises an exogenous polynucleotide encoding HLA-G, HLA-E or its variant; and / or comprises one or both of CD54 and CD58. Deletion or destruction. In some embodiments, HLA-I lacks the deletion or destruction of at least one of the following: B2M, TAP1, TAP2 and TAP-related proteins; or HLA-II lacks the deletion or destruction of at least one of the following: CIITA, RFX5, RFXAP and RFXANK.

[0013] In various embodiments of the cell or its population, the derived cell: (a) comprises a derived CD34+ cell, a derived hematopoietic stem cell and progenitor cell, a derived hematopoietic multipotent progenitor cell, a derived T cell progenitor cell, a derived NK cell progenitor cell, a derived T cell, a derived NKT cell, a derived NK cell, a derived B cell, or a derived effector cell having one or more functional characteristics that are not present in the corresponding primary T cell, NK cell, NKT cell and / or B cell; or an allogeneic effector cell, wherein the effector cell is a derived NK cell or a derived T cell having at least one of the following characteristics: Cells, the properties of which include: (i) improved persistence and / or survival, (ii) increased resistance to activated recipient immune cells, (iii) increased cytotoxicity, (iv) improved tumor penetration, (v) enhanced or acquired ADCC, (vi) enhanced ability of bystander immune cells to migrate to and / or be activated or recruited to tumor sites, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability to rescue tumor antigen escape, and (ix) reduced apoptosis and / or fratricide, compared to their native counterparts obtained from peripheral blood, umbilical cord blood or other donor tissues.

[0014] In some embodiments of a cell or a population thereof, wherein the cell comprises an exogenous polynucleotide encoding CD16 or a variant thereof, the exogenous CD16 comprises at least one of the following: (a) high affinity non-cleavable CD16 (hnCD16) or a variant thereof; (b) F176V and S197P in the extracellular domain of CD16; (c) all or part of the 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 C 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.

[0015] In some embodiments of a cell or a population thereof, wherein the cell comprises 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 dimerizing CAR; (v) a separate CAR; (vi) a multi-chain CAR; (vii) an inducible CAR; (viii) optionally co-expressed in a separate construct or in a bicistronic construct with a cytokine signaling complex comprising a cell surface expressed exogenous cytokine and / or a partial or complete peptide of its receptor; (ix) optionally co-expressed in a separate construct or in a bicistronic construct with a checkpoint inhibitor; and / or (x) optionally inserted at (1) a TRAC or TRBC locus, and / or driven by an endogenous promoter of the TCR, and / or the TCR is knocked out by CAR insertion; (2) a safe harbor locus; or (3) a locus intended for disruption.In some embodiments, the CAR: (i) is specific for at least one of: 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) is specific for any of: 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, 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 kinase erb-B2, 3,4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit α-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, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms' tumor protein (WT-1), and pathogen antigens.

[0016] In some embodiments of a cell or a population thereof, wherein the cell comprises an exogenous polynucleotide encoding a cytokine signaling complex, the cytokine signaling complex comprises: (a) a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, comprising at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 or their corresponding receptors; or (b) at least one of the following: (i) co-expression of IL15 and IL15Rα with a self-cleaving peptide therebetween; (ii) a fusion protein of IL15 and IL15Rα; (iii) an IL15 / IL15Rα fusion protein (IL15Δ) in which the intracellular domain of IL15Rα is truncated; (iv) a membrane-bound Sushi domain of IL15 and IL15Rα (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and a co-receptor γC, wherein the co-receptor γC is native or modified; and (vii) a homodimer of IL15Rβ; wherein any one of (b)(i) to (vii) is capable of being co-expressed with CAR in a separate construct or in a bicistronic construct; or (c) at least one of the following: (i) a fusion protein of IL7 and IL7Rα; (ii) a fusion protein of IL7 and a co-receptor γC, wherein the co-receptor γC is native or modified; and (iii) a homodimer of IL7Rβ, wherein any one of (c)(i)-(iii) is optionally co-expressed with CAR in a separate construct or in a bicistronic construct; and optionally, (d) is transiently expressed.

[0017] In various embodiments of the cell or its population, the derived cell is a derived NK cell or a derived T cell, wherein the derived NK cell is capable of recruiting and / or migrating T cells to the tumor site, and wherein the derived NK cell or the derived T cell is capable of reducing 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, and the checkpoint molecules include PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AR, 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 inhibitory KIR. In some embodiments, the one or more checkpoint inhibitors include: (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents; or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0018] In various embodiments of a cell or a population thereof, the cell comprises: (i) one or more exogenous polynucleotides integrated in a safe harbor locus or a locus intended for destruction; or (ii) more than two exogenous polynucleotides integrated in different safe harbor loci or loci intended for destruction. In some embodiments, one or more safe harbor loci comprise at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1; or wherein the one or more loci intended for destruction comprise at least one of B2M, TAP1, TAP2, TAP-associated protein, 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.

[0019] In some embodiments of the cell or its population, the cytoplasmic signaling domain of the Fas redirector in the cell comprises at least one of the following: the full or partial length of the intracellular domain (ICD) of MyD88 and CD40 represented by SEQ ID NO: 2; or the full or partial length of the intracellular domain (ICD) of CD27 represented by SEQ ID NO: 3; or the full or partial length of the intracellular domain (ICD) of OX40 represented by SEQ ID NO: 5. In some other embodiments, the cell or its population comprising the cytoplasmic signaling domain further comprises an exogenous polynucleotide encoding a fusion protein of a partial or complete peptide of IL7 and a partial or complete peptide of IL7Rα.

[0020] In another aspect, the present invention provides a composition comprising a cell or a colony thereof as described herein. In various embodiments, the composition further comprises one or more therapeutic agents. In some embodiments, one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, carriers comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties or immunomodulatory drugs (IMiDs). In some embodiments in which the therapeutic agent is a checkpoint inhibitor, (i) the checkpoint inhibitor includes: (a) one or more antagonist checkpoint molecules, the one or more antagonist checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AR, 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, lirimumab, monalizumab, nivolumab, pembrolizumab and their derivatives or functional equivalents; (c) at least one of atezolizumab, nivolumab and pembrolizumab; or (ii) the therapeutic agent includes one or more of venetola, azacitidine and pomalidomide. In some embodiments in which 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, veltuzumab, ofatumumab, utuuximab, okatuzumab, obtuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, detuximab, avelumab, daclizumab, or a combination thereof. Monoclonal antibody, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, bivacizumab, RG7356, G44-26, 7G3, CSL362, etozizumab and their humanized or Fc-modified variants or fragments and their functional equivalents and biosimilars thereof.In some embodiments in which the therapeutic agent is an adaptor, the adaptor comprises: (i) a bispecific T cell adaptor (BiTE); (ii) a bispecific killer cell adaptor (BiKE); or (iii) a trispecific killer cell adaptor (TriKE); or the adaptor comprises: (a) a first binding domain that recognizes an extracellular portion of CD3, CD5, CD16, CD28, CD32, CD33, CD64, CD89, NKG2C, NKG2D, or any functional variant thereof of the cell or an alternative immune effector cell; and (b) a second binding domain that is specific for an antigen comprising any of: B 7H3, 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.

[0021] In another aspect, the present invention provides a therapeutic use of a composition provided herein by introducing the composition into a subject in need of adoptive cell therapy, wherein the subject suffers from an autoimmune disorder, a hematological malignancy, a solid tumor, a cancer, or a viral infection.

[0022] In another aspect, the present invention provides a master cell bank (MCB) comprising the clonal iPSCs provided herein.

[0023] In another aspect, the present invention provides a method for making a derivative cell provided herein, wherein the derivative cell is an effector cell, and the method comprises: differentiating a genetically engineered iPSC, wherein the genetically engineered iPSC comprises an exogenous polynucleotide encoding a Fas redirector, which redirects Fas signaling when bound to a FAS agonist, thereby providing the effector cell with improved apoptosis resistance and / or exhaustion resistance. In some embodiments of the manufacturing method, the Fas redirector comprises: (a) a partial or complete peptide comprising an extracellular domain (ECD) of a Fas receptor (FAS), or an extracellular Fas binding domain of a variant or allele thereof; and (b) a cytoplasmic signaling domain comprising a partial or complete peptide of an intracellular domain (ICD) of one or more costimulatory molecules; and wherein the genetically engineered iPSC is a single cell, a clonal cell, or a cell line cell. In some embodiments of the manufacturing method, (i) the Fas redirector further comprises a transmembrane region, which comprises a transmembrane domain or a portion thereof of a transmembrane protein; (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 do not include 41BB; (iv) the FAS agonist includes Fas ligand (FasL); or (v) the genetically engineered iPSC comprising the Fas redirector further comprises one or more of the following: (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (b) an exogenous polynucleotide encoding CD16 or a variant thereof; (c) CD38 knockout; and (d) an exogenous polynucleotide encoding a cytokine signaling complex, which comprises a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface.

[0024] In some embodiments of the manufacturing method, the transmembrane region of the Fas redirector comprises (i) all or at least a portion 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) all or a portion of the transmembrane domain of FAS. In some embodiments, (i) the Fas redirector further comprises a transmembrane region comprising a transmembrane domain or a portion thereof of a transmembrane protein; (ii) the one or more co-stimulatory molecules comprise CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof; (iii) the one or more co-stimulatory molecules do not include 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 the following: (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (b) an exogenous polynucleotide encoding CD16 or a variant thereof; (c) CD38 knockout; and (d) an exogenous polynucleotide encoding a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface. In some embodiments, the transmembrane region of the Fas redirector comprises (i) all or at least a portion 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) all or a portion 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 OX40 represented by SEQ ID NO: 6. NO:6; or (f) the full length or a portion of the intracellular domain (ICD) of IL18R1 represented by SEQ ID NO:7; or (g) the full length or a portion of the intracellular domain (ICD) of IL21R represented by SEQ ID NO:8; or (iii) the Fas reorientor comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any one of SEQ ID Nos:9-15.

[0025] In various embodiments of the manufacturing method, the genetically engineered iPSC further comprises one or more edits that produce: (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-cleavable HLA-G, or knockout of one or both of CD58 and CD54; (iv) B2M, CIITA, TAP1, TAP2, TAP-associated protein, NLRC 5, disruption of at least one of RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; and / or (v) disruption of at least one of HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AR, antigen-specific TCR, chimeric fusion receptor (CFR), Fc receptor, antibody or its functional variant or fragment, check point inhibitor, adapter and for the introduction of at least one of surface triggering receptors coupled to bispecific or multispecific or universal adapter. In some embodiments, the cell comprises HLA-I deficiency and / or HLA-II deficiency; and optionally, wherein the cell comprises exogenous polynucleotides encoding HLA-G, HLA-E or variants thereof; or comprises a deletion or destruction of one or both of CD54 and CD58.

[0026] In various embodiments of the manufacturing method, the method further includes: genome engineering iPSC to knock in: (a) polynucleotides encoding signaling redirection receptors; and optionally, (b) exogenous polynucleotides encoding chimeric antigen receptors (CAR); and optionally, (c) exogenous polynucleotides encoding CD16 or its variants; and optionally also including genome engineering iPSC to: (i) knock out CD38, (ii) knock out one or both of B2M and / or CIITA, (iii) knock out one or both of CD58 and CD54, and / or (iv) introduce HLA-G or non-cleavable HLA-G and / or a signaling complex comprising a portion or complete peptide of an exogenous cytokine expressed on the cell surface and / or its receptor. In some embodiments, the genome engineering includes targeted editing. In some embodiments, the targeted editing includes deletion, insertion, or insertion / deletion, and wherein the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variant of these methods.

[0027] In another aspect, the present invention provides a method of improving effector cell persistence or preventing cell death of effector cells in adoptive cell therapy provided to a subject in need thereof by obtaining effector cells as provided herein.

[0028] In another aspect, the present invention provides a method for improving the efficacy of adoptive cell therapy provided to a subject in need, the method comprising administering the effector cell to the subject, wherein the effector cell comprises a derivative cell or a colony thereof as provided herein. In some embodiments, the method further comprises administering one or more therapeutic agents to the subject. In some embodiments, one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, carriers comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties or immunomodulatory drugs (IMiDs). In some embodiments in which the therapeutic agent is a checkpoint inhibitor, (i) the checkpoint inhibitor includes: (a) one or more antagonist checkpoint molecules, the one or more antagonist checkpoint molecules comprising PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AR, 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, lirimumab, monalizumab, nivolumab, pembrolizumab and their derivatives or functional equivalents; (c) at least one of atezolizumab, nivolumab and pembrolizumab; or (ii) the therapeutic agent includes one or more of venetola, azacitidine and pomalidomide. In some embodiments in which 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, veltuzumab, ofatumumab, utuuximab, okatuzumab, obtuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, detuximab, avelumab, daclizumab, or a combination thereof. Monoclonal antibody, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, bivacizumab, RG7356, G44-26, 7G3, CSL362, etozizumab and their humanized or Fc-modified variants or fragments and their functional equivalents and biosimilars thereof.In some embodiments in which the therapeutic agent is an adaptor, the adaptor comprises: (i) a bispecific T cell adaptor (BiTE); (ii) a bispecific killer cell adaptor (BiKE); or (iii) a trispecific killer cell adaptor (TriKE); or the adaptor comprises: (a) a first binding domain that recognizes an extracellular portion of CD3, CD5, CD16, CD28, CD32, CD33, CD64, CD89, NKG2C, NKG2D, or any functional variant thereof of the cell or an alternative immune effector cell; and (b) a second binding domain that is specific for an antigen comprising any of: B 7H3, 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.

[0029] In another aspect, the present invention provides a method of treating a subject in need of adoptive cell therapy, wherein the method comprises infusing effector cells into the subject, wherein the effector cells comprise a combination of a derivative cell or a population thereof as provided herein and an exogenous cytokine.

[0030] Various objects and advantages of the compositions and methods as provided herein will become apparent from the following description taken in conjunction with the accompanying drawings in which are set forth by way of illustration and example certain embodiments of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1A-Figure 1D showed that iPSC-derived CAR T cells can be transduced with Fas redirecting constructs and expanded in a scalable manufacturing process.

[0032] Figure 2A and Figure 2B Figure 3 FasMFI expression on transduced iPSC-derived CAR T cells throughout T cell differentiation ( Figure 2A ) and Thy1.2 positive percentage ( Figure 2B ).

[0033] Figure 3A and Figure 3BThe Fas redirecting construct is shown to enhance CAR iT killing of engineered SKOV3 expressing CAR-specific antigens. Error bars indicate standard error of the mean; N=6.

[0034] Figure 4 The Fas redirecting construct is shown to enhance CAR iT cell expansion and retention after stimulation against engineered SKOV3 expressing a CAR-specific antigen. Error bars indicate standard error of the mean; N=6.

[0035] Figure 5 FAS redirection constructs are shown to enhance CAR iT cell killing of NALM6 target cells. Error bars indicate standard error of the mean; N=6.

[0036] Figure 6 The Fas redirecting construct was shown to enhance CAR iT cell expansion and retention after stimulation against NALM6 target cells.

[0037] Figure 7 The Fas redirecting construct was shown to provide protection to CAR iT cells against FAS agonist-induced apoptosis.

[0038] Figure 8 The Fas redirecting construct was shown to promote downstream NF-κB signaling.

[0039] Fig. 9 Table showing the sample list used for bulk RNAseq analysis.

[0040] Figures 10A-10D Shown are exemplary experimental results of differential gene expression analysis comparing non-transduced cells to Fas redirected iT cells.

[0041] Figures 11A-11D Shown are exemplary experimental results of differential gene expression analysis comparing CAR-iT cells expressing a Fas dominant-negative construct to CAR-iT cells expressing a Fas redirecting construct.

[0042] Fig. 12A and Fig. 12B Exemplary experimental results showing that Fas-MyD88 / CD40 expressing CD19-CAR_IL7RF exhibited enhanced long-term killing against engineered SKOV3 expressing CD19 are provided.

[0043] Fig.13A and Fig. 13B Exemplary experimental results showing that Fas-MyD88 / CD40 expressing CD19-CAR_IL7RF exhibits enhanced retention against engineered SKOV3 expressing CD19 are provided.

[0044] Fig.14 Exemplary experimental results showing that Fas-MYD88 / CD40 enhances tumor clearance against the SKOV3.CD19 tumor model are provided.

[0045] Fig.15 Exemplary experimental results showing that Fas-MyD88 / CD40 enhances CAR retention and T cell activation in the tumor microenvironment are provided. DETAILED DESCRIPTION

[0046] The genome modification of iPSC (induced pluripotent stem cell) includes polynucleotide insertion, deletion and substitution.Exogenous gene expression in genome engineered iPSC is generally encountered problems, such as after the long-term clonal expansion of initial genome engineered iPSC, after cell differentiation and in the dedifferentiation cell type of the cell derived from genome engineered iPSC, gene silencing or gene expression reduction.On the other hand, direct engineering of primary immune cells such as T cells or NK cells is challenging, and there are obstacles to the preparation and delivery of engineered immune cells for adoptive cell therapy.In various embodiments, the present invention provides effective, reliable and targeted methods for stably integrating one or more exogenous genes (including suicide genes and other functional modes) into iPSC derived cells, the one or more exogenous genes provide and transplant, transport, homing, migration, cytotoxicity, vitality, maintenance, expansion, life span, self-renewal, retention and / or survival-related improved therapeutic characteristics, these iPSC derived cells include but are not limited to HSC (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells and NK cells.

[0047] definition

[0048] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those skilled in the art. In addition, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0049] It should be understood that the present invention is not limited to the specific methodology, protocols and reagents etc. described herein and may vary accordingly. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0050] As used herein, the articles "a", "an" and "the" herein refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0051] The use of alternatives such as "or" should be understood to mean either one, both, or any combination of the alternatives.

[0052] The term "and / or" should be understood to mean one or both of the alternatives.

[0053] As used herein, the term "about" or "approximately" refers to a range of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0054] As used herein, the term "substantially" or "essentially" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "substantially the same" or "essentially the same" refers to a range of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, amounts, weights, or lengths that are about the same as a reference amount, level, value, number, frequency, percentages, dimensions, sizes, amounts, weights, or lengths.

[0055] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition (e.g., a cell population or culture medium), refer to a composition that is free of a specified substance or a source thereof, e.g., 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable, as measured by conventional means. The term "free" or "essentially free" of a certain ingredient or substance in a composition also means that (1) such ingredient or substance is not included in any concentration in the composition, or (2) such ingredient or substance is included in a functionally inert, low concentration in the composition. Similar meanings can be applied to the term "lacking", which refers to the lack of a specific substance or a source thereof in a composition.

[0056] Throughout this specification, unless the context requires otherwise, the word "comprising" should be understood to imply the inclusion of a stated step or element or a group of steps or elements, but not the exclusion of any other step or element or a group of steps or elements. In specific embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0057] The phrase “consisting of is intended to include and be limited to whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or necessary, and that no other elements may be present.

[0058] By "consisting essentially of is meant to include any of the elements listed after the phrase, and is limited to other elements that do not interfere with or affect the activity or function of the listed elements as specified in the present disclosure. Thus, the phrase "consisting essentially of indicates that the listed elements are required or necessary, but other elements are optional and may be present or absent depending on whether they affect the activity or function of the listed elements.

[0059] Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner.

[0060] The term "ex vivo" generally refers to an activity that occurs outside an organism, such as an experiment or measurement performed in or on a living tissue in an artificial environment outside an organism, preferably wherein the change in natural conditions is minimal. In a specific embodiment, an "ex vivo" procedure involves obtaining living cells or tissues from an organism and cultivating them in a laboratory device under sterile conditions, and typically cultivating for a few hours or up to about 24 hours, but including up to 48 hours or 72 hours or longer, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and thawed later to carry out ex vivo processing. Tissue culture experiments or procedures that use living cells or tissues for a duration longer than a few days are generally considered "in vitro", but in certain embodiments, this term can be used interchangeably with ex vivo.

[0061] The term "in vivo" generally refers to activities that take place inside an organism.

[0062] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potency" or "increasing developmental potency" refers to a method of increasing cell potency or dedifferentiating cells into a less differentiated state. For example, cells with increased cell potency have greater developmental plasticity (i.e., can differentiate into more cell types) than the same cells in a non-reprogrammed state. In other words, reprogrammed cells are cells in a less differentiated state than the same cells in a non-reprogrammed state.

[0063] As used herein, the term "differentiation" is a process by which unspecialized ("unspecialized") or weakly specialized cells obtain the characteristics of specialized cells (e.g., blood cells or muscle cells). Differentiated cells or differentiation-inducible cells are cells that have been in a more specialized ("specialized") position within a cell lineage. The term "specialization" refers to a cell in a differentiation process that has progressed to a point in the differentiation path where it will continue to differentiate into a subpopulation of a specific cell type or cell type under normal circumstances and where it cannot differentiate into different cell types or revert to a weaker differentiated cell type under normal circumstances. As used herein, the term "pluripotent" refers to the ability of cells to form all lineages of a body or soma (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell that can form three germ layers: cells of each of the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potency ranging from incomplete or partially pluripotent cells that cannot give rise to a whole organism (e.g., epiblast stem cells or EpiSCs) to more primitive, more potent cells that can give rise to a whole organism (e.g., embryonic stem cells).

[0064] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to stem cells generated in vitro from differentiated adult, neonatal or fetal cells using reprogramming factors and / or small molecule chemical drive methods, which have been induced or altered, i.e., reprogrammed, to be capable of differentiating into tissues of all three germ layers or dermis: mesoderm, endoderm and ectoderm. The iPSCs generated do not refer to cells as they are found in nature.

[0065] As used herein, the term "embryonic stem cell" refers to a naturally occurring pluripotent stem cell in the inner cell mass of an embryonic blastocyst. Embryonic stem cells are pluripotent and produce all the derivative cells of the three primary germ layers: ectoderm, endoderm, and mesoderm during development. It does not contribute to the extraembryonic membrane or placenta (i.e., it is not differentiated totipotent).

[0066] As used herein, the term "pluripotent stem cell" refers to a cell with the developmental potential to differentiate into one or more germ layers (ectoderm, mesoderm and endoderm) but not all three germ layers. Therefore, pluripotent cells may 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" means that cells can form many types of cells in a given lineage, rather than cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (erythrocytes, leukocytes, platelets, etc.), but they cannot form neurons. Therefore, the term "pluripotency" refers to a cell state in which the degree of developmental potential is lower than differentiation omnipotence and pluripotency.

[0067] Pluripotency can be determined in part by assessing the pluripotency properties of the cells. Pluripotency properties include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50; (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation composed of the three somatic cell lineages; and (vi) the formation of embryoid bodies composed of cells from the three somatic cell lineages.

[0068] Two types of pluripotency have been described previously: the "stimulated" or "metastable" pluripotency state is equivalent to the epiblast stem cells (EpiSCs) of the late blastocyst, and the "naive" or "basal" pluripotency state is equivalent to the inner cell mass of the early / preimplantation blastocyst. Although both pluripotency states exhibit the properties 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 during single-cell culture; (iii) overall reduction in DNA methylation; (iv) reduced deposition of the H3K27me3 repressive chromatin mark on the promoters of developmentally regulated genes; and (v) reduced expression of differentiation markers relative to pluripotent cells in the stimulated state. It is generally found that the standard method of cell reprogramming (in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or removed from the resulting pluripotent cells) has the characteristics of the stimulated state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the stimulated state unless exogenous transgene expression is maintained (in which the characteristics of the basal state are observed).

[0069] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a small round shape, a high nuclear to cytoplasmic ratio, a prominent nucleolus, and typical intercellular spacing.

[0070] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0071] "Pluripotency factor" or "reprogramming factor" refers to an agent that can enhance the developmental efficacy of a cell alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can improve the developmental efficacy of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0072] "Cultivation" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "medium" (in each case, the singular form is "medium"), "supplement" and "medium supplement" refer to a nutritional composition for growing cell cultures.

[0073] "Cultivating" or "maintaining" refers to the maintenance, propagation (growth) and / or differentiation of cells outside of a tissue or body (e.g., in a sterile plastic (or coated plastic) cell culture dish or flask). "Cultivating" or "maintaining" can use a culture medium as a source of nutrients, hormones and / or other factors that aid in the propagation and / or maintenance of cells.

[0074] As used herein, the term "mesoderm" refers to one of the three germ layers that appears during early embryogenesis and gives rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, bone, and other supporting and connective tissues.

[0075] As used herein, the term "determined hemogenic endothelial cells" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelial cells" (iHE) refers to a subpopulation of endothelial cells that give rise to hematopoietic stem cells and progenitor cells in a process known as the endothelial-to-hematopoietic transition. Hematopoietic cell development in the embryo proceeds sequentially: from lateral plate mesoderm to angioblasts to definitive hemogenic endothelial cells and hematopoietic progenitor cells.

[0076] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells" or "hematopoietic precursor cells" refer to cells that are specialized in the hematopoietic lineage but are capable of further differentiation into hematopoiesis, and include multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). As used herein, the term "permanent hematopoietic stem cell" refers to a CD34 + Hematopoietic cells, which are capable of giving rise to mature myeloid cell types and lymphocyte cell types, include cells of the T-lineage, NK-lineage, and B-lineage. Hematopoietic cells also include various subsets of primitive hematopoietic cells, which give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0077] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that completes maturation in the thymus and has multiple roles in the immune system, including identifying specific foreign antigens in the body and activating and deactivating other immune cells in an MHC class I restricted manner. The T cell can be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal. The T cell can be a CD3 + T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD8 +T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, initial T cells, regulatory T cells, gamma delta T cells (γδT cells), etc. Other types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Other 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" may also refer to genetically engineered T cells, such as T cells that are modified to express T cell receptors (TCR) or chimeric antigen receptors (CAR). T cells or T cell-like effector cells may also be differentiated by stem cells or progenitor cells ("derived T cells" or "derived T cell-like effector cells", or collectively referred to as "derived T lineage cells"). Derived T cell-like effector cells may have T cell lineages in some aspects, but at the same time have one or more functional characteristics that are not present in primary T cells. In the present 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".

[0078] "CD4 + "T cells" refers to a subpopulation of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a secretory profile upon stimulation that may include secretion of cytokines such as IFN-γ, TNF-α, IL2, IL4, and IL10. The "CD4" molecule is a 55-kD glycoprotein that was originally defined as a differentiation antigen on T lymphocytes but is also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is indicated as a relevant recognition element in major histocompatibility complex (MHC) class II restricted immune responses. On T lymphocytes, it defines a subset of helper / inducer factors.

[0079] "CD8 + "T cells" refers to a subpopulation of T cells that express CD8 on their surface, are restricted to MHC class I, and act as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a relevant recognition element in major histocompatibility complex class I-restricted interactions.

[0080] As used herein, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). The NK cell can be any NK cell, such as a cultured NK cell, e.g., a primary NK cell, or an NK cell from a cultured or expanded NK cell or a cell line NK cell, e.g., NK-92, or an NK cell obtained from a healthy or diseased mammal. As used herein, the term "adaptive NK cell" is interchangeable with "memory NK cell" and refers to a subset of NK cells whose phenotype is CD3 - and CD56 + , expresses at least one of NKG2C and CD57 and optionally CD16, but lacks expression of one or more of: PLZF, SYK, FceRγ, and EAT-2. In some embodiments, the isolated CD56 + NK cell subsets include expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. + Can be weaker or stronger expression.NK cell or NK cell-like effector cell can be differentiated by stem cell or progenitor cell (" derived NK cell " or " derived NK cell-like effector cell ", or be collectively referred to as " derived NK lineage cell "). Derived NK cell-like effector cell can have NK cell lineage in some aspects, but have one or more functional characteristics that are not present in primary NK cell at the same time. In the present application, NK cell, NK cell-like effector cell, derived NK cell, derived NK cell-like effector cell or derived NK lineage cell are collectively referred to as " NK lineage cell ".

[0081] As used herein, the term "NKT cell" or "natural killer T cell" or "NKT lineage cell" refers to a T cell restricted to CD1d that expresses 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. Constant or type I NKT cells express a very limited TCR repertoire: a combination of a typical α chain (Vα24-Jα18 in humans) and a limited spectrum of β chains (Vβ11 in humans). The second NKT cell population, called non-classical or non-constant type II NKT cells, shows a more uneven TCRαβ utilization. Type I NKT cells are considered suitable for immunotherapy. Adaptive or constant (type I) NKT cells can be identified by the expression of one or more of the following markers: TCR Va24-Ja18, Vb11, CDld, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0082] The term "effector cell" is generally applicable to certain cells that perform a specific activity in response to stimulation and / or activation in the immune system, or to cells that perform a specific function when activated. As used herein, the term "effector cell" includes immune cells, "differentiated immune cells", and primary or differentiated cells that are edited and / or regulated to perform a specific activity in response to stimulation and / or activation, and these terms are interchangeable in some cases. Non-limiting examples of effector cells include primary-derived or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0083] As used herein, the term "separation" etc. refers to a cell or cell group that has been separated from its initial environment, that is, the environment of the separated cell is substantially free of at least one component found in the environment of the "unseparated" reference cell. The term includes cells removed from some or all components, as they are found in their natural environment, such as separated from a tissue or biopsy sample. The term also includes cells removed from at least one, some or all components, as the cells are found in a non-naturally occurring environment, such as separated from a cell culture or a cell suspension. Therefore, "separated cells" are partially or completely separated from at least one component (including other substances, cells or cell groups), as they are found in nature or as they grow, store or survive in a non-naturally occurring environment. Specific examples of separated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in non-naturally occurring culture media. Separated cells can be obtained by separating the desired cell or its group from other substances or cells in the environment or by removing one or more other cell groups or subgroups from the environment.

[0084] As used herein, the term "purified" and the like refers to increased purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

[0085] As used herein, the term "encoding" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in a biological process, which have a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties obtained therefrom. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is consistent with the mRNA sequence and is usually provided in a sequence table) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA.

[0086] "Construct" refers to a macromolecule or molecular complex containing a polynucleotide to be delivered to a host cell in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell, in which the nucleic acid construct can be replicated and / or expressed. Therefore, the term "vector" includes the construct to be delivered. The vector can be a linear or circular molecule. The vector can be an integrating or non-integrating vector. The main types of vectors include, but are not limited to, plasmids, free 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.

[0087] So-called "integration" means that one or more nucleotides of the construct are stably inserted into the cell genome, that is, covalently linked to the nucleic acid sequence in the cell chromosome DNA. "Targeted integration" means that the nucleotides of the construct are inserted into the cell chromosome or mitochondrial DNA at a preselected site or "integration site". As used herein, the term "integration" further refers to a process that involves inserting one or more exogenous sequences or nucleotides of the construct into the integration site when the endogenous sequence or nucleotide is missing or not missing. In the case of a missing insertion site, "integration" may also include replacing the missing endogenous sequence or nucleotide with one or more inserted nucleotides.

[0088] As used herein, the term "exogenous" is intended to mean that a reference molecule or reference activity is introduced into a host cell, or is non-native to a host cell. The molecule can be introduced, for example, by introducing a coding nucleic acid into the host genetic material, such as integrated into a host chromosome, or as a non-chromosomal genetic material, such as a plasmid. Therefore, the term, when used with respect to the expression of a coding nucleic acid, refers to the introduction of a coding nucleic acid into a cell in an expressible form. The term "endogenous" refers to a reference molecule or activity present in a host cell. Similarly, the term, when used with respect to the expression of a coding nucleic acid, refers to the expression of a coding nucleic acid contained in a cell rather than introduced exogenously.

[0089] As used herein, a "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that is transcribed into RNA in vivo and, in some cases, translated into a polypeptide when placed under the control of appropriate regulatory sequences. Genes or polynucleotides of interest may include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode a miRNA, shRNA, a native polypeptide (i.e., a polypeptide found in nature) or fragments thereof; a variant polypeptide (i.e., a mutant of a native polypeptide having less than 100% sequence identity with a native polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.

[0090] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length or its analogs. A polynucleotide sequence is composed of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) (when the polynucleotide is RNA, uracil replaces thymine). A polynucleotide may include a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. "Polynucleotide" also refers to double-stranded and single-stranded molecules.

[0091] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules in which amino acid residues are covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and the maximum number of amino acids of a polypeptide is not limited. As used herein, the terms refer to short chains (also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (commonly referred to in the art as polypeptides or proteins). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins and others. The polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides or combinations thereof.

[0092] As used herein, the term "subunit" as used herein refers to each individual polypeptide chain of a protein complex, wherein each individual polypeptide chain can form a stable folded structure by itself. Many protein molecules are composed of more than one subunit, wherein the amino acid sequence can be consistent, 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, the continuous portion of the polypeptide chain is often folded into a compact, local, semi-independent unit, called a "domain". Many protein domains may also contain independent "structural subunits", also called subdomains, which contribute to the common function of the domain. Therefore, as used herein, the term "subdomain" refers to a protein domain within a larger domain, for example, a binding domain within the extracellular domain of a cell surface receptor; or a stimulatory domain or signaling domain of an intracellular domain of a cell surface receptor.

[0093] "Operably linked" refers to the association of a nucleic acid sequence on a single nucleic acid fragment (or an amino acid in a polypeptide having multiple domains) 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 when it is capable of affecting the expression of the coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. As another example, a receptor binding domain can be operably linked to an intracellular signaling domain such that the binding of a receptor to a ligand transduces a signal in response to the binding.

[0094] As used herein, "fusion protein" or "chimeric protein" is a protein produced by genetic engineering to connect two or more partial or complete polynucleotide coding sequences encoding separate proteins, and the expression of these connected polynucleotides produces a single peptide or multiple polypeptides having functional properties derived from each of the original proteins or fragments thereof. Between two adjacent polypeptides of different origin in a fusion protein, a linker (or spacer) peptide may be added.

[0095] As used herein, the term "genetic imprint" refers to the genetic or epigenetic information that contributes to the preferred therapeutic attributes of source cells or iPSC, and can be retained in source cell derived iPSC and / or iPSC derived hematopoietic lineage cells.As used herein, "source cell" is a non-pluripotent cell that can be used to produce iPSC by reprogramming, and source cell derived iPSC can be further differentiated into specific cell types, including any hematopoietic lineage cells.Depending on the context, source cell derived iPSC and its differentiated cells are sometimes collectively referred to as "derived cells".For example, as used throughout this application, derived effector cells or derived NK cells or derived T cells are cells differentiated by iPSC compared to their primary corresponding cells obtained from natural / primitive sources (such as peripheral blood, umbilical cord blood or other donor tissues).As used herein, the genetic imprinting that gives preferred therapeutic attributes is by reprogramming the selected source cells that are specific to donors, diseases or therapeutic responses or by using genome editing to introduce gene modification patterns into iPSC to be incorporated into iPSC. In the aspect of source cells obtained from a specially selected donor, disease or treatment background, the genetic imprints contributing to the preferred therapeutic attributes may include any background-specific genes or epigenetic modifications that show a phenotype that can be retained, i.e., a preferred therapeutic attribute, which is transferred to the derived cells of the selected source cells, regardless of whether the underlying molecular events are identified or not. Source cells that are specific to donors, diseases or treatment responses may include genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including but not limited to pre-arranged monospecific TCRs, such as from virus-specific T cells or constant natural killer T (iNKT) cells; trackable and desired genetic polymorphisms, such as homotypic for point mutations encoding high-affinity CD16 receptors in selected donors; and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibit haplotypes as the population increases. As used herein, preferred therapeutic attributes include transplantation, transport, homing, vitality, self-renewal, retention, immune response regulation and modulation, survival and cytotoxicity improvements of derived cells. Preferential therapeutic attributes may also be related to antigen targeting receptor expression; HLA presentation or its lack; resistance to the tumor microenvironment; induction and immunomodulation of neighboring immune cells; improved on-target specificity as off-tumor effects decrease; resistance to treatments such as chemotherapy. When derivative cells with one or more therapeutic attributes are obtained by differentiating iPSCs with genetic imprints, such derivative cells are also referred to as "synthetic cells", which are genetically imprinted to confer preferential therapeutic attributes integrated into iPSCs. For example, as used throughout this application, synthetic effector cells or synthetic NK cells or synthetic T cells are cells differentiated from genome-modified iPSCs compared to their primary counterparts obtained from natural / native sources (such as peripheral blood, umbilical cord blood or other donor tissues).In some embodiments, a synthetic cell has one or more non-native cellular functions when compared to its closest corresponding primary cell.

[0096] As used herein, the term "enhanced therapeutic properties" refers to the enhancement of the therapeutic properties of a cell compared to a typical immune cell of the same general cell type. For example, NK cells with "enhanced therapeutic properties" will have enhanced, improved and / or enhanced therapeutic properties compared to typical, unmodified and / or naturally occurring NK cells. The therapeutic properties of immune cells may include, but are not limited to, cell engraftment, transport, homing, vitality, self-renewal, retention, immune response regulation and modulation, survival and cytotoxicity. The therapeutic properties of immune cells are also reflected by: antigen targeting receptor expression; HLA presentation or its lack; resistance to the tumor microenvironment; induction and immunomodulation of neighboring immune cells; improved on-target specificity with reduced off-tumor effects; and / or resistance to treatments such as chemotherapy.

[0097] As used herein, the term "adapter" refers to a molecule, such as a fusion polypeptide, that enables a connection between an immune cell (e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil) and a tumor cell; and activates the immune cell. Examples of adaptors include, but are not limited to, bispecific T cell adaptors (BiTEs), bispecific killer cell adaptors (BiKEs), trispecific killer cell adaptors (TriKEs), or multispecific killer cell adaptors, or universal adaptors compatible with a variety of immune cell types.

[0098] As used herein, the term "surface triggering receptor" refers to a receptor that can trigger or initiate an immune response (e.g., a cytotoxic reaction). Surface triggering receptors can be engineered and can be expressed on effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils). In some embodiments, surface triggering receptors promote bispecific or multispecific antibody engagement between effector cells and specific target cells (e.g., tumor cells), independent of the natural receptors and cell types of effector cells. Using this method, iPSCs containing universal surface triggering receptors can be produced, and then such iPSCs are differentiated into populations of various effector cell types expressing universal surface triggering receptors. "Universal" means that surface triggering receptors can be expressed in any effector cell and activate any effector cell (regardless of the cell type), and all effector cells expressing universal receptors can be coupled or connected to adaptors recognizable by surface triggering receptors (regardless of the tumor binding specificity of the adaptor). In some embodiments, adaptors with the same tumor targeting specificity are used to couple with universal surface triggering receptors. In some embodiments, adaptors with different tumor targeting specificities are used to couple with universal surface triggering receptors. Thus, one or more effector cell types can be engaged, thereby killing one specific type of tumor cell in some cases and killing two or more types of tumors in other cases. Surface triggering receptors typically contain a co-stimulatory domain for effector cell activation and an anti-epitope specific for an epitope of the adaptor. Bispecific adaptors have specificity for an anti-epitope of a surface triggering receptor at one end and specificity for a tumor antigen at the other end.

[0099] As used herein, the term "safety switch protein" refers to an engineered protein that is designed to prevent the potential toxicity of cell therapy or otherwise prevent side effects. In some cases, the expression of the safety switch protein is conditionally controlled to address the safety issues of the engineered cells that have been transplanted, and the gene encoding the safety switch protein has been permanently incorporated into its genome. This conditional regulation can be variable and may include control by small molecule-mediated post-translational activation and tissue-specific and / or temporal transcriptional regulation. The safety switch protein can mediate the induction of apoptosis, the inhibition of protein synthesis or DNA replication, growth arrest, transcription and post-transcriptional genetic regulation and / or antibody-mediated depletion. In some cases, the safety switch protein is activated by an exogenous molecule such as a prodrug, which triggers apoptosis and / or cell death of the treated cells when activated. Examples of safety switch proteins include, but are not limited to, suicide genes, such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product and kills the transduced cells.

[0100] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide that can achieve a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative or palliative properties for a disease and can be administered to improve, alleviate, slow down, reverse or reduce the severity of a disease. Pharmaceutically active proteins also have preventive properties and are used to prevent the onset of disease or to reduce the severity of such diseases or pathological conditions when they manifest. "Pharmaceutically active proteins" include complete proteins or peptides or pharmaceutically active fragments thereof. The term also includes pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides. The term pharmaceutically active protein also refers to a variety of proteins or peptides that act in a collaborative or synergistic manner to provide a therapeutic benefit. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors and / or cytokines.

[0101] As used herein, the term "signaling molecule" refers to any molecule that regulates, participates in, inhibits, activates, reduces or increases cell signal transduction. "Signal transduction" refers to the transmission of molecular signals in a chemically modified form, which is achieved by recruiting protein complexes along the path that ultimately triggers biochemical events in cells. Signal transduction 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, TG point signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0102] As used herein, the term "targeting modality" refers to the genetic incorporation of molecules (e.g., polypeptides) into cells to promote antigen and / or epitope specificity, which includes but is not limited to i) antigen specificity (when it involves a unique chimeric antigen receptor (CAR) or T cell receptor (TCR)); ii) adaptor specificity (when it involves a monoclonal antibody or a bispecific adaptor); iii) targeting transformed cells; iv) targeting cancer stem cells, and v) other targeting strategies in the absence of specific antigens or surface molecules.

[0103] As used herein, the term "specificity" may be used to refer to the ability of a molecule (eg, a receptor or adaptor) to selectively bind to a target molecule, as compared to non-specific or non-selective binding.

[0104] As used herein, the term "adoptive cell therapy" refers to a cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, whether the immune cells are isolated from a human donor or isolated from effector cells obtained by in vitro differentiation of pluripotent cells; whether these immune cells are genetically modified or not; or, whether these immune cells are primary donor cells or cells that have been passaged, expanded or immortalized ex vivo after isolation from a donor.

[0105] As used herein, "lymphoid depletion" and "lymphoid conditioning" are used interchangeably and refer to the destruction of lymphocytes and T cells, usually before immunotherapy. The purpose of lymphoid conditioning before the administration of adoptive cell therapy is to promote the steady-state proliferation of effector cells and eliminate other competing elements of competitive steady-state cytokines in regulatory immune cells and the immune system. Therefore, lymphoid conditioning is usually completed by administering one or more chemotherapeutic agents to the subject before the first dose of adoptive cell therapy. In various embodiments, lymphoid conditioning precedes the first dose of adoptive cell therapy by a few hours to a few days. Exemplary chemotherapeutic agents that can be used for lymphoid conditioning include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, sufficient lymphoid depletion by anti-CD38 mAb can provide an alternative conditioning method for the iNK cell therapy of the present invention, without requiring or requiring a minimal amount of CY / FLU-based lymphoid conditioning process, as further described herein.

[0106] As used herein, "homing" or "transport" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor) or an organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, the function of a homing molecule is to recognize and / or initiate the interaction of a cell with a target site. In some embodiments, a homing molecule is a chemokine receptor.

[0107] As used herein, "therapeutically sufficient amount" includes within its meaning a non-toxic but sufficient and / or effective amount of the specific therapeutic agent and / or pharmaceutical composition to which it refers for providing the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's general health, the patient's age, and the stage and severity of the condition being treated. In certain embodiments, a "therapeutically sufficient amount" is sufficient and / or effective to ameliorate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0108] Differentiation of pluripotent stem cells needs to change culture system, such as changing the stimulant in culture medium or the physical state of cell.Most of conventional strategies use the formation of embryoid bodies (EB) as the common and key intermediate step of starting lineage-specific differentiation."Embryoid bodies" are three-dimensional clusters, which have been shown to simulate embryonic development because they produce multiple lineages in their three-dimensional regions.Through differentiation process, usually a few hours to a few days, simple EB (for example, through inducing differentiable aggregated pluripotent stem cells) continues to mature and develop into cystic EB, at this time, usually further processed for several days to a few weeks to continue differentiation.EB formation is started by making pluripotent stem cells close to each other in three-dimensional multilayer cell clusters.Usually, this is achieved by a method in several methods, including allowing pluripotent cells to settle in droplets, making cells settle in "U" shaped bottom well plates or by mechanical agitation.In order to promote EB development, pluripotent stem cell aggregates need to further differentiate prompts, because the aggregates maintained in pluripotent culture maintenance medium do not form suitable EBs. In the present invention, the pluripotent stem cell aggregates are transferred to the differentiation medium, and the differentiation medium provides the prompting to the selected pedigree. Through the appropriate proliferation in the EB cell cluster, the culture based on EB of the pluripotent stem cell usually causes the differentiated cell group (that is, ectoderm, mesoderm and endodermal germ layer) to produce. Although it has been confirmed that cell differentiation can be promoted, EB has produced heterogeneous cells with variable differentiation states, and the reason is that the cells in the three-dimensional structure are inconsistently exposed to the differentiation prompting in the environment. In addition, the formation of EB and maintenance trouble. In addition, the cell differentiation carried out by EB formation is accompanied by appropriate cell amplification, which also causes the differentiation efficiency to reduce.

[0109] In contrast, "aggregate formation" different from "EB formation" can be used to expand pluripotent stem cell derived cell populations. For example, during the pluripotent stem cell expansion based on aggregates, a culture medium that can maintain proliferation and pluripotency is selected. Cell proliferation usually increases the size of aggregates, thereby forming larger aggregates, which can be dissociated into smaller aggregates by mechanical or enzymatic means, thereby maintaining cell proliferation in culture and increasing cell number. Different from EB culture, cells cultured in aggregates maintaining culture medium maintain pluripotency markers. Pluripotent stem cell aggregates require further differentiation prompts to induce differentiation.

[0110] As used herein, "monolayer differentiation" is a term for a differentiation process that is different from differentiation performed by three-dimensional multilayered cell clusters, i.e., "EB formation". In addition to other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Since monolayer culture does not simulate embryonic development, such as in the case of EB formation, differentiation to a specific lineage is considered minimal compared to all three germ layer differentiations in EB formation.

[0111] As used herein, "dissociated cells" or "single dissociated cells" refer to cells that have been substantially separated or purified from other cells or surfaces (e.g., culture plate surfaces). For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells aggregated in vitro can be enzymatically or mechanically dissociated from each other, such as by dissociating 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 may involve disruption of cellular interactions with the extracellular matrix (ECM) and substrate (e.g., culture surface), or disruption of the ECM between cells.

[0112] As used herein, a "master cell bank" or "MCB" refers to a clonal master engineered iPSC line, which is a clonal population of iPSCs that have been engineered to include one or more therapeutic attributes, have been characterized, tested, qualified, and expanded, and have been shown to reliably function as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing setting. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic variation and / or potential contamination by reducing and / or eliminating the total number of passages, thawing, or handling of the iPS cell line during the manufacturing process.

[0113] As used herein, "feeder cells" or "feeder layers" are terms describing a type of cell that is co-cultured with cells of a second type to provide an environment in which cells of the second type can grow, expand or differentiate, because feeder cells provide stimulation, growth factors and nutrition to support the second cell type. Feeder cells are optionally from species different from the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. When feeder cells are co-cultured with other cells, they can be inactivated by irradiation or treatment with anti-mitotic agents (such as mitomycin) to prevent them from growing beyond the cells they support. Feeder cells can include endothelial cells, stromal cells (such as epithelial cells or fibroblasts) and leukemia cells. Not limited to the foregoing, a specific feeder cell type can be a human feeder layer, such as human skin fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEF). In general, a variety of feeder cells can be used in part to maintain pluripotency, directed differentiation toward a certain lineage, enhance proliferation capacity, and promote maturation into specialized cell types (eg, effector cells).

[0114] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, that is substantially free of feeder or stromal cells, and / or has not been preconditioned by cultivating feeder cells. "Preconditioned" culture medium refers to culture medium collected after feeder cells have been cultivated in the culture medium for a certain period of time (e.g., at least one day). The preconditioned culture medium contains a variety of mediator substances, including growth factors and cytokines secreted by feeder cells cultivated in the culture medium. In some embodiments, the feed-free environment does not contain feeder or stromal cells, and is not preconditioned by cultivating feeder cells.

[0115] As used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, "function" refers to (1) at the genetic level - successful knock-in, knock-out, reduced gene expression, transgenic or controlled gene expression, such as inducible or transient expression at a desired cell developmental stage, achieved by direct genome editing or modification or by "transfer", via differentiation or reprogramming of the starting cell that was initially genome engineered; or (2) at the cellular level - successful removal, addition or alteration of cellular function / property, achieved by: (i) in said cell Gene expression modifications obtained by direct genome editing, (ii) gene expression modifications maintained in the cell by "transmission" through differentiation or reprogramming from the starting cell that originally underwent genome engineering; (iii) downstream gene regulation in the cell as a result of gene expression modifications that only occurred at an earlier developmental stage of the cell or only occurred in the starting cell that generated the cell through differentiation or reprogramming; or (iv) enhanced or newly acquired cellular functions or properties exhibited in a mature cell product that was originally derived from genome editing or modification performed at an iPSC, progenitor cell, or dedifferentiated cell source.

[0116] "HLA deficiency", including HLA class I deficiency, HLA class II deficiency or both, refers to the lack or no longer maintaining the surface expression of a complete MHC complex comprising HLA class I protein heterodimers and / or HLA class II heterodimers or a reduction in the level of surface expression such that the attenuated or reduced level is below the level that can be detected naturally by other cells or can be detected by synthetic methods.

[0117] As used herein, "modified iPSC lacking HLA" refers to iPSC lacking HLA, which is additionally modified by introducing a gene expressing a protein associated with, but not limited to, improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, retention, immune escape, suppression resistance, proliferation, costimulation, 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. "Modified" cells lacking HLA also include cells other than iPSC.

[0118] The term "ligand" refers to a substance that forms a complex with a target molecule to generate a signal by binding to a site on the target. A ligand can be a natural or artificial substance that can specifically bind to a target. A ligand can be in the form of a protein, peptide, antibody, antibody complex, conjugate, nucleic acid, lipid, polysaccharide, monosaccharide, small molecule, nanoparticle, ion, neurotransmitter, or any other molecular entity that can specifically bind to a target. The target bound to the ligand can be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and changes the function of the target to trigger a signal transduction reaction is referred to as "agonism" or "agonist". A ligand that binds to a target and blocks or reduces a signal transduction reaction is referred to as "antagonism" or "antagonist".

[0119] The term "antibody" is used in the broadest sense herein, and generally refers to a molecule that produces an immune response containing at least one binding site that specifically binds to a target, wherein the target can be an antigen or a receptor that can interact with certain antibodies. For example, NK cells can be activated by binding of an antibody or an antibody's Fc region to its Fc-γ receptor (FcγR), thereby triggering ADCC (antibody-dependent cellular toxicity)-mediated effector cell activation. The specific fragment or part of an antigen or receptor or target that binds to an antibody is generally referred to as an epitope or antigenic determinant. The term "antibody" includes, but is not limited to, native antibodies and variants thereof, fragments of native antibodies and variants thereof, peptide bodies and variants thereof, and antibody mimics that simulate the structure and / or function of antibodies or their specific fragments or parts (including single-chain antibodies and fragments thereof). Antibodies can be mouse antibodies, human antibodies, humanized antibodies, camel IgG, single variable new antigen receptors (VNAR), shark heavy chain antibodies (Ig-NAR), chimeric antibodies, recombinant antibodies, single domain antibodies (dAb), anti-idiotypic antibodies, bispecific antibodies, multispecific antibodies or multimeric antibodies, or their antibody fragments. Anti-idiotypic antibodies are specific for binding to the idiotype of another antibody, where the idiotype is an 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 variable region fragments (scFv), tandem scFv (scFv)2, single-chain Fab (scFab), disulfide-stabilized Fv (dsFv), minibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single domain antigen binding fragments (sdAb), camelid heavy chain IgG and Fragments, recombinant antibodies with only heavy chains (VHH) and other antibody fragments that maintain the binding specificity of the antibody.

[0120] "Fc receptors" (abbreviated as FcR) are classified based on the type of antibody they recognize. For example, the receptors that bind the most common class of antibodies (IgG) are called Fc-gamma receptors (FcγRs), the receptors that bind IgA are called Fc-alpha receptors (FcαRs) and the receptors that bind IgE are called Fc-epsilon receptors (FcεRs). The classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγRs) include several members: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a) and FcγRIIIB (CD16b), which have different affinities for their antibodies due to their different molecular structures.

[0121] "Chimeric receptor" is a general term for describing engineered, artificial or hybrid receptor protein molecules, which are prepared to contain two or more parts of an amino acid sequence derived from at least two different proteins. Chimeric receptor proteins have been engineered to give cells the ability to initiate signal transduction and perform downstream functions after agonist ligands bind to receptors. 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.

[0122] "Chimeric Fc receptors", abbreviated as CFcR, are terms used to describe engineered Fc receptors whose native transmembrane and / or intracellular signaling domains are modified or replaced by non-native transmembrane and / or intracellular signaling domains. In some embodiments of chimeric Fc receptors, in addition to one or both of the non-native transmembrane and signaling domains, one or more stimulatory domains may be introduced into the intracellular portion of the engineered Fc receptor to enhance cell activation, amplification, and function after triggering the receptor. Unlike chimeric antigen receptors (CARs) containing antigen binding domains with target antigens, chimeric Fc receptors bind to Fc fragments, or Fc regions of antibodies, or are included in adapters or binding molecules and activate cell function in the case of bringing target cells close or not close to the vicinity. For example, Fcγ receptors can be engineered to include selected transmembrane, stimulatory, and / or signaling domains in intracellular regions that react to binding IgG in the extracellular domain, thereby producing CFcRs. In one example, CFcR is produced by replacing its transmembrane domain and / or intracellular domain by engineered CD16, Fcγ receptors. In order to further improve the binding affinity of CFcR based on CD16, the extracellular domain of high-affinity variants (e.g., F176V) of CD64 or CD16 can be combined. In some embodiments of CFcR related to the extracellular domain of high-affinity CD16, the proteolytic cleavage site containing serine at position 197 is eliminated or replaced, so that the extracellular domain of the receptor is not cleavable, i.e., does not experience shedding, thereby obtaining CFcR based on hnCD16.

[0123] FcγR receptor CD16 has been identified as having two isomers: Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells, which 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 "non-cleavable high-affinity CD16 (abbreviated as hnCD16)" refers to natural or non-natural CD16 variants. Wild-type CD16 has low affinity and undergoes extracellular domain shedding, which is a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes after NK cell activation. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants that change or eliminate cleavage sites (positions 195-198) in regions close to the membrane (positions 189-212) do not experience shedding. The cleavage site and the region close to the membrane are described in detail in WO2015 / 148926, the full disclosure of which is incorporated herein by reference. The CD16 S197P variant is an engineered non-cleavable version of CD16. The CD16 variant comprising F158V and S197P has high affinity and is non-cleavable. Another exemplary high affinity and non-cleavable CD16 (hnCD16) variant is an engineered CD16 comprising an extracellular domain derived from one or more of the three exons of the CD64 extracellular domain.

[0124] "T cell receptor", abbreviated as "TCR", generally refers to a protein complex found on the surface of T cells and is responsible for recognizing fragments of antigenic peptides bound to major histocompatibility complex (MHC) molecules. Binding of the TCR to the antigenic peptide initiates TCR-CD3 intracellular activation, recruitment of a large number of signaling molecules, and branching and integration of signaling pathways, resulting in the mobilization of transcription factors important for gene expression and growth and functional acquisition of typical T cells. A typical TCR contains two highly variable protein chains (α and β), each of which contains a constant region close to the cell membrane and a variable region (i.e., binding domain) that binds to the peptide / MHC.

[0125] I. Cells and compositions suitable for adoptive cell therapy with enhanced properties

[0126] This article provides a strategy that systematically engineers the regulatory circuits of cloned iPSCs, and does not affect differentiation efficacy and the cell developmental biology of iPSCs and their derivatives, while enhancing the therapeutic properties of derived cells differentiated from iPSCs. After the selective pattern combination introduced into cells by genetic engineering at the iPSC level, iPSC-derived cells are functionally improved and suitable for adoptive cell therapy. It is unclear whether the iPSCs comprising one or more of the changes in the gene editing provided still have the ability to intervene in cell development and / or the ability to mature and produce functional differentiated cells while retaining the modified activity and / or characteristics. Unexpected failures during directed cell differentiation from iPSCs are attributed to aspects including but not limited to: developmental stage-specific gene expression or lack of gene expression, the need for HLA complex presentation, protein shedding of the surface expression pattern introduced, and the need to reconfigure the differentiation scheme to achieve phenotypic and / or functional changes in cells. The present application has demonstrated that one or more selected genomic modifications as provided herein do not negatively affect iPSC differentiation efficacy, and that functional effector cells derived from engineered iPSCs have enhanced and / or acquired therapeutic properties attributable to the individual or combined genomic modifications retained in the effector cells following iPSC differentiation.

[0127] 1. Exogenous FAS signaling redirector

[0128] The Fas receptor (Fas) is a death receptor that can induce apoptosis when bound to its corresponding ligand, Fas ligand (FasL). A variety of liquid and solid tumors, as well as activated peripheral B cells, T cells, and NK cells, express high levels of FasL. This elevated FasL expression in recipients represents a major obstacle for adoptive immune cell products. CAR-T and CAR-NK cell therapy products can be Fas-positive by classical expression or by FasL-induced Fas activation, thereby being targeted and killed by FasL-induced apoptosis in the tumor environment or by fratricide of neighboring product cells expressing Fas ligand.

[0129] In order to provide genetic enhancement of adoptive cell products, a number of constructs encoding Fas signaling redirecting receptors (also referred to as "Fas redirectors" or "Fas signaling redirectors") have been designed. The Fas signaling redirecting receptor comprises: a partial or complete peptide comprising the extracellular domain (ECD) of the Fas receptor, or an extracellular Fas binding domain of a variant or allele thereof; and a cytoplasmic signaling domain comprising a partial or complete peptide of the intracellular domain (ICD) of one or more co-stimulatory molecules, wherein the Fas signaling redirecting receptor redirects Fas signaling after FasL binding, providing the cell with improved apoptosis resistance and / or exhaustion resistance. The Fas redirector as described herein also comprises a transmembrane region comprising a transmembrane domain or a portion thereof of a transmembrane protein. In some embodiments, the transmembrane region and the extracellular binding domain of the Fas redirector are both from a Fas receptor. In some embodiments, the transmembrane region of the Fas redirector is from a transmembrane protein that is not a Fas receptor. In some embodiments, the transmembrane region and the cytoplasmic signaling domain of the Fas re-director are from the same protein or from different proteins.

[0130] In some embodiments, the extracellular binding domain of the Fas redirector comprises a sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 1. In some embodiments, the extracellular binding domain of the Fas redirector comprises a sequence that is at least about 90% identical to SEQ ID NO: 1. In some embodiments, the extracellular binding domain of the Fas redirector comprises a sequence that is at least about 95% identical 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 to optimally align the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm recognized in the art.

[0131] SEQ ID NO:1QVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN

[0132] In some embodiments, the transmembrane region of the Fas redirector comprises the full length or at least a portion of a native or modified transmembrane region of a 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 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.

[0133] In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector includes CD27, CD28, CD40, MyD88, OX40, IL12Rβ2, IL18R1, IL21R, or a combination thereof. In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector is not 41BB itself.

[0134] In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full length or partial length of the intracellular domain (ICD) of MyD88 and CD40 represented by SEQ ID NO: 2. In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full length or partial length of the intracellular domain (ICD) of CD27 represented by SEQ ID NO: 3. In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full length or partial length of the intracellular domain (ICD) of CD28 represented by SEQ ID NO: 4. In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full length or partial length of the intracellular domain (ICD) of OX40 represented by SEQ ID NO: 5. In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full length or partial length of the intracellular domain (ICD) of IL12Rβ2 represented by SEQ ID NO: 6. In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full length or partial length of the intracellular domain (ICD) of IL18R1 represented by SEQ ID NO: 7. In some embodiments, the costimulatory molecule providing the cytoplasmic signaling domain of the Fas redirector comprises the full length or partial length of the intracellular domain (ICD) of IL21R represented by SEQ ID NO: 8.

[0135] SEQ ID NO:2

[0136] MAAGGPGAGSAAPVSSTSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIE EDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0137] SEQ ID NO:3

[0138] QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0139] SEQ ID NO:4

[0140] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0141] SEQ ID NO:5

[0142] ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0143] SEQ ID NO:6

[0144] SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ

[0145] SEQ ID NO:7

[0146] YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES

[0147] SEQ ID NO:8

[0148] SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS

[0149] In some embodiments, the Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain comprising 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 the signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced by a 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.

[0150] SEQ ID NO:9

[0151] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN LGWLCLLLLPIPLIVWV MAAGGPGAGSAAPVSSTSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMP KKVAKKPTNKAPHPKQEPQEINFPDDLPG SNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0152] Signal peptide-FAS(ECD)- TM -MyD88(ICD)- CD40(ICD)

[0153] In some embodiments, the Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain comprising 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 the signal peptide sequence and / or transmembrane domain sequence may vary in length or sequence, or may be replaced by sequences of different proteins. 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.

[0154] SEQ ID NO:10

[0155] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN LGWLCLLLLPIPLIVWV QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0156] Signal peptide-FAS(ECD)- TM –CD27 (ICD)

[0157] In some embodiments, the Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain comprising 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 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 by a 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.

[0158] SEQ ID NO:11

[0159] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN LGWLCLLLLPIPLIVWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0160] Signal peptide-FAS(ECD)- TM –CD28 (ICD)

[0161] In some embodiments, the Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain comprising 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, wherein the signal peptide sequence and / or transmembrane domain sequence thereof may vary in length or sequence, or may be replaced by a 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.

[0162] SEQ ID NO:12

[0163] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN LGWLCLLLLPIPLIVWV ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0164] Signal peptide-FAS(ECD)- TM –OX40(ICD)

[0165] In some embodiments, the Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain comprising 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 by a 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.

[0166] SEQ ID NO:13

[0167] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN LGWLCLLLLPIPLIVWV SDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ

[0168] Signal peptide-FAS(ECD)- TM –IL12Rb2(ICD)

[0169] In some embodiments, the Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain comprising 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, wherein the signal peptide sequence and / or transmembrane domain sequence thereof may vary in length or sequence, or may be replaced by a 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.

[0170] SEQ ID NO:14

[0171] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN LGWLCLLLLPIPLIVWV YRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES

[0172] Signal peptide-FAS(ECD)- TM –IL18R1(ICD)

[0173] In some embodiments, the Fas redirector comprising an extracellular Fas binding domain and a cytoplasmic signaling domain comprising 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 by sequences of different proteins. 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 an amino acid sequence of SEQ ID NO: 15.

[0174] SEQ ID NO:15

[0175] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN LGWLCLLLLPIPLIVWV SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVLDAEGPCTWPCSCE DDGYPALDLDAGLEEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS

[0176] Signal peptide-FAS(ECD)- TM –IL21R(ICD)

[0177] Therefore, in various embodiments, the Fas redirector as provided herein can be introduced into induced pluripotent cells (iPSC) using one or more construct designs in the above-mentioned construct designs, and introduced into its derivative cells after the differentiation of the iPSC. In addition to iPSC, cloned iPSC, cloned iPS cell lines and iPSC-derived effector cells comprising at least one engineering transformation mode disclosed herein are also provided. A master cell bank is also provided, which comprises cloned engineered iPSCs of single cell sorting and amplification of Fas redirectors introduced exogenously at least as described in this chapter, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing ready-made, engineered, homogenous cell therapy products, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0178] Therefore, in some embodiments, the present invention provides iPSCs and derived cells thereof comprising an exogenous Fas redirector ("Fas" in Table 1), wherein the cells (including derived T cells and NK cells) can be used to overcome Fas-induced apoptosis or cannibalism, and wherein 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 co-stimulatory molecules. In some embodiments, iPSCs and derived cells thereof comprise one or more additional genome edits as described herein without adversely affecting the differentiation potential of iPSCs and the function of derived effector cells (including derived T cells and NK cells).

[0179] 2. Chimeric Antigen Receptor (CAR) Expression

[0180] Any CAR design known in the art can be suitable for genetically engineered iPSC and its derived effector cells. CAR is a fusion protein, which generally comprises an extracellular domain, a transmembrane domain and an intracellular domain, and the extracellular domain comprises an antigen recognition domain. In some embodiments, the extracellular domain may also include a signal peptide or a leader sequence and / or a spacer. In some embodiments, the intracellular domain may also include a signaling peptide that activates the effector cell expressing CAR. In some embodiments, the intracellular domain may also include a signaling domain, wherein the signaling domain is derived from a cytoplasmic domain of a signal transduction protein that is specific to T and / or NK cell activation or function. In some embodiments, the antigen recognition domain can specifically bind to an antigen. In some embodiments, the antigen recognition domain can specifically bind to an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, wherein the tumor may be a liquid or solid tumor. In some embodiments, CAR is suitable for activating T lineage cells or NK lineage cells expressing CAR. In some embodiments, CAR is a NK cell that is specific to NK-specific signaling components. In certain embodiments, T cells are derived from iPSCs including CARs, and derived T lineage cells may include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβT cells, γδT cells, or combinations thereof. In certain embodiments, NK cells are derived from iPSCs including CARs.

[0181] In certain embodiments, the antigen recognition region / domain comprises a mouse antibody, a human antibody, a humanized antibody, a camel Ig, a shark antibody (VNAR) with only a heavy chain, 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, a single-chain antigen-binding fragment (scFv), (scFv)2, a disulfide-stabilized Fv (dsFv), a mini antibody, a bifunctional antibody, a trifunctional antibody, a tetrafunctional antibody, a single-domain antigen-binding fragment (sdAb, nano antibody), a recombinant antibody with only a heavy chain (VHH), and other antibody fragments that maintain the binding specificity of all antibodies. In some embodiments, the antigen recognition region of CAR is derived from the binding domain of a T cell receptor (TCR) targeting a tumor-associated antigen (TAA).

[0182] Non-limiting examples of antigens that can be targeted by 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, and epithelial glycoprotein-2. (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit α-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, carcinoembryonic 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 are capable of causing disease.

[0183] Therefore, in some embodiments, the genetically engineered iPSCs and their derivative cells contain an exogenous polynucleotide encoding a CAR, wherein the CAR is specific to 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.

[0184] In some embodiments, the transmembrane domain of the CAR comprises all or at least a portion of a native or modified transmembrane region of a 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 T cell receptor polypeptide.

[0185] In some embodiments, the signaling peptide of the intracellular domain (or intracellular domain) comprises 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 (protein containing transmembrane and immunoglobulin domains 2), CD3ζ (T cell surface glycoprotein CD3ζ chain), CD3ζ1XX (CD3ζ variant), DAP10 (hematopoietic cell signaling transducer), DAP12 (TYRO protein tyrosine kinase binding protein), DNAM1 (CD226 antigen), FcERIγ (high affinity immunoglobulin epsilon receptor subunit γ), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit β), IL-2Rγ (cytokine receptor common subunit γ), IL-7R (interleukin-7 receptor subunit α), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-DII type 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) full length or at least a portion of the peptide.

[0186] In some embodiments, the intracellular domain of CAR further comprises a second signaling domain and an optional third signaling domain, wherein each of the first signaling domain, the second signaling domain, and the third signaling domain is different. In a specific embodiment, the second signaling domain and / or the third signaling domain comprises a cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγIL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1 or CD8.

[0187] In certain embodiments, the intracellular domain of CAR further comprises at least one co-stimulatory signaling region. The co-stimulatory signaling region may comprise the full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4 or NKG2D or any combination thereof.

[0188] In some embodiments, a CAR suitable for use in a cell as provided herein comprises a costimulatory domain derived from CD28 and a signaling domain of ITAM1 comprising native or modified CD3 zeta 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 comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 16. In some embodiments, the CAR comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 16. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 16. In additional embodiments, the CAR comprising a co-stimulatory domain derived from CD28 and a native or modified ITAM1 of CD3ζ further comprises a hinge domain and a transmembrane domain derived from CD28, wherein the scFv can be connected to the transmembrane domain by the hinge, and the CAR 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: 17. In some embodiments, the CAR comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 17. In some embodiments, the CAR comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 17. In some embodiments, the CAR comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 17. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 17.

[0189] SEQ ID NO:16

[0190] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0191] (153 amino acids CD28 co-stimulatory + CD3ζ ITAM)

[0192] SEQ ID NO:17

[0193] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0194] (219 amino acids CD28 hinge + CD28 TM + CD28 co-stimulatory + CD3ζ ITAM)

[0195] In various embodiments, a CAR suitable for use in a cell as provided herein comprises a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3 zeta 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 comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 18. In some embodiments, the CAR comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 18. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 18. A CAR comprising a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3ζ may further comprise a CD8 hinge, wherein the amino acid sequence of such structure is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 19. In some embodiments, the CAR comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 19. In some embodiments, the CAR comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 19. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 19.

[0196] SEQ ID NO:18

[0197] SNLFVASWIAVMIIFRIGMAVAIFCCFFFPSWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0198] (NKG2D of 263 amino acids TM +2B4+CD3ζ)

[0199] SEQ ID NO:19

[0200] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIF CCFFFPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0201] (308 amino acids, CD8 hinge + NKG2D TM +2B4+CD3ζ)

[0202] Non-limiting CAR strategies also include: heterodimers, which conditionally activate CAR by dimerizing a pair of intracellular domains (see, e.g., U.S. Pat. No. 9,587,020); separate CARs, in which the antigen binding domain, hinge domain, and intracellular domain are homologously recombined to generate CAR (see, e.g., U.S. Publication No. 2017 / 0183407); multi-chain CARs, which allow non-covalent attachment between two transmembrane domains connected to the antigen binding domain and the signaling domain, respectively (see, e.g., U.S. Publication No. 2014 / 0134142); CARs having bispecific antigen binding domains (see, e.g., U.S. Pat. No. 9,447,194), or having a pair of antigen binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Pat. No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Invest. 2016; Vol. 126, No. 8, pp. 3036-3052); inducible CAR (see, e.g., U.S. Publication Nos. 2016 / 0046700, 2016 / 0058857, 2017 / 0166877); switchable CAR (see, e.g., U.S. Publication No. 2014 / 0219975); and any other design known in the art.

[0203] In another embodiment, iPSCs and their derived effector cells comprising a Fas redirector and optionally a CAR have a CAR inserted into the TCR constant region, resulting in endogenous TCR knockout (TCR KO ), thereby optionally placing CAR expression under the control of an endogenous TCR promoter. In some other embodiments, the CAR inserted in the TCR constant region is specific for a tumor antigen comprising at least one of the following: 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, TAP-associated protein, 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.

[0204] Therefore, aspects of the present invention provide genome-engineered iPSCs and derived cells obtained from differentiating the genome-engineered iPSCs, wherein the iPSCs and derived cells contain exogenous polynucleotides encoding Fas redirectors and CARs, optionally one or more additional modification patterns as provided in Table 1, without adversely affecting the differentiation potential of the iPSCs and the function of derived effector cells (including derived T cells and NK cells). A master cell bank is also provided, comprising single-cell sorted and amplified cloned engineered iPSCs with at least an exogenously introduced Fas redirector and optional CAR, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing ready-made, engineered, homogenous cell therapy products that are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0205] 3.CD16 knock-in

[0206] CD16 has been identified as two isoforms: Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells, which binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is expressed only by human neutrophils. As used herein, "high-affinity CD16", "non-cleavable CD16" or "non-cleavable high-affinity CD16" refers to various CD16 variants. Wild-type CD16 has low affinity and undergoes extracellular domain shedding, which is a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes after NK cell activation. F176V (also referred to as F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity; and the S197P variant is an example of a genetically engineered CD16 non-cleavable form. The engineered CD16 variants comprising both F176V and S197P have high affinity and are non-cleavable, and the variants are described in more detail in International Publication No. WO2015 / 148926, the complete disclosure of which is incorporated herein by reference. In addition, the chimeric CD16 receptor in which the extracellular domain of CD16 is substantially replaced by at least a portion of the extracellular domain of CD64 can also achieve the high affinity and non-cleavable characteristics required for the CD16 receptor capable of ADCC. In some embodiments, the replacement extracellular domain of the chimeric CD16 comprises one or more of the following: EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isomers or polymorphic variants).

[0207] Different from the endogenous CD16 expressed by primary NK cells that are lysed by the cell surface after NK cell activation, CD16 in derived NK cells avoids CD16 shedding and maintains constant expression in various non-cleavable forms. In derived NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicating that cell function is improved. Non-cleavable CD16 also enhances antibody-dependent cell-mediated cytotoxicity (ADCC) and the engagement of bispecific, trispecific or multispecific adapters. ADCC is a mechanism of NK cell-mediated lysis by binding CD16 to antibody-coated target cells. Before administering cells to subjects in need of cell therapy, the additional high-affinity properties of hnCD16 introduced in derived NK cells also allow ADCC antibodies to be loaded in vitro to NK cells by hnCD16. As provided herein, in some embodiments, hnCD16 may comprise F176V and S197P, or may comprise an extracellular domain derived from all or part of the length of 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 a derivative NK cell or cell population thereof, which is pre-loaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use to treat a condition, disease, or infection as further described in detail below.

[0208] Unlike primary NK cells, mature T cells from primary sources (i.e., natural / native sources, such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. Surprisingly, iPSCs containing expressed exogenous non-cleavable CD16 do not impair T cell developmental biology, and are able to differentiate into functional derived T lineage cells that not only express exogenous CD16, but can also perform functions through acquired ADCC mechanisms. This acquired ADCC in derived T lineage cells can be used in addition as a method for dual targeting and / or rescuing antigen escape that often occurs with CAR-T cell therapy, wherein the tumor is reduced or lost with the expression of the antigen targeting CAR-T or the expression of the mutant antigen to avoid being recognized by CAR (chimeric antigen receptor) and recurring. When the derived T lineage cells express ADCC through exogenous CD16 (including functional variants and CD16-based CFcR), and when the antibody targets a tumor antigen different from the antigen targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent the recurrence or reappearance of targeted tumors commonly used in CAR-T therapy. This strategy of reducing and / or preventing antigen escape while achieving dual targeting is also applicable to NK cells expressing one or more CARs.

[0209] Therefore, 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 non-cleavable high-affinity CD16 receptor (hnCD16). In some embodiments, hnCD16 comprises both F176V and S197P; and in some embodiments, F176V is included, and the cleavage region is eliminated. In some other embodiments, hnCD16 comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or any percentage of identity compared to any one of the exemplary sequences SEQ ID NO: 20, 21 and 22, each of which comprises at least a portion of the CD64 extracellular domain. In some embodiments, hnCD16 comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 20-22. In some embodiments, hnCD16 comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 20-22. In some embodiments, hnCD16 comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, hnCD16 comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, hnCD16 comprises the amino acid sequence of SEQ ID NO: 22.

[0210] SEQ ID NO:20

[0211] MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPS YRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKF FHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGL QLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFH YQVSFCLVMVLLFAVDTGLYFSV KTNIRSSTRDWKDHKFKWRKDPQDK

[0212] (340 amino acids CD64 domain-based constructs ;CD16TM; CD16ICD )

[0213] SEQ ID NO:21

[0214] MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPS YRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKF FHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGL QLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGL FFPPGYQVSFCLVMVLLFAVDTGLYFSV KTNIRSSTRDWKDHKFKWRKDPQDK

[0215] (336 amino acids CD64 exon-based constructs ;CD16TM; CD16ICD )

[0216] SEQ ID NO:22

[0217] MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPS YRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKF FHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGL QLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLG FFPPGYQVSFCLVMVLLFAVDTGLYFSV KTNIRSSTRDWKDHKFKWRKDPQDK

[0218] (335 amino acids CD64 exon-based constructs ;CD16TM; CD16ICD )

[0219] Thus, provided herein are iPSCs that have been genetically engineered to include exogenous CD16 ("CD16" in Table 1) among other edits contemplated and described herein. exo ”), i.e., a non-cleavable high affinity CD16 receptor (hnCD16), wherein the genetically engineered iPSCs are capable of differentiating into effector cells comprising the hnCD16 introduced into the iPSCs. In some embodiments, the derived effector cells comprising the Fas redirector and optionally the CAR and / or exogenous CD16 are NK cells. In some embodiments, the derived effector cells comprising the Fas redirector and optionally the CAR and / or exogenous CD16 are T cells.

[0220] The exogenous hnCD16 expressed in iPSC or its derivative cells has high affinity in a bispecific, trispecific or multispecific adapter or binder that not only binds to an ADCC antibody or fragment thereof, but also binds to a CD16 or CD64 extracellular binding domain that recognizes the hnCD16. Bispecific, trispecific or multispecific adapters or binders are further described below in the present application. Therefore, the present application provides a derived effector cell or a cell population thereof, which is preloaded with one or more preselected ADCC antibodies in sufficient amounts for the treatment of a condition, disease or infection as further described below by high affinity binding to the extracellular domain of hnCD16 or its variants expressed on the derived effector cells, wherein the hnCD16 comprises an extracellular binding domain of CD64 or CD16 with F176V and S197P. Therefore, in some embodiments, the derived NK cells are preloaded with antibodies. In some embodiments, the derived NK cells are used in combination therapy with antibodies. In some embodiments, the antibody in the combination therapy or the antibody preloaded by the derived effector cells specifically targets CD38. In some embodiments, the antibody in the combination therapy or the antibody preloaded by the derived effector cells specifically targets an antigen other than CD38. In some embodiments, the anti-CD38 antibody is daratumumab.

[0221] In some other embodiments, the exogenous CD16 expressed in iPSC or its derivative cells comprises a CFcR based on CD16 or its variants. By modifying or replacing the native CD16 transmembrane domain / or intracellular domain, a chimeric Fc receptor (CFcR) comprising a non-native transmembrane domain, a non-native stimulatory domain and / or a non-native signaling domain is generated. The term "non-native" as used herein means that, in addition to the receptor providing the extracellular domain, the transmembrane domain, the stimulatory domain or the signaling domain are derived from different receptors. In the description herein, the CFcR based on CD16 or its variants does not have a transmembrane domain, a stimulatory domain or a 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 CD16-based CFcRs, provided chimeric Fc receptors include a transmembrane domain and a signaling domain both derived from one of the following: IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C or NKG2D polypeptides. A specific exemplary embodiment of a CD16-based chimeric Fc receptor comprises a transmembrane domain of NKG2D, a stimulatory domain of 2B4, and a signaling domain of CD3ζ; wherein the extracellular domain of the CFcR is derived from the full length or partial sequence of the extracellular domain of CD64 or CD16, and wherein the extracellular domain of CD16 comprises F176V and S197P.Another exemplary embodiment of a CD16-based chimeric Fc receptor comprises the transmembrane domain and signaling domain of CD3ζ; wherein the extracellular domain of the CFcR is derived from the full length or partial sequence of the extracellular domain of CD64 or CD16, and wherein the extracellular domain of CD16 comprises F176V and S197P.

[0222] Various embodiments of the chimeric Fc receptor based on CD16 as described above can bind to the Fc region of an antibody or fragment thereof with high affinity; or a bispecific, trispecific or multispecific adapter or binder that recognizes the CD16 or CD64 extracellular binding domain of the chimeric Fc receptor. After binding, the stimulatory domain and / or signaling domain of the chimeric receptor achieves activation of effector cells and cytokine secretion, and kills tumor cells targeted by the antibody or bispecific, trispecific or multispecific adapter or binder having a tumor antigen binding component and an Fc region. Without being limited by theory, CFcR can contribute to the killing ability of effector cells through non-native transmembrane domains, stimulatory domains and / or signaling domains, or through adapters that bind to the extracellular domain of the chimeric Fc receptor based on CD16, while increasing the proliferation and / or amplification potential of effector cells. Antibodies and adapters can bring tumor cells expressing antigens and effector cells expressing CFcRs into close proximity, which also helps to enhance tumor cell killing. Exemplary tumor antigens of bispecific, trispecific, and multispecific adaptors 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 adaptors or binders suitable for engaging effector cells expressing CD16-based CFcRs when attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0223] Therefore, in some embodiments, the present invention provides iPSC and its derivative cells comprising exogenous Fas redirector and exogenous CD16 or its variants, wherein these cells (including derived T cells and NK cells) can be used to overcome Fas-induced apoptosis or cannibalism. In some embodiments, iPSC and its derivative cells include Fas redirector and exogenous CD16 or its variants, and optional CAR. In some embodiments, iPSC and its derivative cells include one or more additional genome editing as described herein, without adversely affecting the differentiation potential of iPSC and the function of derived effector cells (including derived T cells and NK cells). A master cell bank is also provided, which includes single cell sorting and amplification of cloned engineered iPSC with at least exogenously introduced Fas redirector and exogenous CD16 or its variants, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing ready-made, engineered, homogeneous cell therapy products, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0224] 4.CD38 knockout

[0225] The cell surface molecule CD38 is highly upregulated in a variety of hematological malignancies, which are derived from both lymphoid and myeloid lineages, including multiple myeloma and CD20 negative B cell malignancies, and its use makes it an attractive target for antibody therapeutics that deplete cancer cells. Antibody-mediated cancer cell depletion is generally attributable to a combination of direct apoptosis induction and activation of immune effector mechanisms such as ADCC (antibody-dependent cell-mediated cytotoxicity). In addition to ADCC, immune effector mechanisms together with therapeutic antibodies may also include antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0226] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells, NK cells, and activated T and B cells. + It is expressed on stem cells and lineage-specialized progenitor cells of the lymphoid, erythroid, and myeloid lines and during the final stages of maturation that continue up to the plasma cell stage. As a type II transmembrane glycoprotein, CD38 performs cellular functions both as a receptor and as a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 catalyzes the conversion of NAD +The synthesis and hydrolysis of CD38 in the reaction to ADP-ribose generates the secondary messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which is essential for the calcium-dependent process of cell adhesion. As a receptor, CD38 recognizes CD31 and regulates cytokine release and cytotoxicity in activated NK cells. CD38 has also been reported to associate with cell surface proteins in lipid rafts, thereby regulating cytoplasmic Ca 2+ traffic, and mediates signal transduction in lymphocytes and myeloid cells.

[0227] However, in the treatment of malignancies, systemic administration of CD38 antigen-binding receptor-transduced T cells has been shown to cleave CD34 + CD38 on hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells + Part, resulting in incomplete therapeutic response and reduced or eliminated efficacy due to impaired receptor immune effector cell function. In addition, in multiple myeloma patients treated with daratumumab, CD38 specific antibodies, although other immune cell types (such as T cells and B cells) are not affected regardless of their CD38 expression, it is observed that NK cells in both bone marrow and peripheral blood are reduced (Casneuf et al., "Hematological Research Progress (Blood Advances.)" 2017; Vol. 1, No. 23, pp. 2105-2114).

[0228] Without being limited by theory, the present application includes a strategy for making full use of the full potential of cancer treatment targeting CD38 in the following manner: by knocking out CD38 in effector cells, so as to overcome the exhaustion or reduction caused by cannibalism of effector cells induced by CD38-specific antibodies and / or CD38 antigen-binding domains. In addition, because CD38-specific antibodies (such as daratumumab) are used in the recipients of allogeneic effector cells to suppress the activation of these recipient lymphocytes, CD38 is upregulated on activated lymphocytes (such as T cells or B cells), so the host allogeneic rejection for these effector cells will be reduced and / or prevented, so as to increase effector cell survival and retention. Therefore, CD38-specific antibodies, secreted CD38-specific adapters or CD38-CAR (chimeric antigen receptor) for the activation of recipient T cells, Treg cells, NK cells and / or B cells can be used as a substitute for lymphoid depletion using chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine) before adoptive cell transfer. In addition, the present application also discloses that hnCD16a is used in the presence of anti-CD38 antibodies or CD38 inhibitors. + / CD38 - Effector cell targeting CD38 + T cells and pbNK cells, CD38 +Depletion of alloreactive cells increases NAD + (nicotinamide adenine dinucleotide, a substrate for CD38) availability and reduces the binding of NAD + Consumption-associated cell death, among other advantages, enhances effector cell responses in immunosuppressive tumor microenvironments and supports cell regeneration in aging, degenerative or inflammatory diseases.

[0229] Thus, strategies as provided herein also include generating iPSC lines comprising a Fas redirector and a CD38 knockout and optionally one or more of a CAR and an exogenous CD16 or a variant thereof, and obtaining an iPSC line comprising a Fas redirector and a CD38 null (CD38 knockout) by directed differentiation of the engineered iPSC line. - / - ) and optionally CAR and one or more derived effector cells of exogenous CD16 or its variants. In one embodiment, the CD38 knockout in the iPSC line is a biallelic knockout.

[0230] As disclosed herein, in some embodiments, a Fas redirector and CD38 - / - , and optionally one or more of CAR and exogenous CD16, the provided iPSC line can be directed to differentiate to produce functional derived hematopoietic cells, including but not limited to mesodermal cells with permanent hemogenic endothelial cell (HE) potential, permanent HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, bone marrow cells, neutrophil progenitor cells, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutrophils, dendritic cells and macrophages. In some embodiments, when anti-CD38 antibodies are used to induce CD16-mediated enhanced ADCC, or anti-CD38 CARs are used for targeted cell killing, Fas redirectors and CD38 are included. - / -And optionally CAR and exogenous CD16 one or more of iPSC and / or its derived effector cells can target CD38 expression (tumor) cells without causing effector cell elimination, i.e., reducing or depleting CD38 expression effector cells, thereby increasing iPSC and its effector cells in the presence of such therapeutic agents and / or exposure to such therapeutic agents after retention and / or survival. In some embodiments, in the presence of such anti-CD38 therapeutic agents (such as anti-CD38 antibodies) and / or after exposure to such therapeutic agents, effector cells have increased retention and / or survival in vivo. In some embodiments, anti-CD38 antibodies are daratumumab, isatuximab or MOR202. In addition, since CD38 is upregulated on activated lymphoid effector cells such as T cells or B cells, CD38-specific antibodies can be used for lympho-depletion, thereby eliminating those activated lymphocytes, overcoming allogeneic rejection, increasing the survival and retention of CD38-negative effector cells, and there is no cannibalism in the receptors of allogeneic effector cell therapy.

[0231] In some embodiments, the Fas redirector and CD38 - / - And optionally the derived effector cells of one or more of CAR and exogenous CD16 are NK cells derived from iPSC. In some embodiments, the Fas redirector and CD38 - / - And optionally the effector cells of one or more of CAR and exogenous CD16 are T cells derived from iPSC. In some embodiments, the Fas redirector and CD38 - / - And optionally, the iPSCs and / or cells derived therefrom of one or more of CAR and exogenous CD16 further comprise one or more additional genome edits as provided in Table 1 without adversely affecting the differentiation potential of the iPSCs and the function of derived effector cells (including derived T cells and NK cells). Also provided is a master cell bank comprising at least exogenously introduced Fas redirectors and CD38 - / - Single-cell sorting and expansion of clonal engineered iPSCs, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogenous cell therapy products that are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0232] 5. Exogenously introduced cytokines and / or cytokine signaling

[0233] By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicity caused by this practice is reduced, and cytokine-mediated cell autonomy is established. In order to achieve lymphocyte autonomy without the need to apply soluble cytokines in addition, the cytokine signaling complex comprising one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and / or their corresponding receptors can be introduced into the cell, so as to allow cytokine signaling with or without expressing cytokines themselves, so as to maintain or improve cell growth, proliferation, amplification and / or effector function, and reduce the risk of cytokine toxicity. In some embodiments, the introduced cytokines and / or their corresponding native or modified receptors for cytokine signaling (signaling complex) are expressed on the cell surface. In some embodiments, constitutively activate cytokine signaling. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary. In some embodiments, transient / temporal expression of cell surface cytokines / cytokine receptors is by expression constructs carried by retroviruses, Sendai viruses, adenoviruses, episomes, minicircles, or RNA including mRNA.

[0234] Provided herein are various construct designs for introducing a protein complex for one, two or more cytokine signaling into a cell, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18 and IL21. In the embodiment in which the cytokine signaling complex is used for IL15, the transmembrane (TM) domain can be native to the IL15 receptor, or can be modified or replaced by the transmembrane domain of any other membrane-bound protein. In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide to simulate the trans presentation of IL15 without eliminating the cis presentation of IL15. In other embodiments, IL15Rα is fused to IL15 at the C-terminus via a linker, simulating trans presentation without eliminating the cis presentation of IL15 and ensuring IL15 membrane binding. In other embodiments, IL15Rα with a truncated intracellular domain is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation of IL15, maintaining membrane-bound IL15, and by otherwise eliminating cis-presentation and / or any other potential signaling pathways mediated by normal IL15R through its intracellular domain.

[0235] In various embodiments, such truncated constructs comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 23. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 23. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence of SEQ ID NO: 23. In one embodiment of truncated IL15 / IL15Rα, the construct does not comprise the last 4 amino acid residues (KSRQ) of SEQ ID NO: 23, and comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 24. In some embodiments, IL15 / IL15Rα comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, the IL15 / IL15Rα comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 24. In some embodiments, the IL15 / IL15Rα comprises the amino acid sequence of SEQ ID NO:24.

[0236] SEQ ID NO:23

[0237] MDWTWILFLVAAATRVHS GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQ

[0238] (379 amino acids; variable signal sequence and linker peptide are underlined)

[0239] SEQ ID NO:24

[0240] MDWTWILFLVAAATRVHS GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYL

[0241] (375 amino acids; variable signal sequence and linker peptide are underlined)

[0242] In other embodiments, the cytoplasmic domain of IL15Rα can be omitted without negatively affecting the autonomous characteristics of effector cells equipped with IL15. In other embodiments, the entire IL15Rα is substantially removed except for the Sushi domain, which is fused to IL15 at one end and fused to the transmembrane domain at the other end (mb-Sushi), optionally using a linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface through the transmembrane domain of any membrane-bound protein. Therefore, when only the desired trans presentation of IL15 is retained, unnecessary signal transduction through IL15Rα is eliminated, including cis presentation. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 25. In some embodiments, the component comprising IL15 fused to the Sushi domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 25. In some embodiments, the component comprising IL15 fused to a Sushi domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 25. In some embodiments, the component comprising IL15 fused to a Sushi domain comprises the amino acid sequence of SEQ ID NO:25.

[0243] SEQ ID NO:25

[0244] MDWTWILFLVAAATRVHS GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0245] (242 amino acid signaling and linker peptides are underlined)

[0246] In other embodiments, native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to achieve constitutive signaling and maintain IL15 membrane binding and trans-presentation. In other embodiments, native or modified co-receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling and membrane-bound trans-presentation of cytokines. Co-receptor γC is also known as common γ chain or CD132, also known as IL2 receptor subunit γ or IL2RG. γC is a cytokine receptor subunit that is shared with the receptor complex for many interleukin receptors, including but not limited to IL2, IL4, IL7, IL9, IL15 and IL21 receptors. In other embodiments, an engineered IL15Rβ that forms a homodimer in the absence of IL15 is suitable for constitutive signaling of cytokines.

[0247] In other various embodiments, the cytokine signaling complex comprises an IL7 receptor fusion (IL7RF), which comprises a full-length or partial-length IL7 and a full-length or partial-length IL7 receptor (IL7R). The transmembrane (TM) domain can be native to the IL7 receptor, or can be modified or replaced by 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 achieve constitutive signaling and maintain membrane-bound IL7. In some embodiments, such constructs comprise an amino acid sequence with at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 26, wherein 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%.

[0248] SEQ ID NO:26

[0249] DSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0250] (Signal peptide-IL7- Connectors -IL7R; transmembrane domain ( TM ), signal peptides and linkers may vary in length and sequence)

[0251] In one embodiment, the native or modified co-receptor γC is fused to IL7 at the C-terminus via a linker for constitutive and membrane-bound cytokine signaling complexes. The co-receptor γC is also known as the common γ chain or CD132, also known as IL2 receptor subunit γ or IL2RG. γC is a cytokine receptor subunit that is shared with the receptor complex for many interleukin receptors, including but not limited to IL2, IL4, IL7, IL9 and IL21 receptors. In addition, engineered IL7Rs that form homodimers in the absence of IL7 are also suitable for constitutive signaling of cytokines.

[0252] Those of ordinary skill in the art will understand that the above-mentioned signal peptide and linker sequence are illustrative and in no way limit variants thereof suitable for use as signal peptides or linkers. There are many suitable signal peptides or linker sequences known and available to those of ordinary skill in the art. Those of ordinary skill in the art understand that the signal peptide and / or linker sequence can replace another sequence without changing the activity of the functional peptide guided by the signal peptide or connected by the linker. Therefore, these iPSCs and their derivative cells can autonomously maintain or improve cell growth, proliferation, amplification and / or effector function without additional in vitro or in vivo contact with the supplied soluble cytokines.

[0253] In iPSCs and their derivative cells comprising both CAR and cytokine signaling complexes ("IL" in Table 1), CAR and IL can be expressed in separate constructs, or can be co-expressed in bicistronic constructs comprising both CAR and IL. In some embodiments, the cytokine signaling complex can be connected to the 5' end or 3' end of the CAR expression construct by the self-cleavage 2A coding sequence. Therefore, the cytokine signaling complex (e.g., IL7 signaling complex) and CAR can be in 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 expressing CAR-2A-IL or IL-2A-CAR, the self-cleavage 2A peptide allows the expressed CAR and IL to dissociate, and then the dissociated IL can be presented on the cell surface, wherein its transmembrane domain is anchored in the cell membrane. The CAR-2A-IL or IL-2A-CAR bicistronic construct design allows coordinated CAR and IL expression in time and quantity, and under the same control mechanism that can be selected to incorporate, for example, an inducible promoter or a promoter with temporal or spatial specificity to express a single ORF. Self-cleaving peptides are found in members of the Picornaviridae family, including aphthoviruses, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), bright vein flat moth virus (TaV) and porcine Teschen virus-1 (PTV-I) (Donnelly, ML et al., Journal of General Virology (J. Gen. Virol), Vol. 82, 1027-101 (2001); Ryan, MD et al., Journal of General Virology, Vol. 72, pp. 2727-2732 (2001)), and cardioviruses, such as Theiler virus (e.g., Theiler murine encephalomyelitis) and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-I and TaV are sometimes also referred to as "F2A", "E2A", "P2A" and "T2A", respectively.

[0254] Also contemplated as disclosed herein bicistronic CAR-2A-IL or IL-2A-CAR embodiments for expressing any other cytokine or cytokine signaling complex provided herein, such as IL2, IL4, IL6, IL9, IL10, IL11, IL12, IL18 and IL21. In some embodiments, bicistronic CAR-2A-IL or IL-2A-CAR is used to express one or more of IL2, IL4, IL7, IL9, IL15, IL21 and cytokine signaling complexes.

[0255] Therefore, in various embodiments, the cytokine signaling complex as provided herein can be introduced into iPSC using one or more of the above-mentioned construct designs, and introduced into its derivative cells after the iPSC is differentiated. In addition to iPSC, cloned iPSC, cloned iPS cell line or iPSC-derived effector cell comprising at least one engineering transformation mode disclosed herein is also provided. Therefore, in some embodiments, the present invention provides iPSC and its derivative cells, which contain the Fas redirector and exogenous cytokine signaling complex described herein ("IL" in Table 1), and optionally CAR, exogenous CD16 or its variant and CD38 - / - One or more of, wherein the Fas redirector redirects Fas signaling when combined with a FAS agonist, thereby providing cells with improved apoptosis resistance and / or exhaustion resistance, including derived T cells and NK cells. In some embodiments, iPSC and its derived cells include one or more additional genome editing as provided in Table 1, without adversely affecting the differentiation potential of iPSC and the function of derived effector cells (including derived T cells and NK cells). A master cell bank is also provided, which comprises a cloned engineered iPSC with single cell sorting and amplification of a Fas redirector and an exogenously introduced cytokine signaling complex as described in at least this section, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing ready-made, engineered, homogenous cell therapy products, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0256] 6. HLA-I Deficiency and HLA-II Deficiency

[0257] It is necessary to match a variety of HLA class I and class II proteins in allogeneic recipients to obtain tissue compatibility, thereby avoiding allogeneic rejection problems. An iPSC cell line is provided herein, and the expression of one or two HLA class I ("HLA-I") proteins and HLA class II ("HLA-II") proteins is eliminated or significantly reduced. HLA class I deficiency can be achieved by causing any region of HLA class I locus (chromosome 6p21) to have functional loss or by causing HLA class I related genes (including but not limited to β-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene and TAP related protein) to be missing or expression level is reduced. For example, B2M gene encodes the common subunit necessary for the cell surface expression of all HLA class I heterodimers. B2M negative cells are HLA-I deficient.

[0258] HLA II class deficiency can be achieved by causing HLA-II related genes (including but not limited to RFXANK, CIITA, RFX5 and RFXAP) to have functional loss or reduction. CIITA is a transcriptional coactivator, which works by the activation of the transcription factor RFX5 required for class II protein expression. CIITA negative cells are HLA-II deficient. However, lack of HLA I class expression increases the sensitivity to NK cell lysis. Therefore, the application provides iPSC lines and derived cells thereof comprising HLA-I deficiency and / or HLA-II deficiency (for example, lack of B2M and / or CIITA expression), wherein the derived effector cells obtained are eliminated by eliminating the need for MHC (major histocompatibility complex) matching, avoiding host (allogeneic) T cells to identify and kill, to achieve allogeneic cell therapy.

[0259] In addition, the lack of HLA class I expression causes host NK cell-mediated lysis. Therefore, in addition to the above-discussed method of regulating CD38 to remove activated host NK cells expressing CD38, in order to solve the "self-deficiency" reaction, HLA-G can be optionally knocked in to avoid NK cell recognition and killing of effector cells derived from HLA-I lacking engineered iPSC. In one embodiment, the iPSC and its derived cells lacking HLA-I provided also include HLA-G knock-in. Alternatively, or in addition to this, the iPSC and its derived cells lacking HLA-I provided also include one or both of CD58 knockout and CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and promote cell (including immune cells) migration. Previous studies have shown that CD58 and / or CD54 destruction effectively reduces the sensitivity of effector cells derived from iPSC lacking HLA-I to allogeneic NK cell killing. Although CD58 knockout was shown to be more efficient than CD54 knockout in reducing allogeneic NK cell activation, double knockout of both CD58 and CD54 was shown to provide the most enhanced reduction in NK cell activation. In some observations, double knockout of CD58 and CD54 was even more effective than HLA-G overexpression of HLA-I-deficient cells in overcoming the "self-deletion" effect.

[0260] Thus, in some embodiments, the present invention provides a strategy to reduce or prevent allogeneic rejection by generating HLA-I and / or HLA-II deficiency (optionally modified with additional HLA-I proteins) while enhancing effector cell retention and / or survival without adversely affecting the differentiation potential of iPSCs and the function of derived effector cells (including derived T cells and NK cells).

[0261] In some embodiments, the effector cells have increased persistence and / or survival in vivo in the presence of and / or after exposure to a therapeutic agent. Thus, in some embodiments, the iPSCs and cells derived therefrom comprising the Fas reorientator are HLA-I deficient (e.g., B2M negative or B2M negative). - / - ), and optionally further comprising CAR, exogenous CD16 or its variant, CD38 - / - and one or more of an exogenously introduced cytokine signaling complex (IL). In some embodiments, the iPSCs and cells derived therefrom comprising the Fas redirector are HLA-I deficient and HLA-II deficient (e.g., B2M - / - and CIITA negative or CIITA - / - ), and optionally further comprising CAR, exogenous CD16 or its variant, CD38 - / - In some embodiments, the iPSCs and cells derived therefrom comprising the Fas redirector are HLA-I and / or HLA-II deficient, and further comprise an exogenous polynucleotide encoding HLA-G or HLA-E, and optionally CAR, exogenous CD16 or its variant, CD38 - / - In some embodiments, the iPSCs and their derivatives comprising the Fas redirector are HLA-I and / or HLA-II deficient and CD58 negative, and optionally further comprise CAR, exogenous CD16 or its variant, CD38 - / - In some embodiments, the iPSCs and their derivatives comprising the Fas redirector are HLA-I and / or HLA-II deficient and CD54 negative, and optionally further comprise CAR, exogenous CD16 or its variant, CD38 - / - and one or more of IL.

[0262] In some embodiments, iPSCs and cells derived therefrom containing Fas redirectors are HLA-I and / or HLA-II deficient, and are CD58 negative and CD54 negative, and optionally further contain CAR, exogenous CD16 or its variant, CD38 - / - In some embodiments, the present invention comprises one or more of Fas redirector and HLA-I and / or HLA-II deficiency, and optionally CAR, exogenous CD16 or its variant, CD38 - / - In some embodiments, the effector cells of one or more of the NK cells and IL cells are NK cells derived from iPSCs. In some embodiments, the NK cells comprise a Fas redirector and HLA-I and / or HLA-II deficiency, and optionally a CAR, an exogenous CD16 or a variant thereof, a CD38 - / -The effector cells of one or more of the ILs are T cells derived from iPSCs.

[0263] In some embodiments, iPSCs and cells derived therefrom comprise a Fas redirector and an HLA deletion or modification (e.g., HLA-I and / or HLA-II modification, including: HLA-I knockout with or without HLA-II knockout, and optionally one or more of HLA-E or HLA-G knockin or overexpression, CD54 knockout, and CD58 knockout; such HLA-I and / or HLA-II modifications are represented by "HLA" in Table 1) and optionally a CAR, an exogenous CD16 or a variant thereof, CD38 - / - and one or more of IL, said cells further comprising one or more additional genome edits as provided in Table 1, without adversely affecting the differentiation potential of the iPSCs and the function of the derived effector cells (including derived T cells and NK cells).

[0264] Also provided is a master cell bank comprising single cell sorted and expanded clonally engineered iPSCs having at least Fas redirector and HLA deficiency as described in this section, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogenous cell therapy products that are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0265] 7. Genetically engineered iPSC lines and derived cells provided herein

[0266] According to the above content, the present application provides iPSC, cloned iPS cell, iPS cell line cell or its derivative cell, wherein each cell comprises at least a Fas redirector as described herein ("Fas" in Table 1). In some embodiments, these cells comprise an exogenous polynucleotide encoding a signaling redirection receptor, the signaling redirection receptor comprising (a) an extracellular Fas binding domain comprising a partial or complete peptide of the extracellular domain (ECD) of the Fas receptor (FAS) or a variant or allele thereof, and a cytoplasmic signaling domain comprising a partial or complete peptide of the intracellular domain (ICD) of one or more co-stimulatory molecules, wherein the signaling redirection receptor is a Fas redirector, and the Fas redirector redirects Fas signaling when bound to a FAS agonist, thereby providing the cell or the derivative cell with improved apoptosis resistance and / or exhaustion resistance. In some embodiments, the derived cells are hematopoietic lineage cells, including but not limited to mesodermal cells with permanent hemogenic endothelial cell (HE) potential, permanent HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, bone marrow cells, neutrophil progenitor cells, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutrophils, dendritic cells and macrophages. In some embodiments, functional derived hematopoietic cells include effector cells, which have one or more functional characteristics that are not present in corresponding primary T cells, NK cells, NKT cells and / or B cells.

[0267] In some embodiments, the derived cells include NK or T lineage cells. - / - iPSC-derived NK or T lineage cells with one or more of , cytokine signaling complexes, HLA deficiency, and any other patterns (as shown in Table 1) can be used to overcome or reduce Fas-induced apoptosis or fratricide and / or tumor microenvironment suppression associated with solid tumors. As discussed above, derived CAR-T cells expressing hnCD16 have acquired ADCC, providing an additional mechanism for tumor killing in addition to CAR targeting. In some embodiments, the derived cells include NK lineage cells. Containing Fas redirectors and optionally CAR, exogenous CD16, CD38 - / - iPSC-derived NK cells with one or more of the following patterns (as shown in Table 1) have enhanced cytotoxicity and are effective in recruiting bystander cells including T cells to infiltrate and kill tumor cells.

[0268] In the presence of a Fas redirector and optionally CAR, exogenous CD16, CD38- / - In some embodiments of the effector cells of one or more of the cytokine signaling complex, HLA deficiency and any other mode (as shown in Table 1), the cells derived from the engineered iPSC are complete in HLA-II, and still tolerate the allogeneic rejection of activated receptor T, B and NK cells. In some embodiments, iPSC and its derived effector cells are complete in HLA-II, and have synergistically increased retention and / or survival in the presence of activated receptor T cells, B cells and NK cells. In some embodiments, iPSC and its derived effector cells can be used to overcome or reduce the tumor microenvironment suppression associated with solid tumors. When anti-CD38 antibodies are used in combination therapy with the derived effector cells, the cells have synergistically increased retention, survival and effector functions.

[0269] Also provided is an iPSC comprising a Fas redirector and optionally a CAR, exogenous CD16, CD38 - / - , cytokine signaling complex ("IL" in Table 1), HLA deficiency, and one or more of any other patterns (as shown in Table 1), wherein the iPSCs are capable of directing differentiation to produce functional derived hematopoietic cells, optionally with HLA-E or HLA-G knock-in and knock-out of one or both of CD54 and CD58 to overcome alloreactive NK cells.

[0270] Therefore, the present application provides iPSCs and functionally derived hematopoietic cells thereof, comprising any one of the following genotypes in Table 1. In some embodiments, iPSCs and their functionally derived hematopoietic cells have a genotype comprising at least one Fas redirector ("Fas" in Table 1). In addition, "HLA" in Table 1 represents HLA-I and / or HLA-II modification, including: HLA-I knockout with or without HLA-II knockout, and optionally one or more of HLA-E or HLA-G knock-in or overexpression, CD54 knockout, and CD58 knockout. In some embodiments, iPSCs and their functionally derived hematopoietic cells have a genotype comprising at least one Fas redirector and optionally a checkpoint inhibitor ("CI" in Table 1). + ”), adapter (“En + ”) or antibody (“Ab + ”) genotype. Checkpoint inhibitors, adaptors, and antibodies are discussed in more detail below.

[0271] Table 1: Suitable exemplary genotypes of provided cells :

[0272]

[0273]

[0274]

[0275]

[0276]

[0277] 8. Additional Modifications

[0278] In some embodiments, the genetically modified iPSC and its derived cells further comprise the genotype listed in Table 1, wherein the cells may also comprise one or more of the following genetic modification patterns: safety switch proteins, targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote the transplantation, transportation, homing, vitality, self-renewal, retention, immune response regulation and regulation and / or survival of iPSC or its derived cells. In some embodiments, the iPSC and its derived effector cells comprising any of the genotypes in Table 1 may additionally comprise: TAP1, TAP2, TAP-related protein, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and at least one of the deletions or disruptions of any gene in the chromosome 6p21 region; or HLA-E, HLA-G, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention relates to the introduction of at least one of R, TCR, Fc receptor, antibody, adapter and surface triggering receptor for coupling with bispecific, multispecific or universal adapter.

[0279] 8.1 Adaptors

[0280] Adaptor is a fusion protein composed of two or more single-chain variable fragments (scFv) or other functional variants of different antibodies or their fragments, wherein at least one scFv is bound to effector cell surface molecules or surface triggering receptors, and at least another is bound to target cells via target cell specific surface molecules. Adaptor can be bispecific or multispecific. Such bispecific or multispecific adaptors can guide effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages and / or neutrophils) to tumor cells and activate immune effector cells, and have shown great potential for maximizing the benefits of CAR-T cell therapy. The example of adaptor includes but is not limited to bispecific T cell adaptor (BiTE), bispecific killer cell adaptor (BiKE), trispecific killer cell adaptor (TriKE), multispecific killer cell adaptor, or a universal adaptor compatible with a variety of immune cell types.

[0281] In some embodiments, the adapter is used in combination with a population of effector cells described herein by simultaneous or sequential administration, wherein the effector cells comprise a surface molecule or surface triggering receptor recognized by the adapter. In some other embodiments, the adapter ("En " in Table 1 + ”) is a bispecific antibody expressed by derived effector cells by genetically engineering iPSCs as described herein, followed by directed differentiation of the engineered iPSCs. Exemplary effector cell surface molecules or surface triggering receptors that can be used for recognition or conjugation by bispecific or multispecific adaptors include, but are not limited to, CD3, CD5, CD16, CD28, CD32, CD33, CD64, CD89, NKG2C, NKG2D, and chimeric Fc receptors as disclosed herein.

[0282] In some embodiments, the target cell of the adapter is a tumor cell. Exemplary tumor cell surface molecules for bispecific or multispecific adapter 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, Mucl, Mucl6, PDL1, PSMA, PAMA, P-cadherin, ROR1, and VEGF-R2. In various embodiments, the bispecific adapter 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.

[0283] In another embodiment, the bispecific antibody is also included in the joint between the effector cell and the tumor cell antigen binding domain. For example, modified IL15 can be used as a joint of effector NK cells to promote cell expansion (referred to as TriKE or tri-specific killing adapter in some publications). Exemplary TriKE includes but is 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.

[0284] In some embodiments, the surface triggering receptor used for the bispecific or multispecific adaptor can be endogenous to the effector cell, sometimes depending on the cell type. In some other embodiments, the methods and compositions provided herein can be used to introduce one or more exogenous surface triggering receptors into the effector cell, for example, by additional engineering of iPSCs containing the genotypes listed in Table 1, and then directing the differentiation of the iPSCs to T cells, NK cells, or any other effector cells containing the same genotype and surface triggering receptor as the source iPSC.

[0285] 8.2 Antibodies for Immunotherapy

[0286] In some embodiments, in addition to the genome engineered effector cells as provided herein, additional therapeutic agents comprising antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication may be used in combination with these effector cells. In some embodiments, antibodies are used in combination with effector cell populations described herein by simultaneous or sequential administration to a subject. In other embodiments, such antibodies or fragments thereof ("Ab" in Table 1) are used in combination with effector cells. +”) can be expressed by effector cells by genetically engineering iPSCs with an exogenous polynucleotide sequence encoding the antibody or a fragment thereof and directing the differentiation of the engineered iPSCs. In some embodiments, the effector cells additionally express an exogenous CD16 variant, wherein the cytotoxicity of the effector cells is enhanced by the 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, tumor- or virus-specific antigens activate the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, antibodies suitable for combination therapy as an additional therapeutic agent with the administered iPSC-derived effector cells include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, ubrituximab, okalaizumab, 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 humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof.

[0287] In some embodiments, the Fas redirector and optionally CAR, exogenous CD16, CD38 - / - , cytokine signaling complexes, and HLA deficiency, for use in combination therapy comprising said effector cells and anti-CD38 antibodies such as daratumumab, isatuximab, and MOR202. In some embodiments of the combination that can be used to treat liquid tumors or solid tumors, the combination comprises: comprising a Fas redirector and optionally a CAR, exogenous CD16, CD38 - / - , cytokine signaling complex, and HLA deficiency; and daratumumab. In some additional embodiments, the iPSC-derived NK cells included in combination with one of the anti-CD38 antibodies include a Fas redirector, a CAR, exogenous CD16, CD38 - / -, HLA deficiency and IL15 or cytokine signaling complex; wherein IL15 or its signaling complex is co-expressed with CAR or expressed alone. In some additional embodiments, the iPSC-derived T cells included in the combination with one of the anti-CD38 antibodies include Fas redirectors, CAR, exogenous CD16, CD38 - / - , HLA deficiency and IL7 or its cytokine signaling complex. In some specific embodiments, IL7 or its signaling complex is expressed together or separately with CAR.

[0288] In some embodiments, iPSC-derived effector cells, including iPSC-derived NK cells or T cells, comprise a Fas redirector and optionally a CAR, exogenous CD16, CD38 - / - , cytokine signaling complexes, HLA deficiency, and Ab + One or more of the following, where “Ab + " refers to an exogenous polynucleotide sequence encoding an antibody or a fragment thereof, and wherein the antibody or fragment thereof is expressed by an effector cell. In various embodiments, Ab + Contains any one or more of the above-mentioned therapeutic antibodies or functional fragments thereof.

[0289] 8.3 Checkpoint Inhibitors

[0290] Checkpoints are cellular molecules, typically cell surface molecules, that are able to inhibit or downregulate immune responses when not inhibited. It is now clear that tumors co-opt certain immune checkpoint pathways as a primary mechanism of immune resistance, particularly against T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can reduce checkpoint gene expression or gene products, or reduce the activity of checkpoint molecules, thereby blocking inhibitory checkpoints and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, with these agents providing long-term remissions in a variety of indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a major problem.

[0291] Therefore, the present application provides a method of overcoming CI resistance by including a functional derivative cell of genome engineering provided herein in a combination therapy with CI in some embodiments. In some embodiments, the checkpoint inhibitor is used in combination with the effector cell population described herein by administering the effector cell population to the subject simultaneously or continuously. Some embodiments of the combination therapy with the effector cells described herein include at least one checkpoint inhibitor to target at least one checkpoint molecule; wherein the derivative cell has a genotype listed in Table 1. In other embodiments, the checkpoint inhibitor ("CI" in Table 1) is used in combination with the effector cell population described herein. +”) is expressed by effector cells by genetically engineering iPSCs with an exogenous polynucleotide sequence encoding a checkpoint inhibitor or a fragment or variant thereof and directing the differentiation of the engineered iPSCs.

[0292] In some embodiments, the exogenous polynucleotide sequence encoding the check point inhibitor or its fragment is co-expressed with CAR in a separate construct or in a bicistronic construct. In some other embodiments, the sequence encoding the check point inhibitor or its fragment can be connected to the 5' end or 3' end of the CAR expression construct by the self-cleavage 2A coding sequence, and the self-cleavage 2A coding sequence is shown as, for example, CAR-2A-CI or CI-2A-CAR. Therefore, the coding sequence of the check point inhibitor and CAR is in a single open reading frame (ORF). When the check point inhibitor is delivered, expressed and secreted as a payload by the derived effector cells that can infiltrate the tumor microenvironment (TME), it counteracts the inhibitory check point molecules after engaging the TME, allowing the activation of effector cells by, for example, CAR or activation receptor activation mode. 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.

[0293] Checkpoint inhibitors suitable for combination therapy with the derived effector cells provided herein and / or expressed by the derived effector cells provided herein include, but are not limited to, PD-1 (Pdcd1, 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 2A R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and antagonists of inhibitory KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0294] 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 antibody (VNAR) with only a heavy chain, 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), mini antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single-domain antigen-binding fragments (sdAb, nano antibodies), recombinant antibodies with only heavy chains (VHH), and other antibody fragments that maintain the binding specificity of all antibodies, which can be more cost-effective to produce, easier to use, or more sensitive than all antibodies. 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), lirimumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and any derivatives, functional equivalents, or biosimilars thereof.

[0295] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is based on microRNA, because many miRNAs are found as regulatory factors that control the expression of immune checkpoints (Dragomir et al., Cancer Biol Med., 2018, Vol. 15, No. 2, pp. 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.

[0296] In some embodiments, a checkpoint inhibitor (“CI +”) is co-expressed with CAR and inhibits at least one of the following checkpoint molecules: PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2A R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E and inhibitory KIR. In some embodiments, the check point inhibitor co-expressed with CAR in the derivative cells having the genotype listed in Table 1 is selected from the group comprising: atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and their humanized or Fc-modified variants, fragments and their functional equivalents or biosimilars. In some embodiments, the check point inhibitor co-expressed with CAR is atezolizumab or its humanized or Fc-modified variants, fragments or their functional equivalents or biosimilars. In some other embodiments, the check point inhibitor co-expressed with CAR is nivolumab or its humanized or Fc-modified variants, fragments or their functional equivalents or biosimilars. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0297] In some other embodiments of the combination therapy comprising the derived effector cells provided herein and at least one antibody that inhibits checkpoint molecules, the antibody is not produced by or in a derived cell, and is administered before, simultaneously or after the derived cell provided herein is administered. In some embodiments, it is simultaneous or sequential to administer one, two, three or more checkpoint inhibitors in the combination therapy with the derived NK lineage cells or T lineage cells provided. In one embodiment of the combination therapy, the checkpoint inhibitors included in the treatment are atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and their humanization or Fc-modified variants, fragments and their functional equivalents or biosimilars. In some embodiments of the combination therapy, the checkpoint inhibitors included in the treatment are atezolizumab or its humanization or Fc-modified variants, fragments and their functional equivalents or biosimilars. 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, and functional equivalent or biosimilar thereof.

[0298] In some embodiments, iPSC-derived effector cells, including iPSC-derived NK cells or T cells, comprise a Fas redirector and optionally a CAR, exogenous CD16, CD38 - / - , cytokine signaling complexes, HLA deficiency, and CI + One or more of the following, where “CI + " refers to an exogenous polynucleotide sequence encoding a check point inhibitor or a fragment thereof, and wherein the check point inhibitor or fragment thereof is expressed by an effector cell. In various embodiments, CI + Contains any one or more of the above-mentioned therapeutic antibodies or functional fragments thereof.

[0299] II. Methods for Targeted Genome Editing at Selected Loci in iPSCs

[0300] As used interchangeably herein, genome editing or gene editing is a type of genetic engineering, in which DNA insertion, deletion and / or replacement are performed in the genome of the target cell. Targeted genome editing (interchangeable with "targeted genome editing" or "targeted gene editing") can achieve insertion, deletion and / or replacement at preselected sites in the genome. When the endogenous sequence is missing at the insertion site during targeted editing, the endogenous gene containing the affected sequence can be knocked out or reduced due to sequence deletion. Therefore, targeted editing can also be used to accurately interrupt endogenous gene expression. The term "targeted integration" is similarly used herein, which refers to a method involving inserting one or more exogenous sequences when the endogenous sequence is missing or not missing at the insertion site. In contrast, the randomly integrated gene undergoes position effect and silence, so that its expression is unreliable and unpredictable. For example, transgenic silencing is particularly prone to occur in centromere and subtelomeric regions. On the contrary, the newly integrated gene can affect surrounding endogenous genes and chromatin, potentially changing cell properties or facilitating cell transformation. Therefore, insertion of exogenous DNA into a preselected locus, such as a safe harbor locus or genomic safe harbor (GSH), is important for safety, efficiency, copy number control, and reliable genetic response control.

[0301] Targeted editing can be achieved by nuclease-independent methods or by nuclease-dependent methods. In nuclease-independent targeted editing methods, homologous recombination is directed by the enzymatic machinery of the host cell, through homologous sequences flanking the inserted foreign polynucleotide.

[0302] Alternatively, targeted editing can be achieved with a higher frequency by using specific rare-cutting nucleases to specifically introduce double-strand breaks (DSBs). This type of nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without using a donor vector containing exogenous genetic material, NHEJ usually causes random insertion or deletion (insertion / deletion) of a small amount of endogenous nucleotides. In contrast, when a donor vector containing exogenous genetic material flanked by a pair of homologous arms is present, exogenous genetic material can be introduced into the genome by homologous recombination during homology-directed repair (HDR), thereby generating "targeted integration". In some cases, the targeted integration site is intended to be within the coding region of a selected gene, and therefore targeted integration may disrupt gene expression, resulting in simultaneous knock-in and knock-out (KI / KO) in a single editing step.

[0303] Can realize in the locus (GOI) of paying attention to one or more transgenics being inserted in the selected position to knock out the gene simultaneously.The locus suitable for knocking in and knocking out (KI / KO) simultaneously includes but is not limited to B2M, TAP1, TAP2, TAP associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR α or TCR β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3 and TIGIT.Utilize the corresponding site-specific targeting homology arm for positional selective insertion, allow transgenic under endogenous promoter, express at this site, or express under the exogenous promoter included in construct.When two or more transgenics are inserted into the selected position in CD38 locus, the connecting sequence of for example 2A joint or IRES is placed between any two transgenics. The 2A linker encodes a self-cleaving peptide derived from, for example, FMDV, ERAV, PTV-I, or TaV (referred to as "F2A," "E2A," "P2A," and "T2A," respectively), such that separate proteins can be produced from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of silencing of the transgene and / or exogenous promoter. In various embodiments, the exogenous promoter can be CAG or other constitutive, inducible, temporally specific, tissue-specific, and / or cell-type-specific promoters, including but not limited to CMV, EF1α, PGK, and UBC.

[0304] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) systems. In addition, the DICE (double integrase cassette exchange) system utilizing phiC31 and Bxb1 integrases is also a promising tool for targeted integration.

[0305] ZFNs are targeted nucleases that comprise a nuclease fused to a zinc finger DNA binding domain. "Zinc finger DNA binding domain" or "ZFBD" means a polypeptide domain that binds DNA through one or more zinc fingers in a sequence-specific manner. Zinc fingers are domains of about 30 amino acids within a zinc finger binding domain, the structure of which is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A "designed" zinc finger domain is a domain that does not exist in nature, and its design / composition is primarily derived from rational criteria, such as the application of substitution rules and computerized algorithms to process information in databases storing existing ZFP design and binding data information. See, e.g., U.S. Pat. Nos. 6,140,081, 6,453,242, and 6,534,261, see also WO 98 / 53058, WO 98 / 53059; WO 98 / 53060, WO 02 / 016536, and WO 03 / 016496, the entire disclosures of which are incorporated herein by reference. A "selected" zinc finger domain is a domain not found in nature, the production of which is primarily derived from empirical methods, such as phage display, interaction interception, or hybrid selection. ZFNs are described in more detail in U.S. Pat. Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is a fusion of a FokI nuclease with a zinc finger DNA binding domain.

[0306] TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. "Transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain" or "TALE DNA binding domain" means the polypeptide domain of the TAL effector protein responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by Xanthomonas plant pathogens during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences through their DNA binding domains, and activate gene transcription at these sequences through their transactivation domains. The TAL effector DNA binding domain specificity depends on the variable number of effectors of imperfect 34 amino acid repeats, which include polymorphisms at selected repeat positions, called repeat variable diresidues (RVDs). TALEN is described in more detail in U.S. Publication No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of TALEN in the art is a fusion polypeptide of FokI nuclease and TAL effector DNA binding domain.

[0307] Another example of a targeted nuclease used in the methods of the present invention is a targeted Spo11 nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA binding domain (e.g., a zinc finger DNA binding domain specific for a DNA sequence of interest, a TAL effector DNA binding domain, etc.).

[0308] Additional examples of targeted nucleases suitable for use with embodiments of the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used alone or in combination.

[0309] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR-associated nucleases from the family including: cpf, cse, csy, csn, csd, cst, csh, csa, csm and cmr; restriction endonucleases; meganucleases; homing endonucleases, etc.

[0310] Using Cas9 as an example, CRISPR / Cas9 requires two main components: (1) Cas9 endonuclease and (2) crRNA-tracrRNA complex. When co-expressed, the two components form a complex that is recruited to the target DNA sequence, including the PAM and the seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target the selected sequence. These two components can then be delivered to mammalian cells by transfection or transduction.

[0311] DICE-mediated insertion utilizes a pair of recombinases (e.g., phiC31 and Bxb1) to provide unidirectional integration of foreign DNA, which is strictly limited to the small attB and attP recognition sites of each enzyme itself. Since these att targets do not naturally occur in mammalian genomes, they must first be introduced into the genome at the desired integration site. See, e.g., U.S. Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0312] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct also comprises a pair of homology arms specific to the desired integration site, and the targeted integration method comprises introducing the construct into the cell to allow the cell host enzyme mechanism to achieve site-directed homologous recombination. In another embodiment, the method for achieving targeted integration in the 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 to the desired integration site into the cell to achieve ZFN-mediated insertion. In yet another embodiment, the method for achieving targeted integration in the 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 to the desired integration site into the cell to achieve TALEN-mediated insertion. In another embodiment, the method for achieving targeted integration in the cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing a Cas9 expression cassette and a gRNA comprising a guide sequence specific to the desired integration site into the cell to achieve Cas9-mediated insertion. In another embodiment, the method for achieving targeted integration in a cell comprises introducing a construct comprising a pair of one or more att sites for DICE recombinase into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to achieve DICE-mediated targeted integration.

[0313] Sites that are expected to be used for targeted integration include, but are not limited to, safe harbor loci or genomic safe harbors (GSHs), which are intragenic or extragenic regions of the human genome that, in theory, can accommodate predictable expression of newly integrated DNA without adversely affecting host cells or organisms. Applicable safe harbors must allow transgenic expression to be sufficient to produce the desired level of protein or non-coding RNA encoded by the vector. The safe harbor must also not make the cell susceptible to malignant transformation or alter cell function. In order for the integration site to become a potential safe harbor locus, it is ideally necessary to meet criteria including, but not limited to, the following: as judged by sequence annotation, there is no interruption of regulatory elements or genes; it is an intergenic region in a gene-dense region, or a convergence position between two genes transcribed in opposite directions; maintain a distance to minimize the possibility of long-range interactions between the transcriptional activator encoded by the vector and the promoter of the adjacent gene (especially cancer-related and microRNA genes); and have obvious ubiquitous transcriptional activity, as reflected by the expression pattern of sequence tags (ESTs) expressed in a wider space and time, which indicates ubiquitous transcriptional activity. This latter feature is particularly important in stem cells, where during differentiation, chromatin remodeling often leads to silencing of some loci and potential activation of other loci. In regions suitable for exogenous insertion, the exact loci chosen for insertion should be free of repetitive elements and conserved sequences and against which primers for amplifying homology arms can be easily designed.

[0314] Sites suitable for human genome editing or specifically targeted integration include, but are not limited to, adeno-associated virus site 1 (AAVS1), 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 may also be a suitable site for insertion using the targeted integration compositions and methods disclosed herein. In addition, collagen and HTRP loci may also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be necessary, particularly in stem cells for specific integration events, and optimization of insertion strategies, including promoter selection, exogenous gene sequence and configuration, and construct design, is often required.

[0315] The editing site for targeted insertion / deletion is generally contained in an endogenous gene whose expression and / or function is intended to be interrupted. In some embodiments, the endogenous gene comprising the targeted insertion / deletion is associated with immune response regulation and modulation. In some other embodiments, the endogenous gene comprising the targeted insertion / deletion is associated with the following: targeting mode, receptor, signaling molecule, transcription factor, drug target candidate, immune response regulation and modulation, or inhibiting stem cell and / or progenitor cell and its derived cell transplantation, transport, homing, vitality, self-renewal, retention and / or survival protein.

[0316] Thus, one aspect of the invention provides a method for targeted integration in a selected locus, the selected locus comprising a genomic safe harbor or a preselected locus known or confirmed to be safe and fully regulated to achieve continuous or transient gene expression, for example, with respect to the constant region of a T cell, a T cell receptor (TCR). In one embodiment, the genomic safe harbor used in the targeted integration method comprises one or more desired integration sites, comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR or RUNX1, or other loci that meet the genomic safe harbor criteria. In one embodiment, the method for targeted integration in a cell comprises: introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a construct comprising a pair of homologous arms and one or more exogenous sequences that are specific to the desired integration site, to achieve site-directed homologous recombination by a cell host enzyme mechanism, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR or RUNX1, or other loci that meet the genomic safe harbor criteria. Additional integration sites include endogenous loci intended for disruption (such as reduction or knockout) comprising B2M, TAP1, TAP2, TAP-related protein, 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.

[0317] In another embodiment, the 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 achieve ZFN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, 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 another embodiment, the 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 achieve TALEN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, 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, the method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, introducing into the cell a Cas9 expression cassette and a gRNA comprising a guide sequence specific to a desired integration site to achieve Cas9-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, 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, the method for targeted integration in a cell comprises introducing one or more att sites comprising a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for the DICE recombinase to achieve DICE-mediated targeted integration, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, 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.

[0318] In addition, as provided herein, the above methods for targeted integration in safe harbors are used to insert any polynucleotides of interest, such as polynucleotides encoding: safety switch proteins, targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and proteins that promote stem cell and / or progenitor cell transplantation, transportation, homing, vitality, self-renewal, retention and / or survival. In some other embodiments, the construct comprising one or more exogenous polynucleotides also comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the present invention is a suicide gene encoding a safety switch protein. Suicide gene systems suitable for inducing 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 specific to cell types, such as genetic modification of T lymphocytes using the B cell molecule CD20 to allow them to be eliminated after administration of mAb rituximab. In addition, when the genetically engineered cells are exposed to cetuximab, modified EGFR containing epitopes recognized by cetuximab can be used to deplete the cells. Therefore, one aspect of the present invention provides a method for targeted integration of one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR and B cell CD20.

[0319] In some embodiments, one or more exogenous polynucleotides integrated by the methods described herein are driven by an exogenous promoter that is operably linked and contained in a construct for targeted integration. The promoter may be inducible or structural and may be time-specific, tissue-specific, or cell-type specific. Structural promoters suitable for the methods of the invention include, but are not limited to, cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0320] The exogenous polynucleotides integrated by the methods herein can be driven at the integration site by an endogenous promoter in the host genome. In one embodiment, the methods described herein are used to target one or more exogenous polynucleotides to be integrated into the AAVS1 locus in the cell genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the methods described herein are used to target the ROSA26 locus integrated into the cell genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the methods described herein are used to target the H11 locus integrated into the cell genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the methods described herein are used to target the collagen locus integrated into the cell genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the methods described herein are used to target the HTRP locus integrated into the cell genome. In one embodiment, at least one integrated polynucleotide is driven by the endogenous HTRP promoter. In theory, only the correct insertion into the desired position can achieve gene expression of the exogenous gene driven by the endogenous promoter.

[0321] In some embodiments, one or more exogenous polynucleotides included in the construct of the targeted integration method are driven by a promoter. In some embodiments, the construct comprises one or more joint sequences located between two adjacent polynucleotides driven by the same promoter, so that the physical spacing between the parts is larger and the feasibility of the enzyme mechanism is maximized. The joint peptide of the joint sequence can be composed of amino acids selected to produce physical spacing between the parts (exogenous polynucleotides, and / or proteins or peptides encoded therefrom), which are softer or harder depending on the relevant functions. The joint sequence can be cleaved by protease or chemically cleaved to produce a separate part. Examples of enzyme cleavage sites in the joint include cleavage sites of proteolytic enzymes (such as enterokinase, factor Xa, trypsin, collagenase and thrombin). In some embodiments, the protease is a protease naturally produced by the host or introduced by an exogenous source. Alternatively, the cleavage site in the joint can be a site that can be cleaved after being exposed to a selected chemical substance (such as cyanogen bromide, hydroxylamine or low pH). The optional joint sequence can serve the purpose of providing a cleavage site. The linker sequence should allow the part to be effectively positioned relative to another adjacent part so that the part functions correctly. The linker can also be a simple amino acid sequence that is long enough to prevent any steric hindrance between the parts. In addition, the linker sequence can achieve post-translational modifications, including but not limited to, for example, phosphorylation sites, biotinylation sites, sulfation sites, γ-carboxylation sites, etc. In some embodiments, the linker sequence is soft so that the biologically active peptide cannot maintain a single undesirable conformation. The linker can mainly contain amino acids with small side chains, such as glycine, alanine and serine, to provide flexibility. In some embodiments, about 80 or 90% or more of the linker sequence contains glycine, alanine or serine residues, especially glycine and serine residues. In several embodiments, the G4S linker peptide separates the terminal processing domain and the endonuclease domain of the fusion protein. In other embodiments, the 2A linker sequence allows a single translation to produce two separate proteins. Suitable linker sequences can be easily identified empirically. In addition, the suitable size and sequence of the linker sequence can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some embodiments, any two adjacent linker sequences are different.

[0322] The method of introducing the construct comprising the exogenous polynucleotide to be targeted for integration into the cell can be realized using the method known to transfer genes to cells. In one embodiment, the construct comprises a viral vector, such as adenoviral vector, adeno-associated viral vector, retroviral vector, slow viral vector, Sendai viral vector skeleton. In some embodiments, plasmid vectors are used to deliver exogenous polynucleotides to target cells and / or to express exogenous polynucleotides in target cells (such as pAl-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo) etc. In some other embodiments, free-type vectors are used to deliver exogenous polynucleotides to target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used for genetic engineering to introduce insertion, deletion or substitution by homologous recombination. Unlike slow viruses, rAAV is not integrated into the host genome. In addition, free-type rAAV vectors mediate homologous directional genes, which are targeted with much higher ratios compared to the 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, genome-modified iPSCs and their derivative cells obtained using the methods and compositions described herein comprise at least one genotype listed in Table 1.

[0323] III. Methods for obtaining and maintaining genome-engineered iPSCs

[0324] In various embodiments, the present invention provides a method for obtaining and maintaining genome engineering iPSC, the method includes one or more targeted editing performed at one or more desired sites, wherein one or more targeted editing is maintained intact and functional in amplified genome engineering iPSC or iPSC derived non-pluripotent cells, at corresponding selected editing sites. Targeted editing introduces iPSC and its derivative cells into the genome (i.e., introducing targeted integration and / or insertion / deletion at selected sites). Compared with the peripheral blood origin primary effector cells of direct engineering patient sources, many benefits of genome engineering derived cells obtained by editing and differentiation as provided herein include but are not limited to: the source of engineered effector cells is not limited; there is no need to repeatedly manipulate effector cells, especially when involving multiple engineering modes; the obtained effector cells are regenerated due to having elongated telomeres and experiencing less exhaustion; the effector cell group is uniform with respect to editing sites, copy number and lack of allele variants, random mutations and expression variegation, mainly due to the ability to perform clonal selection in the engineered iPSC as provided herein.

[0325] In a specific embodiment, the genome engineering iPSC comprising one or more targeted editing at one or more selected sites is maintained, passaged and expanded for a long time in a cell culture medium as a fate maintenance medium (FMM) as shown in Table 2 as a single cell, wherein the iPSC retains targeted editing and functional modification at the selected site. The components of the culture medium can be present in the culture medium in an amount within the optimal range shown in Table 2. iPSCs cultured in FMM have been shown to continue to maintain their undifferentiated and basic or initial characteristics; provide genomic stability without the need for culture cleaning or selection; and differentiate via in vitro embryoid bodies or monolayers (without forming embryoid bodies); and differentiation via in vivo teratoma formation easily produces all three somatic cell lineages. See, for example, International Publication No. WO2015 / 134652, the disclosure of which is incorporated herein by reference.

[0326] Table 2: Exemplary culture media for iPSC reprogramming and maintenance

[0327]

[0328]

[0329] 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 being free or substantially free of a TGFβ receptor / ALK5 inhibitor, wherein the iPSCs retain intact and functional targeted edits at the selected site.

[0330] Another aspect of the present invention provides a method for producing genome-engineered iPSCs, which is by targeted editing of iPSCs; or firstly, genome-engineered non-pluripotent cells are produced by targeted editing, and then the selected / separated genome-engineered non-pluripotent cells are reprogrammed to obtain iPSCs comprising the same targeted editing as non-pluripotent cells. Another aspect of the present invention provides genome-engineered non-pluripotent cells, which undergo reprogramming simultaneously by introducing targeted integration and / or targeted insertion / deletion into cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and wherein the reprogramming conditions include contacting non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneously carrying out genome engineering and reprogramming, targeted integration and / or targeted insertion / deletion can be introduced into non-pluripotent cells by contacting non-pluripotent cells with one or more reprogramming factors and optionally one or more small molecules before initiating reprogramming or substantially simultaneously.

[0331] In some embodiments, in order to simultaneously perform genome engineering and reprogramming of non-pluripotent cells, targeted integration and / or insertion / deletion can also be introduced into non-pluripotent cells after initiating a multi-day reprogramming process by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and the vector carrying the construct is introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes (including but not limited to SSEA4, Tra181 and CD30).

[0332] In some embodiments, reprogramming is maintained and expanded by treating non-pluripotent cells with at least one reprogramming factor and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 2). In some embodiments, the genome-engineered iPSC produced by any of the above methods is further maintained and expanded using a mixture (FMM; Table 2) comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor.

[0333] In some embodiments of the method of generating genome-engineered iPSCs, the method comprises: genome-engineering iPSCs by introducing one or more targeted integrations and / or indels into iPSCs to obtain genome-engineered iPSCs having at least one genotype listed in Table 1. Alternatively, the method of generating genome-engineered iPSCs comprises: (a) introducing one or more targeted edits into non-pluripotent cells to obtain genome-engineered non-pluripotent cells comprising targeted integrations and / or indels at selected sites, and (b) contacting the genome-engineered non-pluripotent cells with one or more reprogramming factors and optionally a small molecule composition to obtain genome-engineered iPSCs comprising targeted integrations and / or indels at selected sites, the small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor. Alternatively, the method for producing genome-engineered iPSCs includes: (a) contacting non-pluripotent cells with one or more reprogramming factors and optional small molecule compositions to initiate non-pluripotent cell reprogramming, the small molecule compositions comprising TGFβ receptor / ALK inhibitors, MEK inhibitors, GSK3 inhibitors and / or ROCK inhibitors; (b) introducing one or more targeted integrations and / or insertions / deletions into reprogrammed non-pluripotent cells for genome engineering; and (c) obtaining cloned genome-engineered iPSCs comprising targeted integrations and / or insertions / deletions at selected sites. Any of the above methods may also include single cell sorting of genome-engineered iPSCs to obtain cloned iPSCs. By clonal expansion of genome-engineered iPSCs, a master cell bank is generated to include cloned engineered iPSCs of single cell sorting and amplification with at least one phenotype as provided herein. The master cell bank is subsequently cryopreserved, thereby providing a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogenous cell therapy products that are well-defined and uniform in composition and can be produced on a large scale in a cost-effective manner.

[0334] 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. WO2015 / 134652 and WO 2017 / 066634, the disclosure of which is incorporated herein by reference. One or more reprogramming factors may be in the form of polypeptides. Reprogramming factors may also be in the form of polynucleotides encoding reprogramming factors, and thus may be introduced into non-pluripotent cells by vectors (e.g., retroviruses, Sendai viruses, adenoviruses, episomes, plasmids and minicircles). In a specific embodiment, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by a combination of plasmids. See, for example, International Publication No. WO2019 / 075057A1, the disclosure of which is incorporated herein by reference.

[0335] In some embodiments, non-pluripotent cells utilize a variety of constructs comprising different exogenous polynucleotides and / or different promoters, through a variety of vector transfections for targeted integration at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or genes encoding proteins that promote iPSC or its derived cells to transplant, transport, home, vitality, self-renewal, retention and / or survival. In some embodiments, exogenous polynucleotides encode RNA, including but not limited to siRNA, shRNA, miRNA and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters, which are selected from the group consisting of: constitutive promoters, inducible promoters, time-specific promoters and tissue or cell type-specific promoters. Therefore, when, for example, in the presence of an inducer or under conditions where a promoter is activated in a specific differentiated cell type, the polynucleotide is expressible. In some embodiments, the polynucleotides are expressed in iPSC and / or in cells differentiated from iPSC. In one embodiment, one or more suicide genes are driven by a constitutive promoter, such as Capase-9 driven by CAG. These constructs comprising different exogenous polynucleotides and / or different promoters can be transfected into non-pluripotent cells simultaneously or continuously. Non-pluripotent cells undergoing targeted integration of multiple constructs can contact one or more reprogramming factors simultaneously to start reprogramming with genetic engineering, thereby obtaining iPSCs containing multiple targeted integrated genome engineering in the same cell pool. Therefore, this stable method realizes simultaneous reprogramming and engineering strategies, thereby obtaining iPSCs with clones of multiple modes integrated into one or more selected target sites. In some embodiments, iPSCs and their derivative cells obtained using the genome modification of the methods and compositions herein include at least one genotype listed in Table 1.

[0336] IV. Methods for obtaining genetically engineered effector cells by differentiating genome-engineered iPSCs

[0337] Another aspect of the present invention provides a method for differentiating genome-engineered iPSCs in vivo by teratoma formation, wherein differentiated cells derived from genome-engineered iPSCs in vivo retain complete and functional targeted editing, including targeted integration and / or insertion / deletion at the desired site. In some embodiments, genome-engineered iPSCs form differentiated cells derived from vivo via teratoma formation and include one or more inducible suicide genes integrated at one or more desired sites, the sites including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, β-2 microglobulin, CD38, GAPDH, TCR or RUNX1, or other loci that meet the genomic safe harbor criteria. In some other embodiments, genome-engineered iPSCs form differentiated cells derived from vivo via teratoma formation and include polynucleotides encoding targeted patterns or encoding proteins that promote the transport, homing, vitality, self-renewal, retention and / or survival of stem cells and / or progenitor cells. In some embodiments, the genome engineered iPSCs are differentiated cells derived in vivo via teratoma formation, and comprise one or more inducible suicide genes, and further comprise one or more insertions / deletions in endogenous genes associated with immune response regulation and mediation. In some embodiments, the insertion / deletion is contained in one or more endogenous checkpoint genes. In some embodiments, the insertion / deletion is contained in one or more endogenous T cell receptor genes. In some embodiments, the insertion / deletion is contained in one or more endogenous MHC class I inhibitory genes. In some embodiments, the insertion / deletion is contained in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, insertion / deletion is included in one or more endogenous genes, including but not limited to AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-related protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, 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 iPSC of genome engineering transformation comprising one or more exogenous polynucleotides at the selected site also comprises targeted editing in the B2M (β-2-microglobulin) encoding gene.

[0338] In a specific embodiment, the iPSC of genome engineering transformation comprising one or more genetic modifications as provided herein is used for in vitro derivation of hematopoietic cell lineages or any other specific cell type, wherein the derived non-pluripotent cells retain functional genetic modifications, including targeted editing at selected sites. In one embodiment, the iPSC-derived cells of genome engineering transformation include but are not limited to mesodermal cells with permanent hemogenic endothelial cell (HE) potential, permanent HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, bone marrow cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells and macrophages, wherein the cells derived from the genome engineering transformation iPSC retain functional genetic modifications, including targeted editing at the desired site.

[0339] Differentiation methods and compositions suitable for obtaining iPSC-derived hematopoietic cell lineages include, for example, differentiation methods and compositions described in International Publication No. WO2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, the method and composition for producing hematopoietic cell lineages is under serum-free, feeder-free and / or matrix-free conditions and in expandable and monolayer culture platforms without the need for EB formation by permanent hemogenic endothelial cells (HE) derived from pluripotent stem cells (including iPSC). The range of cells that can be differentiated according to the provided method is pluripotent stem cells to progenitor cells that are specialized in specific terminal differentiated cells and transdifferentiated cells and directly turn to hematopoietic fate without experiencing a variety of lineage cells of pluripotent intermediates. Similarly, the range of cells produced by stem cell differentiation is pluripotent stem cells or progenitor cells to terminal differentiated cells, and all intermediate hematopoietic cell lineages.

[0340] The method for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture comprises contacting pluripotent stem cells with BMP pathway activators and optionally bFGF. As provided, pluripotent stem cell-derived mesodermal cells are obtained and expanded without forming embryoid bodies from pluripotent stem cells. The mesodermal cells are then contacted with BMP pathway activators, bFGF, and WNT pathway activators to obtain expanded mesodermal cells with permanent hemogenic endothelial (HE) potential without forming embryoid bodies from pluripotent stem cells. By subsequently contacting with bFGF and optionally present ROCK inhibitors and / or WNT pathway activators, mesodermal cells with permanent HE potential differentiate into permanent HE cells, and during differentiation, the permanent HE cells are also expanded.

[0341] The methods provided herein for obtaining cells of the hematopoietic lineage are advantageous over EB-mediated differentiation of pluripotent stem cells because: EB formation produces modest to minimal cell expansion; does not allow monolayer culture, which is important for many applications that require uniform expansion and uniform differentiation of cells in a population; and is laborious and inefficient.

[0342] The monolayer differentiation platform provided promotes differentiation to permanent hemogenic endothelial cells, thereby obtaining hematopoietic stem cells and differentiation offspring, such as T cells, B cells, NKT cells and NK cells. The monolayer differentiation strategy achieves a combination of enhanced differentiation efficiency and large-scale expansion, and can deliver a number of pluripotent stem cell-derived hematopoietic cells in different therapeutic applications. In addition, the monolayer culture performed using the method provided herein produces functional hematopoietic lineage cells, which achieve a full range of in vitro differentiation, in vitro regulation and long-term hematopoietic self-renewal, reconstruction and transplantation in vivo. As provided, iPSC-derived hematopoietic lineage cells include but are not limited to permanent hemogenic endothelial cells, hematopoietic multipotent progenitor cells, hematopoietic stem cells and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages and neutrophils.

[0343] Thus, in various embodiments, a method for directing differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage comprises: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate differentiation and expansion of mesodermal cells from pluripotent stem cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor to initiate differentiation and expansion of mesodermal cells with definitive HE potential from mesodermal cells, wherein the composition is optionally free of a TGFβ receptor / ALK inhibitor; (iii) contacting mesodermal cells with definitive HE potential with a composition to initiate differentiation and expansion of definitive hemogenic endothelial cells from pluripotent stem cell-derived mesodermal cells with definitive hemogenic endothelial cell potential, the composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; and optionally a Wnt pathway activator, wherein the composition is optionally free of a TGFβ receptor / ALK inhibitor.

[0344] 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 to inoculate and expand the pluripotent stem cells, wherein the composition does not contain a TGFβ receptor / ALK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs, or initial iPSCs, or iPSCs comprising one or more genetic imprints; and the one or more genetic imprints contained in the iPSCs are retained in the hematopoietic cells differentiated therefrom. In some embodiments for guiding the differentiation of pluripotent stem cells into hematopoietic lineage cells, the differentiation of pluripotent stem cells into hematopoietic lineage cells lacks the production of embryoid bodies and is in a monolayer culture form.

[0345] In some embodiments of the above methods, the resulting pluripotent stem cell-derived definitive hemogenic endothelial cells are CD34 + In some embodiments, the resulting definitive hemogenic endothelial cells are CD34 + CD43 - In some embodiments, the permanent hemogenic endothelial cells are CD34 + CD43 - CXCR4 - CD73 - In some embodiments, the permanent hemogenic endothelial cells are CD34 + CXCR4 - CD73 - In some embodiments, the permanent hemogenic endothelial cells are CD34 + CD43 - CD93 - In some embodiments, the permanent hemogenic endothelial cells are CD34 + CD93 - .

[0346] In some embodiments of the above method, the method further comprises (i) contacting the pluripotent stem cell-derived permanent hemogenic endothelial cells with a composition to initiate differentiation of the permanent hemogenic endothelial cells into pre-T cell progenitor cells, the 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 an optional BMP activator; and optionally, (ii) contacting the pre-T cell progenitor cells with a composition to initiate differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells, the composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L and IL7, but not VEGF, bFGF, TPO, BMP activator and ROCK inhibitor. In some embodiments of the method, the pluripotent stem cell-derived T cell progenitor cells are CD34 + CD45+ CD7 + In some embodiments of the method, the pluripotent stem cell-derived T cell progenitor is a CD45 + CD7 + .

[0347] In some further embodiments of the above-mentioned method for directing pluripotent stem cells to differentiate into cells of the hematopoietic lineage, the method further comprises: (i) contacting pluripotent stem cell-derived permanent hemogenic endothelial cells with a composition to initiate differentiation of permanent hemogenic endothelial cells into pre-NK cell progenitor cells, the 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; and optionally, (ii) contacting pluripotent stem cell-derived pre-NK cell progenitor cells with a composition to initiate differentiation of pre-NK cell progenitor cells into NK cell progenitor cells or NK cells, the composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium does not contain one or more of VEGF, bFGF, TPO, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cell-derived NK progenitor cells are CD3 - CD45 + CD56 + CD7 + In some embodiments, the pluripotent stem cell-derived NK cells are CD3 - CD45 + CD56 + , and optionally further NKp46 + 、CD57 + and CD16 + definition.

[0348] Thus, using the above differentiation methods, one or more iPSC-derived hematopoietic cell populations can be obtained: (i) CD34 +HE (iCD34), using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2 and iNK-B2; (ii) permanent hemogenic endothelial cells (iHE), using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2 and iNK-B2; (iii) permanent HSC, using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2 and iNK -B2; (iv) multipotent progenitor cells (iMPP), using iMPP-A; (v) T cell progenitor cells (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 progenitor cells (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 culture medium:

[0349] a.iCD34-C contains ROCK inhibitor, selected from bFGF, VEGF, SCF, IL6,

[0350] One or more growth factors and cytokines from the group consisting of IL11, IGF and EPO, and optionally a Wnt pathway activator; and no TGFβ receptor / ALK inhibitor;

[0351] b. iMPP-A contains BMP activator, ROCK inhibitor and a group consisting of TPO, IL3,

[0352] One or more growth factors and cytokines from the group consisting of GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L and IL11;

[0353] 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;

[0354] d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L and IL7;

[0355] e.iNK-A2 comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7 and IL15; and optionally a BMP activator, and

[0356] f. iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15.

[0357] In some embodiments, the genome-engineered iPSC-derived cells obtained by the above method include one or more inducible suicide genes integrated at one or more desired integration sites, wherein the one or more desired integration sites include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, TAP-related protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, 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, or other loci that meet genomic safe harbor criteria. In some other embodiments, the genome-engineered iPSC-derived cells comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transport, homing, viability, self-renewal, retention, and / or survival of stem and / or progenitor cells. In some embodiments, the genome-engineered iPSC-derived cells comprising one or more suicide genes further comprise one or more insertions / deletions comprised 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 inhibitory genes.

[0358] In addition, methods and compositions applicable to the dedifferentiation of genome-engineered hematopoietic cells of the first fate to genome-engineered hematopoietic cells of the second fate include, for example, methods and compositions described in publication number WO2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided therein allow the initial non-pluripotent cells to be partially reprogrammed into non-pluripotent intermediate cells by: limiting endogenous Nanog gene expression during reprogramming; and subjecting non-pluripotent intermediate cells to conditions for differentiating intermediate cells into desired cell types. 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.

[0359] V. Therapeutic Use of Immune Cells Derived from Genetically Engineered iPSCs with Functional Patterns

[0360] In some embodiments, the present invention provides a composition comprising a population or subpopulation of functionally enhanced derived immune cells separated, which are differentiated by genome-engineered iPSC using the disclosed methods and compositions. In some embodiments, iPSC includes one or more targeted gene editing that can be retained in iPSC-derived effector cells, wherein the genetically engineered iPSC and its derived cells are suitable for cell-based adoptive therapy. In one embodiment, the population or subpopulation of genetically engineered effector cells separated includes iPSC-derived CD34 cells. In one embodiment, the population or subpopulation of genetically engineered effector cells separated includes iPSC-derived HSC cells. In one embodiment, the population or subpopulation of genetically engineered effector cells separated includes iPSC-derived proT cells or T cells. In one embodiment, the population or subpopulation of genetically engineered effector cells separated includes iPSC-derived proNK cells or NK cells. In one embodiment, the population or subpopulation of genetically engineered effector cells separated includes iPSC-derived immune regulatory cells or bone marrow-derived suppressor cells (MDSC). In some embodiments, the effector cells of iPSC-derived genetic engineering are further regulated in vitro to improve therapeutic potential. In one embodiment, the population or subpopulation of the separation of the effector cells of the genetic engineering of iPSC includes the initial T cells, stem cell memory T cells and / or central memory T cells of the number or ratio of increase. In one embodiment, the population or subpopulation of the separation of the effector cells of the genetic engineering of iPSC includes the I type NKT cells of the number or ratio of increase. In another embodiment, the population or subpopulation of the separation of the effector cells of the genetic engineering of iPSC includes the adaptive NK cells of the number or ratio of increase. In some embodiments, the population or subpopulation of the separation of CD34 cells, HSC cells, T cells, NK cells or bone marrow-derived suppressor cells derived from the genetic engineering of iPSC is allogeneic. In some other embodiments, the population or subpopulation of the separation of CD34 cells, HSC cells, T cells, NK cells or MDSC derived from the genetic engineering of iPSC is autologous.

[0361] In some embodiments, the iPSCs used for differentiation contain genetic imprints selected to convey desired therapeutic properties in derived effector cells, with the proviso that the cell developmental biology is not disrupted during differentiation, and with the proviso that the genetic imprints are retained and functional in differentiated hematopoietic cells derived from said iPSCs.

[0362] In some embodiments, the genetic imprint in pluripotent stem cells comprises (i) one or more gene modification patterns acquired by genomic insertions, deletions, or substitutions in the genome of pluripotent cells during or after reprogramming of non-pluripotent cells to iPSCs; or (ii) one or more retainable therapeutic attributes of source-specific immune cells that are donor-specific, disease-specific, or therapeutic response-specific, and wherein the pluripotent cells are reprogrammed from source-specific immune cells, wherein the iPSCs retain the source therapeutic attributes, and the iPSC-derived hematopoietic lineage cells also comprise the source therapeutic attributes.

[0363] In some embodiments, the genetic modification pattern comprises one or more of the following: a safety switch protein, a targeting pattern, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate; or a protein that promotes the transplantation, transport, homing, viability, self-renewal, retention, immune response regulation and modulation, and / or survival of iPSCs or their derivatives. In some embodiments, the genetically modified iPSCs and their derivatives comprise the genotypes listed in Table 1. In some other embodiments, the genetically modified iPSCs and their derivative cells comprising the genotypes listed in Table 1 further comprise an additional genetic modification pattern comprising (1) one or more of the deletion or disruption of B2M, TAP1, TAP2, TAP-associated protein, 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) one or more of the deletion or disruption of 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 for coupling with bispecific or multispecific or universal adapters.

[0364] In yet other embodiments, iPSC-derived hematopoietic lineage cells comprise therapeutic properties of source-specific immune cells associated with a combination of at least two of the following: (i) expression of one or more antigen-targeting receptors; (ii) modified HLA; (iii) resistance to the tumor microenvironment; (iv) recruitment of bystander immune cells and immune regulation; (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 resistance to apoptosis and / or exhaustion.

[0365] In some embodiments, the iPSC-derived hematopoietic cells comprise a genotype listed in Table 1, and the cells express a Fas redirector ("Fas" in Table 1), wherein the Fas redirector comprises (a) an extracellular Fas binding domain comprising a partial or complete peptide of the extracellular domain (ECD) of the Fas receptor (FAS), or 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 co-stimulatory molecules, wherein the Fas redirector redirects Fas signaling when bound to a FAS agonist, thereby providing the cell with improved resistance to apoptosis and / or resistance to exhaustion. In various embodiments, the Fas redirector further comprises a transmembrane region comprising a transmembrane domain of a transmembrane protein or a portion thereof. In some embodiments, the one or more co-stimulatory molecules include 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 includes Fas ligand (FasL). In some embodiments, the cell comprising the Fas redirector also includes one or more of the following: (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (b) an exogenous polynucleotide encoding CD16 or its variants; (c) CD38 knockout; and (d) an exogenous polynucleotide encoding a cytokine signaling complex, the cytokine signaling complex comprising an exogenous cytokine expressed on the cell surface and / or a portion or complete peptide of its receptor.

[0366] Specifically, the present application provides a method for improving the efficacy of adoptive cell therapy provided to a subject in need thereof, wherein the method comprises administering a derived effector cell or a population thereof as described herein. In various embodiments, the derived effector cell comprises a Fas redirector, wherein the Fas redirector comprises (a) an extracellular Fas binding domain comprising a partial or complete peptide of an extracellular domain (ECD) of a Fas receptor (FAS) or a variant or allele thereof, and (b) a cytoplasmic signaling domain comprising a partial or complete peptide of an intracellular domain (ICD) of one or more co-stimulatory molecules, and wherein the Fas redirector redirects Fas signaling when bound to a FAS agonist, thereby providing the cell with improved resistance to apoptosis and / or resistance to exhaustion. In various embodiments, the derived effector cell optionally further comprises a CAR, exogenous CD16, CD38 as shown in Table 1 - / -, cytokine signaling complexes, HLA deficiency / modification, antibodies, checkpoint inhibitors, adapters, and any other modality. In various embodiments, the transmembrane region of the Fas redirector comprises (i) all or at least a portion 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) all or a portion 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 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 OX40 represented by SEQ ID NO: 6. NO:6; or (f) the full length or a portion of the intracellular domain (ICD) of IL18R1 represented by SEQ ID NO:7; or (g) the full length or a portion of the intracellular domain (ICD) of IL21R represented by SEQ ID NO:8; or (iii) the Fas reorientor comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any one of SEQ ID NOs:9-15.

[0367] In various embodiments of the method for improving the efficacy of adoptive cell therapy, effector cells are administered to subjects as a 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, before or after the administration of the derived effector cells. Therefore, in some embodiments, the antibody is used in combination with the effector cell colonies described herein by being administered to the subject simultaneously or continuously. In other embodiments, such antibodies or fragments thereof can be expressed by effector cells by genetically engineering iPSC using an exogenous polynucleotide sequence encoding the antibody or fragment thereof and guiding the differentiation of the engineered iPSC, as described herein. In some embodiments of the method, the effector cell is an iPSC-derived hematopoietic cell. In some embodiments of the method, the effector cell is an iPSC-derived T cell, NK cell or NKT cell.

[0368] In another embodiment of the method for improving the efficacy of the adoptive cell therapy provided to a subject in need, the method also includes administering antibodies specific to the surface proteins of the same or different upregulations targeted by CAR, and / or one or more additional therapeutic agents. In some embodiments of the method, the antibody includes anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, anti-CD38 antibodies or any humanization or Fc-modified variants or fragments, functional equivalents or biosimilars thereof. In some embodiments of the therapeutic agent used in the method, the therapeutic agent includes peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNA, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, carriers, antibodies, chemotherapeutic agents or radioactive moieties or immunomodulatory drugs (IMiD) comprising one or more polynucleic acids of interest.

[0369] A variety of diseases can be improved by introducing the derived effector cells of the present invention into subjects suitable for adoptive cell therapy. In some embodiments, iPSC-derived hematopoietic cells as provided herein are used for allogeneic adoptive cell therapy. In other embodiments, iPSC-derived hematopoietic cells provided herein are used to prevent or reduce tumor microenvironment suppression associated with solid tumors. In other embodiments, iPSC-derived hematopoietic cells as provided herein are used to improve the efficacy of adoptive cell therapy. In addition, in some embodiments, the present invention provides the therapeutic use of the above-mentioned therapeutic composition and / or combination therapy in the following manner: the composition is introduced into a subject suitable for adoptive cell therapy, wherein the subject suffers from an autoimmune disorder; a hematological malignancy; a solid tumor; or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0370] Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML)), lymphomas, non-Hodgkin's lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndrome. Examples of solid cancers include, but are not limited to, brain cancer, prostate cancer, breast cancer, lung cancer, colon cancer, uterine cancer, skin cancer, liver cancer, bone cancer, pancreatic cancer, ovarian cancer, testicular cancer, bladder cancer, kidney cancer, head cancer, neck cancer, stomach cancer, cervical cancer, rectal cancer, laryngeal cancer, and esophageal cancer. Examples of various autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (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 ( syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV (human immunodeficiency virus), HSV (herpes simplex virus), KSHV (Kaposi's sarcoma-associated herpes virus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), VZV (varicella zoster virus), adenovirus, lentivirus, BK polyomavirus-associated disorders.

[0371] Treatment using the derived hematopoietic lineage cells of the embodiments disclosed herein can be performed after the onset of symptoms or used to prevent recurrence. The term "treatment" and the like are generally used herein to mean obtaining the desired pharmacological and / or physiological effects. For the disease and / or the adverse effects attributable to the disease, the effect can be preventive and therapeutic in terms of complete or partial prevention of the disease, and / or in terms of partial or complete cure, it can be therapeutic. As used herein, "treatment" covers any intervention in the subject's disease, and includes: preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; inhibiting the disease, that is, blocking its development; or alleviating the disease, that is, causing the regression of the disease. The therapeutic agent or composition can be administered before, during or after the onset of the disease or injury. The treatment of developing diseases is also of great concern, wherein the treatment stabilizes or reduces the patient's undesirable clinical symptoms. In a specific embodiment, the subject in need of treatment suffers from a disease, condition and / or injury that can contain, improve and / or improve at least one related symptom by cell therapy. Certain embodiments contemplate that subjects in need of cell therapy include, but are not limited to, bone marrow or stem cell transplant candidates, subjects who have received chemotherapy or radiation therapy, subjects who have or are at risk of developing a hyperproliferative disorder or cancer (e.g., a hyperproliferative disorder or a hematopoietic cancer), subjects who have or are at risk of developing a tumor (e.g., a solid tumor), subjects who have or are at risk of developing a viral infection or a disease associated with a viral infection.

[0372] In assessing responsiveness to a therapy comprising a derived hematopoietic lineage cell of the embodiments disclosed herein, the response may be measured by criteria comprising at least one of the following: clinical benefit rate, survival until death, pathological complete response, semiquantitative measure of pathological response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, reduction in circulating tumor cells, circulating marker response, and response evaluation criteria in solid tumors ( R esponse E valuation C riteria I n S olid T umors; RECIST).

[0373] Therapeutic compositions comprising iPSC-derived hematopoietic lineage cells as disclosed herein may be administered to a subject before, during, and / or after other treatments. Therefore, the method of combined therapy may involve administering or preparing iPSC-derived effector cells before, during, and / or after using another therapeutic agent. As provided above, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components, or their replacement factors, carriers comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents, or radioactive moieties or immunomodulatory drugs (IMiDs). Administration of iPSC-derived immune cells and administration of other therapeutic agents may be spaced a few hours, a few days, or even a few weeks in time. Additionally or alternatively, administration may be combined with other bioactive agents or patterns, such as, but not limited to, anti-tumor agents, non-drug therapies, such as surgery.

[0374] In some embodiments of combined cell therapy, the therapeutic combination comprises iPSC-derived hematopoietic lineage cells and another therapeutic agent provided herein, and the other therapeutic agent is an antibody or antibody fragment. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody can 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, tumor or virus-specific antigens activate the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability. In some embodiments, antibodies suitable for combination therapy with the administered iPSC-derived hematopoietic lineage cells as an additional therapeutic agent include, but are not limited to, anti-CD20 (e.g., rituximab, veltuzumab, ofatumumab, utoximab, okatuzumab, obtuzumab), anti-HER2 (e.g., trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (e.g., cetuximab), anti-GD2 (e.g., dietuximab), anti-PDL1 (e.g., avelumab), anti-CD38 (e.g., daratumumab, isatuximab, MOR202), anti-CD123 (e.g., 7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof.

[0375] In some embodiments, the additional therapeutic agent includes one or more checkpoint inhibitors. Checkpoint refers to a cell molecule, typically a cell surface molecule, that can inhibit or downregulate an immune response when not inhibited. Checkpoint inhibitors are antagonists that can reduce checkpoint gene expression or gene products or reduce the activity of checkpoint molecules. Suitable checkpoint inhibitors for combination therapy with derived effector cells are provided above, and the derived effector cells include NK cells or T cells.

[0376] Some embodiments of the combination therapy comprising the provided derived effector cells further include at least one inhibitor of a targeted checkpoint molecule. Some other embodiments of the combination therapy with the provided derived effector cells include two, three or more inhibitors, so as to target two, three or more checkpoint molecules. In some embodiments, the effector cells for the combination therapy as described herein are derived NK cells as provided. In some embodiments, the effector cells for the combination therapy as described herein are derived T cells. In some embodiments, as provided herein, the derived NK cells or T cells for the combination therapy are functionally enhanced. In some embodiments, two, three or more checkpoint inhibitors can be administered in the combination therapy while, before or after the derived effector cells are administered. In some embodiments, two or more checkpoint inhibitors are administered simultaneously or one at a time (in sequence).

[0377] 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 antibody (VNAR) with only a heavy chain, 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), mini antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, single-domain antigen-binding fragments (sdAb, nano antibodies), recombinant antibodies with only heavy chains (VHH), and other antibody fragments that maintain the binding specificity of all antibodies, which can be more cost-effective to produce, easier to use, or more sensitive than all antibodies. 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.

[0378] Combination therapies comprising derived effector cells and one or more checkpoint inhibitors are useful for treating both liquid and solid cancers, including but not limited to cutaneous T-cell lymphoma, non-Hodgkin's lymphoma (NHL), mycosis fungoides, Paget-like reticulosis, Say's syndrome, granulomatous cutis laxa, lymphomatoid papulosis, pityriasis lichenoides chronicus, pityriasis lichenoides acne acute, CD30 + Cutaneous T-cell lymphoma, secondary cutaneous CD30 + Large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennart's lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, Hodgkin's lymphoma (HL), head and neck tumors; squamous cell carcinoma, rhabdomyosarcoma, Lewis lung carcinoma (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, liver cancer, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastatic carcinoma and nasopharyngeal carcinoma.

[0379] In some embodiments, in addition to the derived effector cells as provided herein, the combination for therapeutic use comprises one or more additional therapeutic agents, which include a chemotherapeutic agent or a radioactive moiety. A chemotherapeutic agent refers to a cytotoxic anti-tumor agent, i.e., a chemical agent that preferentially kills tumor cells or interrupts the cell cycle of rapidly proliferating cells, or is found to eradicate cancer stem cells and is used therapeutically to prevent or reduce tumor cell growth. Chemotherapeutic agents are sometimes also referred to as anti-tumor or cytotoxic drugs or agents, and are well known in the art.

[0380] In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, dichloromethane, mephalin, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vinca

[0013] The present invention relates to an antibiotic comprising: an antibiotic (daunorubicin, doxorubicin, mitoxantrone, biguanide, actinomycin D, plicamycin, puromycin, and gramicidine D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine. Additional agents include gminoglutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzumab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, dimethyltestosterone (calusterone), capecitabine, celecoxib,Cetuximab, cladribine, cytarabine, dacarbazine, denileukin diftitox, diethlstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, nitrogen mustard, megestrol, melphalan, mercaptopurine, methylamine pterin, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegademase, pegasparagase, pentostatin, pipobroman, plicamycin, polyphenylpropenol polifeprosan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene,Tositumomab, trastuzumab, tretinoin, uracilmustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those that are approved as chemotherapeutic agents or radiotherapeutic agents and are known in the art. Such agents may be referenced by any of a number of standard physician and oncologist references (e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill, NY, 1995) or by the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated from time to time. ,

[0381] Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide stimulate both NK cells and T cells. As provided herein, IMiDs can be used in cancer treatment together with iPSC-derived therapeutic immune cells.

[0382] In addition to the isolated population of iPSC-derived hematopoietic lineage cells included in the therapeutic composition, the composition suitable for administration to the patient may also include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable culture media, such as cell culture media) or other pharmaceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition administered and the specific method for administering the therapeutic composition. Therefore, there are a variety of suitable formulations for the therapeutic compositions of embodiments of the present invention (see, for example, Remington's Pharmaceutical Sciences, 17th edition, 1985, the disclosure of which is hereby incorporated by reference in its entirety).

[0383] In one embodiment, the therapeutic composition comprises pluripotent cell-derived T cells produced using the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived NK cells produced using the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived CD34 T cells produced using the methods and compositions disclosed herein. +HE cells. In one embodiment, the therapeutic composition comprises HSC derived from pluripotent cells prepared using the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises MDSC derived from pluripotent cells prepared using the methods and compositions disclosed herein. The therapeutic composition comprising the iPSC-derived hematopoietic lineage cell population as disclosed herein can be administered separately or in combination with other suitable compounds by intravenous, intraperitoneal, enteral or tracheal administration to achieve the desired therapeutic goal.

[0384] These pharmaceutically acceptable carriers and / or diluents may be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffer may comprise up to about 5% (weight / weight) of the total composition. Electrolytes, such as but not limited to sodium chloride and potassium chloride, may also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, in the range of about 6 to about 9, or in the range of about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH in the pH range. In another embodiment, the pH of the therapeutic composition is about 7. Alternatively, the pH of the therapeutic composition is in the range of about 6.8 to about 7.4. In yet another embodiment, the pH of the therapeutic composition is about 7.4.

[0385] The present invention also provides, in part, the use of a pharmaceutically acceptable cell culture medium in a specific composition and / or culture of an embodiment of the present invention. Such compositions are suitable for administration to human subjects. In general, any culture medium that supports the maintenance, growth and / or health of iPSC-derived effector cells according to an embodiment of the present invention is suitable for use as a pharmaceutical cell culture medium. In a specific embodiment, a pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free culture medium. In various embodiments, the serum-free culture medium is animal-free and may optionally be protein-free. Optionally, the culture medium may contain a biopharmaceutically acceptable recombinant protein. An animal-free culture medium refers to a culture medium in which the components are derived from non-animal sources. Recombinant proteins replace protozoan proteins in animal-free culture mediums and nutrition is obtained from synthetic, plant or microbial sources. In contrast, a protein-free culture medium is defined as being substantially protein-free. Those skilled in the art will appreciate that the above culture medium examples are illustrative and in no way limit the culture medium formulations applicable to the present invention, and there are many known and suitable culture media available for use by those skilled in the art.

[0386] The iPSC-derived hematopoietic lineage cells may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34 + HE cells, HSCs, B cells, 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, proT cells, proNK cells, CD34 + HE cells or myeloid derived suppressor cells (MDSC). In some embodiments, the present invention provides a therapeutic composition having purified T cells or NK cells, for example, having about 95% T cells, NK cells, proT cells, proNK cells, CD34 + The invention relates to compositions of isolated populations of HE cells or myeloid-derived suppressor cells (MDSC) for treating a subject in need of cell therapy.

[0387] As will be understood by those of ordinary skill in the art, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs based on the methods and compositions herein can be used in cell therapies as described above. For autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA matched relative to the patient. In another embodiment, the derived hematopoietic lineage cells are not HLA matched to the subject, wherein the derived hematopoietic lineage cells are NK cells or T cells comprising a Fas redirector, wherein the Fas redirector comprises: an extracellular Fas binding domain comprising a partial or complete peptide of the extracellular domain (ECD) of the Fas receptor (FAS) or a variant or allele thereof, and a cytoplasmic signaling domain comprising a partial or complete peptide of the intracellular domain (ICD) of one or more costimulatory molecules, wherein the Fas redirector redirects Fas signaling when bound to a FAS agonist, and wherein the NK cells or T cells optionally comprise one or more patterns listed in Table 1.

[0388] 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 8cells, at least 1×10 9 cells or at least 5×10 9 In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1×10 5 cells to about 1×10 6 cells; approximately 0.5×10 per dose 6 cells to about 1×10 7 cells; approximately 0.5×10 per dose 7 cells to about 1×10 8 cells; approximately 0.5×10 per dose 8 cells to about 1×10 9 cells; approximately 1×10 per dose 9 cells to about 5×10 9 cells; approximately 0.5×10 per dose 9 cells to about 8×10 9 cells; approximately 3×10 per dose 9 cells to about 3×10 10 cells, or any range therebetween. Generally speaking, 1×10 8 cells / dose translates to 1.67 × 10 6 cells / kg.

[0389] In one embodiment, the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a portion or a single umbilical cord blood, or is 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 6cells / 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 8 cells / kg body weight or 1×10 9 cells / kg body weight.

[0390] In one embodiment, a dose of derived hematopoietic lineage cells is delivered to a subject. In an exemplary embodiment, an effective amount of cells provided to a 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, including all intermediate cell doses.

[0391] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg or about 10×10 6 cells / kg or more, including all intermediate cell doses.

[0392] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg to about 10×10 6 cells / kg, about 3×10 6 cells / kg to about 10×10 6cells / kg, about 4×10 6 cells / kg to about 10×10 6 cells / kg, about 5×10 6 cells / kg to about 10×10 6 cells / kg, 2×10 6 cells / kg to about 6×10 6 cells / kg, 2×10 6 cells / kg to about 7×10 6 cells / kg, 2×10 6 cells / kg to about 8×10 6 cells / kg, 3×10 6 cells / kg to about 6×10 6 cells / kg, 3×10 6 cells / kg to about 7×10 6 cells / kg, 3×10 6 cells / kg to about 8×10 6 cells / kg, 4×10 6 cells / kg to about 6×10 6 cells / kg, 4×10 6 cells / kg to about 7×10 6 cells / kg, 4×10 6 cells / kg to about 8×10 6 cells / kg, 5×10 6 cells / kg to about 6×10 6 cells / kg, 5×10 6 cells / kg to about 7×10 6 cells / kg, 5×10 6 cells / kg to about 8×10 6 cells / kg or 6×10 6 cells / kg to about 8×10 6 cells / kg, including all intermediate cell doses.

[0393] In some embodiments, the therapeutic use of derived hematopoietic lineage cells is a single dose treatment. In some embodiments, the therapeutic use of derived hematopoietic lineage cells is a multiple dose treatment. In some embodiments, multiple dose treatment is a dose once a 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 or every 50 days or any number of days during.

[0394] Compositions comprising derived hematopoietic lineage cell groups of the present invention can be sterile, and can be suitable for administration and ready for administration (i.e., can be administered without any further treatment) to human patients / subjects. Cell-based compositions prepared for administration mean that the composition does not require any additional processing or manipulation before transplantation or administration to a subject. In other embodiments, the present invention provides a separate population of derived hematopoietic lineage cells that are amplified and / or regulated before administration with one or more agents including small chemical molecules. Compositions and methods for regulating immune cells (including iPSC-derived effector cells) are described in more detail in, for example, International Publication No. WO2017 / 127755, and its related disclosures are incorporated herein by reference. For derived hematopoietic lineage cells that are genetically engineered to express recombinant TCR or CAR, cells can be activated and amplified using methods such as, for example, described in U.S. Patent No. 6,352,694.

[0395] In certain embodiments, different schemes can be used to provide pr...

Claims

1. A cell or a population thereof, wherein: (i) the cell is (a) an immune cell; (b) an induced pluripotent cell (iPSC), a cloned iPSC or an iPS cell line cell; or (c) a derivative cell obtained by differentiating the cell of (b); (ii) the cell comprises an exogenous polynucleotide encoding a signal transduction redirection receptor, wherein the signal transduction redirection receptor comprises: (a) a peptide comprising a portion or the entire extracellular domain (ECD) of the Fas receptor (FAS), or an extracellular Fas binding domain of a variant or allele thereof; and (b) a cytoplasmic signaling domain comprising a portion or the entirety of a peptide of the intracellular domain (ICD) of one or more costimulatory molecules, Wherein the signaling redirecting receptor is a Fas redirector, which redirects Fas signaling when bound to a FAS agonist, thereby providing improved apoptosis resistance and / or exhaustion resistance to the cell or the derived cell.

2. The cell or population thereof according to claim 1, wherein (i) the Fas reorientator further comprises a transmembrane region, wherein the transmembrane region comprises a transmembrane domain of a transmembrane protein or a portion 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 do not include 41BB; (iv) the FAS agonist comprises Fas ligand (FasL); or (v) the cell further comprises one or more of the following: (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (b) an exogenous polynucleotide encoding CD16 or a variant thereof; (c) CD38 knockout; and (d) an exogenous polynucleotide encoding a cytokine signaling complex, wherein the cytokine signaling complex comprises a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface.

3. The cell or population thereof according to claim 2, wherein the transmembrane region of the Fas reorientator comprises: (i) all or at least a portion of a native or modified transmembrane region of a 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 T cell receptor polypeptide; or (ii) the full length or a portion of the transmembrane domain of FAS.

4. The cell or population thereof according to claim 1, wherein: (i) the extracellular binding domain of the Fas re-director comprises a sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 1; (ii) the cytoplasmic signaling domain of the Fas re-director 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 reorientor comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any one of SEQ ID NOs: 9-15.

5. The cell or population thereof according to any one of claims 1 to 4, wherein the cell further comprises one or more of the following: (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 uncleavable 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, TAP-associated protein, 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 its functional variant or fragment, check point inhibitor, adapter and at least one of surface triggering receptor for coupling with bispecific or multispecific or universal adapter.

6. The cell or population thereof according to any one of claims 1 to 5, wherein the cell comprises HLA-I deficiency and / or HLA-II deficiency; and optionally, The cells comprise an exogenous polynucleotide encoding HLA-G, HLA-E or a variant thereof; and / or comprise a deletion or disruption of one or both of CD54 and CD58.

7. The cell or population thereof according to claim 6, wherein the HLA-I deficiency comprises a deletion or disruption of at least one of: B2M, TAP1, TAP2, and TAP-related protein; or wherein the HLA-II deficiency comprises a deletion or disruption of at least one of: CIITA, RFX5, RFXAP and RFXANK.

8. The cell or population thereof according to any one of claims 1 to 7, wherein the derivative cell: (a) Including derived CD34 + cells, derived hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional characteristics that are not present in the corresponding primary T cells, NK cells, NKT cells and / or B cells; or (b) is an allogeneic effector cell, wherein the effector cell is a derived NK cell or a derived T cell having at least one of the following properties, the properties comprising: Compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or other donor tissues, (i) improved retention and / or survival; (ii) increased resistance to activated recipient immune cells; (iii) increased cytotoxicity; (iv) improved tumor penetration; (v) enhanced or acquired ADCC; (vi) enhanced ability of bystander immune cells to migrate to tumor sites and / or to become activated or recruited to tumor sites; (vii) enhanced ability to reduce tumor immunosuppression; (viii) Improved ability to rescue tumor antigen escape; as well as (ix) Reduced apoptosis and / or fratricide.

9. The cell or population thereof according to claim 2, wherein the exogenous CD16 comprises at least one of: (a) high affinity uncleavable CD16 (hnCD16) or a variant thereof; (b) F176V and S197P in the extracellular domain of CD16; (c) derived from all or part of the extracellular domain of CD64; (d) non-native (or non-CD16) transmembrane domain; (e) non-native (or non-CD16) intracellular domain; (f) non-native (or non-CD16) signaling domain; (g) a non-native stimulatory domain; and (h) Transmembrane domains, signaling domains and stimulatory domains that are not derived from CD16 and are derived from the same or different polypeptides.

10. The cell or population thereof according to claim 9, wherein: (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 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 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.

11. The cell or population thereof of claim 1, wherein the CAR is: (i) T cell-specific or NK cell-specific; (ii) bispecific antigen-binding CAR; (iii) switchable CAR; (iv) dimerized CAR; (v) separating CAR; (vi) multi-chain CAR; (vii) inducible CAR; (viii) co-expression with a cytokine signaling complex comprising partial or complete peptides of exogenous cytokines and / or their receptors expressed on the cell surface, optionally in separate constructs or in a bicistronic construct; (ix) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct; and / or (x) optionally inserted at: (1) TRAC or TRBC loci, and / or driven by the endogenous promoter of TCR, and / or the TCR is knocked out by insertion of the CAR; (2) safe harbor loci; or (3) Genetic loci targeted for disruption.

12. The cell or population thereof of claim 1, wherein the CAR is: (i) is specific for at least one of: CD19, BCMA, B7H3, CD20, CD22, CD38, CD52, CD79b, CD123, EGFR, EGP2 / EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MR1, MSLN, Mucl, Mucl6, NYESO1, VEGF-R2, PSMA, and PDL1; and / or (ii) is specific for any of the following: 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, CLEC12 A. 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 kinase erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human Epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), mucin 1 (Muc-1), mucin 16 (Muc- 16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NYESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms' tumor protein (WT-1), and pathogen antigens.

13. The cell or population thereof of claim 2, wherein the cytokine signaling complex comprises: (a) a partial or complete peptide of an exogenous cytokine expressed on the cell surface and / or its receptor, which comprises at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 or their corresponding receptors; or (b) At least one of the following: (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 with truncated intracellular domain of IL15Rα (IL15Δ); (iv) fusion protein of the membrane-bound Sushi domain of IL15 and IL15Rα; (v) fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and co-receptor γC, wherein the co-receptor γC is native or modified; and (vii) homodimer of IL15Rβ; wherein any of (b)(i) to (vii) can be co-expressed with CAR in a separate construct or in a bicistronic construct; or (c) At least one of the following: (i) fusion protein of IL7 and IL7Rα; (ii) a fusion protein of IL7 and co-receptor γC, wherein the co-receptor γC is native or modified; and (iii) IL7Rβ homodimer, wherein any of (c)(i)-(iii) is optionally co-expressed with CAR in a separate construct or in a bicistronic construct; and optionally, (d) Transient expression.

14. The cell or population thereof according to claim 1, wherein the derived cell is a derived NK cell or a derived T cell, wherein the derived NK cell is capable of recruiting and / or migrating T cells to a tumor site, and wherein the derived NK cell or the derived T cell is capable of reducing tumor immunosuppression in the presence of one or more check point inhibitors.

15. The cell or population thereof according to claim 11 or 14, wherein the one or more checkpoint inhibitors are antagonists against one or more checkpoint molecules, the checkpoint molecules comprising 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 inhibitory KIR.

16. The cell or population thereof of claim 15, wherein the one or more checkpoint inhibitors comprise: (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

17. The cell or population thereof according to claim 1, wherein the cell comprises: (i) one or more exogenous polynucleotides integrated into a safe harbor locus or a locus targeted for disruption; or (ii) More than two exogenous polynucleotides integrated in different safe harbor loci or loci targeted for disruption.

18. The cell or population thereof of claim 17, wherein the one or more safe harbor loci comprise at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1; or wherein the one or more loci intended for disruption comprise at least one of B2M, TAP1, TAP2, TAP-associated protein, 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.

19. The cell or population thereof of claim 1, wherein 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 or partial length of the intracellular domain (ICD) of OX40 represented by SEQ ID NO:

5. 20 . The cell or the population thereof according to claim 19 , 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.

21. A composition comprising the cell or population thereof according to any one of claims 1 to 20.

22. The composition of claim 21 further comprising one or more therapeutic agents.

23. The composition of claim 22, wherein the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor 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).

24. The composition of claim 23, wherein: (i) The checkpoint inhibitors include: (a) one or more antagonists of 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, 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, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents; (c) at least one of atezolizumab, nivolumab, and pembrolizumab; or (ii) the therapeutic agent comprises one or more of venetoclax, azacitidine and pomalidomide.

25. The composition of claim 23, wherein the antibody comprises: (a) anti-CD20 antibody, anti-HER2 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-CD123 antibody, anti-GD2 antibody or anti-PDL1 antibody; or (b) rituximab, veltuzumab, ofatumumab, utoximab, okatuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dietuximab, avelumab, daclizumab, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, bivacizumab, RG7356, G44-26, 7G3, CSL362, etozuzumab and their humanized or Fc-modified variants or fragments and their functional equivalents and biosimilars thereof.

26. The composition of claim 23, wherein the adaptor comprises: (i) Bispecific T cell engagers (BiTEs); (ii) bispecific killer cell engager (BiKE); or (iii) Trispecific Killer Cell Engager (TriKE); or wherein the adaptor 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 the cell or an alternative immune effector cell; and (b) a second binding domain that is specific for an antigen comprising any of the following: 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.

27. The therapeutic use of a composition according to any one of claims 21 to 26, achieved by introducing the composition into a subject in need of adoptive cell therapy, wherein the subject suffers from an autoimmune disorder, a hematological malignancy, a solid tumor, a cancer or a viral infection.

28. A master cell bank (MCB) comprising the clonal iPSCs according to any one of claims 1 to 20.

29. A method of preparing a derivative cell according to any one of claims 1 to 20, wherein the derivative cell is an effector cell, and the method comprises: differentiating genetically engineered iPSCs, wherein the genetically engineered iPSCs comprise an exogenous polynucleotide encoding a Fas redirector that redirects Fas signaling when bound to a FAS agonist, Thereby providing the effector cells with improved resistance to apoptosis and / or exhaustion.

30. The method of claim 29, wherein the Fas redirector comprises: (a) a peptide comprising part or all of the extracellular domain (ECD) of the Fas receptor (FAS), or an extracellular Fas-binding domain of a variant or allele thereof; and (b) a cytoplasmic signaling domain comprising a portion or the entirety of a peptide of the intracellular domain (ICD) of one or more costimulatory molecules; and The genetically engineered iPSCs are single cells, cloned cells or cell line cells.

31. The method of claim 30, wherein: (i) the Fas reorientator further comprises a transmembrane region, wherein the transmembrane region comprises a transmembrane domain of a transmembrane protein or a portion 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 do not include 41BB; (iv) the FAS agonist comprises Fas ligand (FasL); or (v) the genetically engineered iPSC comprising the Fas reorientator further comprises one or more of the following: (a) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (b) an exogenous polynucleotide encoding CD16 or a variant thereof; (c) CD38 knockout; and (d) an exogenous polynucleotide encoding a cytokine signaling complex, wherein the cytokine signaling complex comprises a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface.

32. The method of claim 31, wherein the transmembrane region of the Fas reorientor comprises: (i) all or at least a portion of a native or modified transmembrane region of a 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 T cell receptor polypeptide; or (ii) the full length or a portion of the transmembrane domain of FAS.

33. The method of claim 31, wherein: (i) the extracellular binding domain of the Fas re-director comprises a sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 1; (ii) the cytoplasmic signaling domain of the Fas re-director 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 reorientator comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any one of SEQ ID Nos: 9-15.

34. The method of any one of claims 29 to 33, wherein the genetically engineered iPSC further comprises one or more edits that produce: (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 uncleavable HLA-G, or knockout of one or both of CD58 and CD54; (iv) disruption of at least one of B2M, CIITA, TAP1, TAP2, TAP-associated protein, 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, check point inhibitor, adapter and at least one of surface triggering receptor for coupling with bispecific or multispecific or universal adapter.

35. The method according to any one of claims 29 to 34, wherein the cell comprises HLA-I deficiency and / or HLA-II deficiency; and optionally, wherein the cell comprises an exogenous polynucleotide encoding HLA-G, HLA-E or a variant thereof; Or comprise a deletion or disruption of one or both of CD54 and CD58.

36. The method of claim 31, further comprising: Genome engineering of iPSCs to knock in: (a) the polynucleotide encoding the signaling redirecting receptor; and 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 further comprising genome engineering the iPSCs to: (i) Knockout of CD38, (ii) knocking out one or both of B2M and CIITA, (iii) knocking out one or both of CD58 and CD54, and / or (iv) introducing HLA-G or uncleavable HLA-G and / or the signaling complex comprising the partial or complete peptide of the exogenous cytokine and / or its receptor expressed on the cell surface.

37. The method of claim 36, wherein the genome engineering comprises targeted editing.

38. The method of claim 37, wherein the targeted editing comprises a deletion, an insertion, or an insertion / deletion, and wherein the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variant of these methods.

39. A method of improving effector cell persistence or preventing cell death of effector cells in adoptive cell therapy provided to a subject in need thereof by obtaining said effector cells according to any one of claims 29 to 38.

40. A method of improving the efficacy of adoptive cell therapy provided to a subject in need thereof, the method comprising administering to the subject effector cells, wherein the effector cells comprise the derivative cell or population thereof according to any one of claims 1 to 20.

41. The method of claim 40, further comprising administering one or more therapeutic agents to the subject.

42. The method of claim 41, wherein the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor 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).

43. The method of claim 42, wherein: (i) The checkpoint inhibitors include: (a) one or more antagonists of 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, 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, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab; or (ii) the one or more therapeutic agents include one or more of venetoclax, azacitidine, and pomalidomide.

44. A method of treating a subject in need of adoptive cell therapy, wherein the method comprises infusing effector cells into the subject, wherein the effector cells comprise a derivative cell or population thereof according to any one of claims 1 to 20 in combination with an exogenous cytokine.

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