Invisible strategies involved in immune recognition pathways for allogeneic 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. The derived cells provided herein have stable and functional genome editing that delivers improved or enhanced therapeutic effects. Also provided are therapeutic compositions comprising the functionally enhanced derived effector cells alone, or functionally enhanced derived effector cells treated in combination with an antibody or a checkpoint inhibitor, and uses thereof.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 328,730 filed on April 7, 2022; U.S. Provisional Application Serial No. 63 / 341,970 filed on May 13, 2022; U.S. Provisional Application Serial No. 63 / 382,620 filed on November 7, 2022; and U.S. Provisional Application Serial No. 63 / 386,880 filed on December 9, 2022, the disclosures of which are hereby incorporated by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The sequence listing entitled 184143-642601_SL.xml, created on April 5, 2023 and 85,569 bytes in size, 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, such as the tumor microenvironment and associated immunosuppression, recruitment, trafficking, and infiltration.

[0008] The purpose of the present disclosure is to produce 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 one or more of 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] In some embodiments, 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 modification stipulates that the developmental potential of iPSC in directed differentiation will not be adversely affected by the engineering modification mode in iPSC, and also requires that the engineering modification mode generally plays a role in the derivative cells as expected.In addition, such strategies overcome 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 engineering such cells generally lack reproducibility and uniformity, so that cells show the poor cell retention with high cell death and low cell amplification. Furthermore, the strategies disclosed herein can avoid the generation of heterogeneous effector cell populations that would otherwise be obtained using a starting heterogeneous source of primary cells.

[0010] Some aspects of the invention provide genome-engineered iPSCs obtained using a method comprising (I), (II) or (III), reflecting respectively the strategy of genome engineering after, simultaneously with or before the reprogramming process:

[0011] (I): Genetically engineering iPSCs using one or both of (i) and (ii) in any order: (i) introducing one or more constructs into iPSCs to allow targeted integration at a selected site; (ii) (a) introducing one or more double-strand breaks into iPSCs at a selected site using one or more nucleases capable of recognizing the selected site; and (b) culturing the iPSCs in step (I)(ii)(a) to allow endogenous DNA repair to generate targeted insertions / deletions at the selected site; thereby obtaining genome-engineered iPSCs capable of differentiating into partially or fully differentiated cells.

[0012] (II): Genetically engineering reprogrammed non-pluripotent cells in the following manner to obtain genome-engineered iPSCs, comprising: (i) contacting non-pluripotent cells with one or more reprogramming factors and optionally a small molecule composition to initiate reprogramming of non-pluripotent cells, the small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor; and (ii) introducing one or both of (a) and (b) into the reprogrammed non-pluripotent cells in step (II)(i) in any order: (a) one or more constructs allowing targeted integration at a selected site; (b) one or more double-strand breaks at a selected site using at least one nuclease capable of recognizing a selected site, and then culturing the cells in step (II)(ii)(b) to allow endogenous DNA repair, thereby generating targeted insertions / deletions at the selected site; the resulting genome-engineered iPSCs thus comprise at least one functional targeted genome edit, and the genome-engineered iPSCs are capable of differentiating into partially or fully differentiated cells.

[0013] (III): A method for genetically engineering reprogrammed non-pluripotent cells to obtain genome-engineered iPSCs comprises (i) and (ii): (i) introducing one or both of (a) and (b) into non-pluripotent cells in any order: (a) one or more constructs that allow targeted integration at a selected site; (b) one or more double-strand breaks at a selected site using at least one nuclease capable of recognizing the selected site, wherein the cells in step (III)(i)(b) are cultured to allow endogenous DNA repair, thereby generating targeted insertions / deletions at the selected site; and (ii) contacting the cells in step (III)(i) with one or more reprogramming factors and an optional small molecule composition to obtain genome-engineered iPSCs comprising targeted editing at the selected site, the small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor; thereby obtaining genome-engineered iPSCs comprising at least one functional targeted genome edit, and the genome-engineered iPSCs are capable of differentiating into partially differentiated cells or fully differentiated cells.

[0014] In one embodiment of the above method, at least one targeted genome editing at one or more selected sites comprises inserting one or more exogenous polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the migration, trafficking, homing, viability, self-renewal, persistence and / or survival of genome-engineered iPSCs or their derivatives. In some embodiments, the exogenous polynucleotides for insertion are operably linked to (1) one or more exogenous promoters, including CMV, EF1α, PGK, CAG, UBC or other constitutive, inducible, temporal-specific, tissue-specific or cell-type-specific promoters; or (2) one or more endogenous promoters contained in the selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other loci that meet genomic safe harbor criteria. In some embodiments, the genome-engineered iPSCs generated using the above methods contain one or more different exogenous polynucleotides encoding proteins comprising cysteine ​​proteases, thymidine kinases, cytosine deaminases, modified EGFRs, or B cell CD20, wherein when the genome-engineered iPSCs contain two or more suicide genes, the suicide genes are integrated into different safe harbor loci, and the safe harbor loci contain AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotides encode part or all of the peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors. In some embodiments, a portion or complete peptide of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and / or their corresponding receptors encoded by an exogenous polynucleotide is in the form of a fusion protein.

[0015] In some other embodiments, the iPSCs of genome engineering generated using the methods provided herein include insertions / deletions at one or more endogenous genes associated with the following items: targeting patterns, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins that inhibit iPSC or its derivative cell transplantation, transportation, homing, vitality, self-renewal, retention and / or survival. In some embodiments, the endogenous genes for interruption include at least one of the following: B2M, TAP1, TAP2, TAP-associated protein (Tapasin), NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.

[0016] In yet other embodiments, the genome-engineered iPSCs generated using the methods provided herein comprise an exogenous polynucleotide encoding a caspase located at the AAVS1 locus and an exogenous polynucleotide encoding a thymidine kinase located at the H11 locus.

[0017] In some other embodiments, methods (I), (II) and / or (III) further comprise: contacting the genome-engineered iPSC with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor to maintain the pluripotency of the genome-engineered iPSC. In one embodiment, the genome-engineered iPSC obtained comprising at least one targeted genome editing is functional, can effectively differentiate and can differentiate into non-pluripotent cells comprising the same functional genome editing.

[0018] The present invention also provides the following in various aspects and embodiments.

[0019] One aspect of the present application provides a cell or a population thereof, wherein (i) the cell is (a) an immune cell; (b) an induced pluripotent cell (iPSC); or (c) a derivative cell (e.g., a derived effector cell) obtained from a differentiated iPSC; and (ii) the cell comprises: (a) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); and optionally (b) one or both of CD38 knockout and endogenous TCR knockout; or one or more of CD38 knockout, endogenous TCR knockout, and one or both of CD58 and CD54 knockout. In some embodiments, the cell has improved resistance to host immune alloreactivity compared to cells without exogenous polynucleotides. In various embodiments of a cell or a population thereof, (i) iPSC is a cloned iPSC, a single cell dissociated iPSC, an iPSC cell line cell, or an iPSC master cell bank (MCB) cell; or (ii) the derived cell includes a derived CD34 +cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells or derived B lineage cells; or (iii) the derived cells include derived effector cells having one or more functional characteristics, and the one or more functional characteristics are not present in the corresponding primary T cells, NK cells, NKT cells and / or B cells.

[0020] In some embodiments of a cell or its population, the ADR is specific to 41BB or to CD38. In some embodiments, the ADR comprises a 41BB-specific ligand (i) operably connected to a signaling domain that promotes effector cell activation, or (ii) a CD38 binding domain operably connected to a signaling domain that promotes effector cell activation. In one embodiment of a cell or its population, the 41BB-specific ligand is 4-1BBL, an antibody or fragment thereof targeting 4-1BB, or a 4-1BBL-Fc fusion. In some embodiments of a cell or its population, the signaling domain comprises CD3ζ or its functional fragment, Fc receptor, or a combination thereof from DAP12. In various embodiments of a cell or its population, the ADR further comprises one, two, three, or more costimulatory domains. In some embodiments of a cell or its population, one, two, three, or more costimulatory domains are from intracellular signaling domains of CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, or CD40. In some embodiments, the ADR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:8.

[0021] In various embodiments of the cell or its population, the cell further comprises one or more of the following: (i) a chimeric antigen receptor (CAR); (ii) a knockout of one or both of CD58 and CD54; (iii) an exogenous CD16 or a variant thereof; (iv) a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface; (v) at least one of the genotypes listed in Table 2; (vi) disruption of at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (vii) disruption of at least one of CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AR, antigen-specific TCR, Fc receptor, antibody or its functional variant or fragment, checkpoint inhibitor, adapter and at least one of the surface triggering receptors for coupling with agonists. In some embodiments of the cell or its population, the cell has therapeutic properties including one or more of the following compared to the corresponding primary cells of cells without the same gene editing obtained from peripheral blood, umbilical cord blood or any other donor tissue: (i) increased cytotoxicity; (ii) improved retention and / or survival; (iii) enhanced ability to migrate neighboring immune cells to the tumor site and / or activate or recruit neighboring immune cells; (iv) improved tumor penetration; (v) enhanced ability to reduce tumor immunosuppression; (vi) improved ability to rescue tumor antigen escape; (vii) controlled apoptosis; (viii) enhanced or obtained ADCC; and (ix) ability to avoid cannibalism.

[0022] In some embodiments of the cell or its population, the exogenous CD16 or its variant comprises at least one of the following: (a) high affinity uncleavable CD16 (hnCD16); (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 of the cell or its population, the CAR is: (i) T cell specific or NK cell specific; (ii) bispecific antigen binding CAR; (iii) switchable CAR; (iv) dimerization CAR; (v) separate CAR; (vi) multi-chain CAR; (vii) inducible CAR; (viii) co-expressed with another CAR; (ix) co-expressed with a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, optionally in a separate construct or in a bicistronic construct; (x) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct; (xi) specific for at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA and PDL1;and / or (xii) 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, CLEC12A, an antigen of cytomegalovirus (CMV) infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase erb B2,3,4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit 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, mucin 1 (Muc-1 ), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGFR2), Wilms tumor protein (WT-1) and pathogen antigen; and optionally, wherein the CAR of any one of (i) to (xii) is inserted at the TCR locus and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by CAR insertion. ;

[0023] In some embodiments of the cell or its population, the cytokine signaling complex: (a) comprises a partial or complete peptide of at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and their corresponding receptors; or (b) comprises at least one of the following: (i) co-expression of IL15 and IL15Rα by using a self-cleaving peptide; (ii) a fusion protein of IL15 and IL15Rα; (iii) an IL15 / IL15Rα fusion protein with a truncated intracellular domain of IL15Rα; (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) ) a fusion protein of IL15 and 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) can be co-expressed with CAR in a separate construct or in a bicistronic construct; or (c) comprises 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 expression cassette; and optionally, (d) is transiently expressed. In some embodiments of the cell or its population, the cell is an NK lineage cell or a T lineage cell, wherein (i) the NK lineage cell or the T lineage cell has improved infiltration and / or retention at the tumor site; (ii) the NK lineage cell is able to recruit T cells and / or migrate T cells to the tumor site; or (iii) in the presence of one or more checkpoint inhibitors, the NK lineage cell or the T lineage cell is able to reduce tumor immunosuppression. In some embodiments of the cell or its population, the checkpoint inhibitor is an antagonist to one or more checkpoint molecules, which 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, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some embodiments, the checkpoint inhibitor comprises: (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents; or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0024] 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 selected locus; or (ii) more than two exogenous polynucleotides integrated in different safe harbor loci or two or more selected loci. In some embodiments, the safe harbor locus includes at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH or RUNX1; or wherein the selected locus is one of B2M, TAP1, TAP2, TAP-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3 or TIGIT; and / or wherein the integration of the exogenous polynucleotide knocks out the expression of the gene in the locus. In some embodiments, the TCR locus is the constant region of TCRα and / or TCRβ.

[0025] In one embodiment of a cell or its group, the cell comprises: (i) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); (ii) CD38 knockout and exogenous CD16; and (iii) TCR knockout; and wherein compared to cells without all (i), (ii) and (iii), the cell or its group has improved resistance to host immune alloreactivity. In one embodiment of a cell or its group, the cell comprises: (i) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); (ii) CD38 knockout; (iii) exogenous CD16 or its variant; (iv) some or all of the peptides of IL15 and some or all of the peptides of the IL15 receptor; and (v) CAR; wherein compared to cells without all (i), (ii) and (iii), the cell or its group has improved resistance to host immune alloreactivity. In one embodiment, CAR is specific to at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1. In one embodiment, CAR is specific to CD19.

[0026] In another aspect, the present application provides a method for improving the resistance of effector cells to host immune alloreactivity, wherein the method comprises: (i) obtaining engineered iPSCs comprising an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR), and optionally one or both of CD38 knockout and endogenous TCR knockout; or one or more of CD38 knockout, endogenous TCR knockout, and knockout of one or both of CD58 and CD54, and (ii) differentiating the iPSCs into effector cells, thereby producing effector cells with improved resistance to host immune alloreactivity compared to corresponding cells without the exogenous polynucleotides.

[0027] In some embodiments of the method for improving effector cell resistance to host immune alloreactivity, (i) an induced pluripotent cell (iPSC) is engineered to generate a genome-edited iPSC comprising one or more exogenous polynucleotides encoding an allogeneic immune defense receptor (ADR) and optionally a knockout of one or both of CD38 and an endogenous TCR, or one or more of a knockout of CD38, an endogenous TCR, and a knockout of one or both of CD58 and CD54; or (ii) an induced pluripotent cell (iPSC) is engineered to generate a genome-edited iPSC comprising one or more exogenous polynucleotides encoding an allogeneic immune defense receptor (ADR) and optionally a knockout of one or both of CD38 and an endogenous TCR; Into the polynucleotide encoding allogeneic immune defense receptor (ADR) and optionally knock out CD38 and endogenous TCR one or both, or CD38 knockout, endogenous TCR knockout and CD58 and CD54 one or both of the knockout of one or more to engineer immune cells, to produce ADR and optional CD38 knockout and endogenous TCR knockout one or both, or CD38 knockout, endogenous TCR knockout and CD58 and CD54 one or both of the knockout of one or more of the genome editing effector cells. In some embodiments, ADR is specific to 41BB or to CD38. In some embodiments of the method for improving effector cell resistance, ADR includes (i) and SEQ ID NO:8 has at least 75%, 80%, 85%, 90%, 95% or 99% identity of the amino acid sequence, or (ii) CD38 binding domain. In some embodiments of the method for improving effector cell resistance, the engineered iPSC further comprises one or more edits compared to a corresponding primary cell of the engineered iPSC obtained from peripheral blood, umbilical cord blood, or any other donor tissue without the same gene edits, resulting in: (i) a chimeric antigen receptor (CAR); (ii) knockout of one or both of CD58 and CD54; (iii) introduction of exogenous CD16 or a variant thereof; (iv) a cytokine signaling complex comprising an exogenous cell surface expressed (v) at least one of the genotypes listed in Table 2; (vi) disruption of at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (vii) disruption of CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AR, antigen-specific TCR, Fc receptor, antibody or its functional variant or fragment, check point inhibitor, adapter and at least one of surface triggering receptor for coupling with agonist. In some embodiments of the method for improving effector cell resistance, the improved effector cell resistance to host immune alloreactivity is in vivo.

[0028] In another aspect, the present application provides a method for improving the in vivo resistance of CAR-T cells to host immune alloreactivity according to the methods provided herein.

[0029] In another aspect, the application provides a composition comprising a cell or a colony thereof provided herein. In some embodiments of the composition, the composition further comprises one or more therapeutic agents. In some embodiments of the composition, one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, effectors, antibodies or functional variants or fragments thereof, 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, chemotherapeutic agents or radioactive moieties or immunomodulatory drugs (IMiDs). In some embodiments of the composition in which the therapeutic agent is a checkpoint inhibitor, the checkpoint inhibitor comprises: (i) 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, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor α), TLR3, VISTA, NKG2A / H LA-E or inhibitory KIR; (ii) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and their derivatives or functional equivalents; or (iii) at least one of atezolizumab, nivolumab and pembrolizumab; or (b) the therapeutic agent includes one or more of venetola, azacitidine and pomalidomide. In some embodiments of the composition in which the therapeutic agent is an antibody, the antibody includes: (a) anti-CD20 antibody, anti-HER2 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-CD123 antibody, anti-GD2 antibody, anti-PDL1 antibody and / or anti-CD38 antibody; (b) rituximab, veltuzumab, ofatumumab, utoximab, okatuzumab, obinutuzumab, isbetumomab, ocrelizumab, inotuzumab, mosetuzumab, epa The invention relates to an antibody comprising: (a) daratumumab, (b) trastuzumab, (c) pertuzumab, (d) alemtuzumab, (d) cetuximab, (d) dituximab, (d) avelumab, (e) daratumumab, (f) isatuximab, (g) MOR202, (g) 7G3, (g) CSL362, (h) etozik, and (h) humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof; or (c) daratumumab, and wherein the derived effector cells comprise a CD38 knockout, and optionally expression of CD16 or a variant thereof.

[0030] In some embodiments of the composition, the composition further comprises a sensitizer. In some embodiments, the sensitizer comprises at least one of a chemotherapeutic agent, external beam radiation, brachytherapy, and a radiopharmaceutical. In some embodiments, the sensitizer increases chemokine secretion and / or surface expression of tumor cells when in contact with tumor cells. In some embodiments, the sensitizer includes: (i) at least one of calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90; or (ii) paclitaxel.

[0031] In another aspect, the present application provides therapeutic use of the composition provided herein by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject suffers from: an autoimmune disorder, a hematological malignancy, a solid tumor, a cancer, or a viral infection.

[0032] In another aspect, the present application provides a master cell bank (MCB) comprising clonal iPSCs as provided herein.

[0033] In another aspect, the present application provides a method for making derived effector cells, which derived effector cells contain an allogeneic immune defense receptor (ADR) and optionally one or both of CD38 knockout and endogenous TCR knockout, or one or more of CD38 knockout, endogenous TCR knockout, and one or both of CD58 and CD54 knockout, wherein the method comprises differentiating genetically engineered iPSCs into derived effector cells, wherein the genetically engineered iPSCs contain an exogenous polynucleotide encoding the ADR and optionally one or both of CD38 knockout and endogenous TCR knockout. In some embodiments, the genetically engineered iPSC further comprises one or more of the following: (i) CAR; (ii) exogenous CD16 or a variant thereof; (iii) a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface; (iv) at least one of the genotypes listed in Table 2; (v) disruption of at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (vi) disruption of at least one of CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, 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 agonists. In some embodiments of the manufacturing method, (i) the iPSC is a cloned iPSC, a single cell dissociated iPSC, an iPSC cell line cell or an iPSC master cell bank (MCB) cell; or (ii) the derived cells include derived CD34+ cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells or derived B lineage cells; or (iii) the derived cells include derived effector cells with one or more functional characteristics, and the one or more functional characteristics are not present in the corresponding primary T cells, NK cells, NKT cells and / or B cells.

[0034] In some embodiments of the manufacturing method, the exogenous CD16 or its variant includes at least one of the following: (a) high-affinity uncleavable CD16 (hnCD16); (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 of the manufacture, the CAR is: (i) T cell specific or NK cell specific; (ii) bispecific antigen binding CAR; (iii) switchable CAR; (iv) dimerization CAR; (v) separate CAR; (vi) multi-chain CAR; (vii) inducible CAR; (viii) co-expressed with another CAR; (ix) co-expressed with a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, optionally in a separate construct or in a bicistronic construct; (x) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct; (xi) specific for at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA and PDL1;and / or (xii) 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, CLEC12A, an antigen of cytomegalovirus (CMV) infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase erb B2,3,4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1) , mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1) and pathogen antigen; and optionally, wherein the CAR of any one of (i) to (xii) is inserted at the TCR locus and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by CAR insertion. ;

[0035] In some embodiments of the manufacturing method, the method also includes genome engineering the cloned iPSC to knock in a polynucleotide encoding ADR; and optionally: (i) knocking out one or both of CD38 and endogenous TCR, (ii) knocking out one or both of CD58 and CD54, and / or (iii) introducing one or more of the following: exogenous CD16 or its variant, CAR and / or a cytokine signaling complex comprising a portion or complete peptide of an exogenous cytokine expressed on the cell surface and / or its receptor. In some embodiments of the manufacturing method, 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.

[0036] In another aspect, the application provides a method for producing a cloned master engineered iPSC system using CRISPR, ZFN or TALEN-mediated editing of cloned iPSC, wherein editing includes knocking in polynucleotides encoding allogeneic immune defense receptors (ADR), and one or both of optional CD38 knockouts and endogenous TCR knockouts, thereby producing engineered iPSC. In some embodiments of the production method, ADR is inserted into one of the following loci: B2M, TAP1, TAP2, TAP-associated proteins, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3 or TIGIT; and wherein the insertion knocks out the expression of the gene in the locus.

[0037] In some embodiments of the production method, the method also includes sorting engineered iPSC to obtain the iPSC of single cell dissociation, and the iPSC of the single cell dissociation includes one or both of the polynucleotides encoding ADR and optional CD38 knockout and endogenous TCR knockout, or CD38 knockout, endogenous TCR knockout and CD58 and CD54 one or more of the knockout of one or both. In some embodiments of the production method, the method also includes amplifying the iPSC of single cell dissociation to produce the iPSC colony of clone master engineering. In some embodiments of the production method, the method also includes freezing and preserving the iPSC system of clone master engineering produced. In some embodiments of the production method, the method also includes analyzing the off-target editing and / or karyotype of the engineered iPSC.

[0038] In another aspect, the present application provides a clonal master engineered iPSC line generated using the methods provided herein.

[0039] In another aspect, the present application provides a method of treating a disease or condition, the method comprising administering a composition provided herein to a subject in need thereof.

[0040] In another aspect, the application provides a method for treating a disease or condition, the method comprising administering a cell or a colony thereof as provided herein to a subject in need thereof. In various embodiments of the method of treatment, the method further comprises administering a sensitizer to the subject, thereby pre-treating the tumor cells in the subject. In various embodiments of the method of treatment, the method further comprises administering one or more therapeutic agents to the subject.

[0041] In another aspect, the present application provides a method for treating a subject, comprising: (a) administering a sensitizer to a subject to pretreat tumor cells in the subject; and (b) administering cells disclosed herein or a population thereof to the subject after administering the sensitizer, wherein the subject suffers from an autoimmune disorder; hematological malignancies; solid tumors; cancer or viral infection. In various embodiments of the method of treatment, the sensitizer includes at least one of a chemotherapeutic agent, external beam radiation, brachytherapy, and a radiopharmaceutical. In some embodiments, the sensitizer increases the secretion and / or surface expression of 4-1BB and / or CD38 of tumor cells when in contact with tumor cells. In some embodiments, the sensitizer includes: (i) at least one of x-ray radiation, gamma radiation, photon radiation, proton radiation, and neutron radiation; or (ii) at least one of calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90; or (iii) paclitaxel.

[0042] In some embodiments of the method of treatment, the method further comprises administering one or more therapeutic agents. In some embodiments, the one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, effectors, antibodies or functional variants or fragments thereof, 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, chemotherapeutic agents or radioactive moieties or immunomodulatory drugs (IMiDs). In some embodiments of the method of treatment in which the therapeutic agent is a checkpoint inhibitor, the checkpoint inhibitor comprises: (i) one or more antagonist checkpoint molecules, including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2A R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor α), TLR3, VISTA, NKG2A / H LA-E or inhibitory KIR; (ii) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and their derivatives or functional equivalents; or (iii) at least one of atezolizumab, nivolumab and pembrolizumab; or (b) the therapeutic agent includes one or more of venetola, azacitidine and pomalidomide. In some embodiments of the method of treatment in which the therapeutic agent is an antibody, the antibody includes: (a) anti-CD20 antibody, anti-HER2 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-CD123 antibody, anti-GD2 antibody, anti-PDL1 antibody and / or anti-CD38 antibody; (b) rituximab, veltuzumab, ofatumumab, utoximab, okatuzumab, obinutuzumab, isbetumomab, ocrelizumab, inotuzumab, mosetuzumab, edoxaban, oxalutuzumab ... One or more of palituzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dituximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, etozuzumab and humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof; or (c) daratumumab, and wherein the derived effector cells comprise CD38 knockout, and optionally expression of CD16 or a variant thereof.

[0043] In another aspect, the present application provides a method for reducing or preventing the alloreactivity of host cells to allogeneic effector cells in adoptive cell therapy provided to a subject in need, wherein the allogeneic effector cells include cells or populations thereof provided herein, and wherein the method optionally includes CD38 regulation. In various embodiments, the host cell includes alloreactive immune cells, which include primary T cells, B cells and / or NK cells. In some embodiments, CD38 regulation: (i) including administering CD38 antagonists to the subject before, during or after allogeneic effector cells are infused to the subject; or (ii) including preloading CD38 antagonists to allogeneic effector cells in vitro before allogeneic effector cells are infused to the subject. In some embodiments, CD38 antagonists include: (i) anti-CD38 antibodies or CD38-CAR; (ii) daratumumab, isatuximab or MOR202; and / or (iii) daratumumab. In some embodiments, the method: (i) reduces or prevents host cell alloreactivity to allogeneic effector cells; (ii) eliminates or reduces the number of alloreactive host cells; (iii) prolongs the survival and persistence of allogeneic effector cells; (iv) delays host immune reconstitution; and / or (v) prevents leakage protection of allogeneic effector cells to host cell alloreactivity via overexpression of HLA-G or HLA-E. In another aspect, the invention provides a method of treating a subject in need of adoptive cell therapy according to the methods provided herein.

[0044] In another aspect, the present invention provides a cell or a colony thereof, wherein: (i) the cell is (a) an immune cell; (b) an induced pluripotent cell (iPSC); or (c) a derived effector cell obtained from differentiating the iPSC; and (ii) the cell comprises: an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); an exogenous polynucleotide encoding an exogenous CD16 or a variant thereof; an exogenous polynucleotide encoding a cytokine signaling complex comprising an exogenous cytokine expressed on the cell surface and / or part or all of its receptor peptides; and CD38 knockout, and wherein the cell has improved resistance to host immune alloreactivity compared to cells without exogenous polynucleotides. In various embodiments, the cell further comprises CAR. In various embodiments, the cell further comprises a double knockout of CD58 and CD54.

[0045] In some embodiments of a cell or its population, the ADR comprises a 41BB-specific ligand operably linked to a signaling domain that promotes effector cell activation. In some embodiments, the 41BB-specific ligand is 4-1BBL, an antibody or fragment thereof targeting 4-1BB, or a 4-1BBL-Fc fusion. In some embodiments, the signaling domain comprises CD3ζ or a functional fragment thereof, an Fc receptor, or a combination thereof from DAP12. In some embodiments, the ADR further comprises one, two, three or more co-stimulatory domains. In some embodiments, one, two, three or more co-stimulatory domains are from the intracellular signaling domain of CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM or CD40. In some embodiments, the ADR comprises (i) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 8; or (ii) a CD38 binding domain.

[0046] In some embodiments of the cell or its population, the cytokine signaling complex comprises a fusion protein of IL7 and IL7Rα, or an IL15 / IL15Rα fusion protein with a truncated intracellular domain of IL15Rα. In some embodiments, the exogenous CD16 or its variant is a high-affinity non-cleavable CD16 (hnCD16). In some embodiments, the high-affinity non-cleavable CD16 (hnCD16) comprises an extracellular domain domain of CD16 with F176V and S197P. In some embodiments, (i) an exogenous polynucleotide encoding an exogenous CD16 or its variant; and (ii) at least one of the exogenous polynucleotides encoding the cytokine signaling complex is inserted into the CD38 locus, resulting in CD38 knockout.

[0047] In another aspect, the invention provides a method of treating a subject, the method comprising administering a cell or a population thereof as described herein to a subject in need thereof, wherein the subject has an autoimmune disorder; a hematological malignancy; a solid tumor; a cancer or a viral infection. In various embodiments, the method does not include administering a Cy / Flu-based lymphodepletion therapy.

[0048] In another aspect, the invention provides a method of reducing or preventing host cell alloreactivity against allogeneic effector cells in adoptive cell therapy provided to a subject in need thereof, wherein the allogeneic effector cells include cells described herein or a population thereof, wherein the method optionally includes CD38 modulation. In some embodiments, the method does not include administering Cy / Flu-based lymphodepletion therapy.

[0049] 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

[0050] Figures 1A to 1E Exemplary stealth editing showing that iPSC-derived effector cells passively evade or selectively deplete alloreactive immune cells.

[0051] Figure 2A and Figure 2B ADR was shown to enhance iPSC-derived effector cells by selectively targeting 4-1BB-positive alloreactive immune cells to prolong functional persistence and proliferation.

[0052] Figure 3A and Figure 3B It is shown that distinct populations of alloreactive T cells present before and after Cy / Flu treatment are eliminated by ADR-equipped effector cells.

[0053] Figure 4A and Figure 4B showed that effector cells equipped with ADRs exhibited enhanced functional persistence in mixed lymphocyte reactions.

[0054] Figure 5A and Figure 5B Display ADR + Effector cells inhibit the expansion of alloreactive T cells and NK cells.

[0055] Fig. 6A and Figure 6B Display ADR + Effector cells selectively deplete alloreactive immune cells expressing 4-1BB.

[0056] Fig. 7A and Figure 7B Display ADR + Effector cells selectively target CD4 + and CD8 + Alloreactive T cell subsets.

[0057] FIG. 8A to FIG. 8C Display ADR + Effector cells exhibited enhanced functional persistence in the presence of daily tumor challenge.

[0058] Fig. 9 We show that iPSCs engineered to carry TRAC-driven CAR and ADR transgenes can be successfully differentiated into T cells with intracellular CD3 expression and robust co-expression of CAR and ADR.

[0059] Fig.10 We show that ADR expression by iPSC-derived iT cells provides protection against initiated allogeneic T-cell rejection and depletes allogeneic T cells.

[0060] Fig.11 We showed that effector cells ± ADR exhibited similar cytotoxicity against CD19-expressing tumor targets in vitro.

[0061] Fig. 12A and Fig. 12B Display ADR + Effector cells demonstrated unimpaired tumor control in vivo in the presence of host alloreactive T cells.

[0062] FIG. 13A to FIG. 13D We show that peripheral blood NK self-deletion responses are evaded in iNKs with dual co-stimulatory ligand deficiency.

[0063] Fig.14A and Fig. 14B showed that CD38 knockout in iT cells abrogated anti-CD38 ADCC when combined with peripheral blood NK cells.

[0064] Fig.15 It was shown that pretreatment with anti-CD38 monoclonal antibody protected CAR-iT cells from exhaustion.

[0065] Fig.16 Allogeneic T cells and NK cells were shown to be depleted in a daratumumab- and CAR-iT-dependent manner.

[0066] Fig.17A and Fig. 17B Display CD38 + T cells and pbNK cells were depleted in the presence of daratumumab. Fig. 17C and Fig.17D Display CD38 + T cells and pbNK cells were depleted in a daratumumab-dependent manner.

[0067] Fig.18 Figure 3 shows the expression of CD38 and 4-1BB in CD3 cells from 10 days of co-culture (8:1 iNK to PBMC ratio) and analysis of CD38 and 4-1BB in CD3 + Representative FACS plots of expression in T cells.

[0068] Fig.19A and Fig.19B CD38-negative iNK cells were shown to be resistant to alloreactive pbNK attack in the presence of daratumumab.

[0069] FIG. 20A to FIG. 20CWe show that ADR-equipped CD19-CAR effector cells have unimpaired antitumor efficacy in an in vivo disseminated Nalm6 leukemia model in an alloreactive setting.

[0070] Fig.21 showed that the addition of anti-CD38 antibody to lymphoid depletion conditioning (LDC) delayed host immune reconstitution and extended the window of opportunity for adoptive cell therapy in patients treated with engineered CD38KO hnCD16 iNK cells in combination with daratumumab compared with patients treated with engineered iNK cells without daratumumab.

[0071] Fig. 22 Uniform Manifold Approximation and Projection (UMAP) visualization showing lymphocyte distribution in lymphoma patients and multiple myeloma patients treated with engineered iNK cells in combination with daratumumab. DETAILED DESCRIPTION

[0072] The genome modification of iPSC (induced pluripotent stem cell) may include one or more of polynucleotide insertion, deletion, substitution and their combination.Exogenous gene expression in genome engineered iPSC is generally problematic, such as after long-term clonal expansion of initial genome engineered iPSC, after cell differentiation and in dedifferentiated cell types from cells 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 some embodiments, the present invention provides efficient, reliable and targeted methods for stably integrating one or more exogenous genes (including suicide genes and other functional modes) into iPSC-derived cells, which one or more exogenous genes provide improved therapeutic properties related to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, expansion, lifespan, self-renewal, persistence and / or survival, including but not limited to HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T lineage cells, NKT lineage cells, NK lineage cells and immune effector cells having one or more functional characteristics that are not present in primary NK cells, T cells and / or NKT cells.

[0073] definition

[0074] 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.

[0075] 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.

[0076] 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.

[0077] The use of alternatives such as "or" should be understood to mean either one, both, or any combination of the alternatives.

[0078] The term "and / or" should be understood to mean one or both of the alternatives.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] The term "in vivo" generally refers to activities that take place inside an organism.

[0088] 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.

[0089] 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).

[0090] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a stem cell produced in vitro from differentiated adult, neonatal or fetal cells that has been induced or altered, i.e., reprogrammed to be capable of differentiating into all three germ layers or dermis: mesoderm, endoderm, and ectoderm tissues. In some embodiments, the reprogramming process uses reprogramming factors and / or small molecule chemical drive methods. The iPSC produced does not refer to cells as they are found in nature.

[0091] 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.

[0092] As used herein, the term "pluripotent stem cell" refers to a cell with the developmental potential to differentiate into one or more germ layers (i.e., 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 known in the art, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells." Pluripotent" 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, and the degree of its developmental potential is lower than differentiation omnipotence and pluripotency.

[0093] 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.

[0094] 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).

[0095] 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.

[0096] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock or other domesticated animal.

[0097] "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.

[0098] "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.

[0099] "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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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". In some embodiments, the derived T lineage cells are iPSC-derived T cells obtained by differentiating iPSCs, which are also referred to as "iT" cells herein.

[0104] "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.

[0105] "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.

[0106] 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 the following: 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. + It can be weaker or stronger expression.NK cells or NK cell-like effector cells can be differentiated by stem cells or progenitor cells ("derived NK cells" or "derived NK cell-like effector cells", or collectively referred to as "derived NK lineage cells"). Derived NK cell-like effector cells may have NK cell lineages in some aspects, but have one or more functional characteristics that are not present in primary NK cells at the same time. In the present application, NK cells, NK cell-like effector cells, derived NK cells, derived NK cell-like effector cells or derived NK lineage cells are collectively referred to as "NK lineage cells". In some embodiments, derived NK lineage cells are iPSC-derived NK cells obtained by differentiating iPSC, which are also referred to as "iNK" cells herein.

[0107] 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, αGalCer, CD161, and CD56.

[0108] 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.

[0109] 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.

[0110] 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%.

[0111] 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.

[0112] "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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] "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.

[0120] 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.

[0121] 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. Priority 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; and / or 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.

[0122] 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.

[0123] 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.

[0124] 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-antigenic determinant that is specific for an antigenic determinant 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.

[0125] 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.

[0126] 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.

[0127] 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 the cell. Examples of 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, toll gate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0128] As used herein, the term "targeting modality" refers to the genetic incorporation of molecules (e.g., polypeptides) into cells to promote antigen and / or antigenic determinant 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.

[0129] 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.

[0130] As used herein, the term "adoptive cell therapy" refers to a cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes (e.g., T cells, B cells and / or NK cells), whether the immune cells are isolated from a human donor or 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.

[0131] 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.

[0132] As used herein, "radiation" refers to the emission or transmission of energy in the form of waves or particles. Exemplary forms of radiation include, but are not limited to, electromagnetic radiation (e.g., radio waves, microwaves, infrared, visible light, ultraviolet light, x-rays, and gamma radiation), particle radiation (e.g., alpha radiation, beta radiation, proton radiation, and neutron radiation), and acoustic radiation (e.g., ultrasound, sound, and seismic waves). In various embodiments, the amount of radiation is measured as Gray (Gy), which is defined as one joule of radiation energy absorbed per kilogram of matter. In radiotherapy, the amount of radiation applied varies according to the type and stage of the cancer being treated. For cure, typical doses for epithelial solid tumors range from 60Gy to 80Gy, while lymphomas are typically treated with 20Gy to 40Gy. Preventive (adjuvant) doses are typically about 45Gy-60 Gy, in 1.8Gy–2Gy fractions each time (e.g., for breast cancer, head cancer, neck cancer). In various embodiments, radiation can be used as a sensitizer disclosed herein.

[0133] As used herein, "radiotherapy" refers to a class of cancer treatments that involves the use of radiation to damage cells by destroying the genetic material that controls how cells grow and divide. Although both healthy cells and cancer cells are damaged by radiotherapy, the goal of radiotherapy is to destroy normal healthy cells as little as possible. The term "radiotherapy" generally refers to external beam radiotherapy, in which high-energy beams (e.g., x-rays, gamma rays, photons, protons, neutrons, ions, and any other energy form suitable for such treatment) are produced by a machine outside the subject being treated, and are aimed at a precise point on the subject's body. However, the term "radiotherapy" also includes brachytherapy, in which seeds, ribbons, or capsules containing or otherwise connected to a radioactive source are placed in or near a tumor or cancer cell in the subject's body. Brachytherapy includes low-dose rate implants, high-dose rate implants, and permanent implants. The term "radiotherapy" also includes systemic radiation therapy, in which a radioactive drug (e.g., a radiopharmaceutical or a radionuclide, including a radioactive peptide) is given orally or intravenously to the subject, and is concentrated in the tumor or region where the cancer cell is located in the subject's body. Similar to antibody-drug candidates, in which an antibody that binds to a tumor antigen is linked to a toxic drug, a radiopharmaceutical incorporates a radioactive compound linked to a targeting molecule (e.g., an antibody) that specifically binds to a tumor antigen. Examples of radioactive compounds that can be used for radiopharmaceuticals include, but are not limited to, calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and at least one of yttrium-90. In various embodiments, radiation therapy can be used as a sensitizer as disclosed herein.

[0134] As used herein, "homing" or "transport" refers to the active navigation (migration) of cells to a target site (e.g., a cell, a tissue (e.g., a tumor) or an organ). "Homing molecules" refer to molecules that direct cells 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. As used herein, "chemokine receptor" refers to a cell surface molecule that binds a chemokine. Chemokine receptors may include naturally occurring or recombinant chemokine receptors or variants thereof. Exemplary chemokine receptors include, but are not limited to, a CXC chemokine receptor (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), a CX3C chemokine receptor (e.g., CX3CR1), an XC chemokine receptor (e.g., XCR1), or variants thereof.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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).

[0142] 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.

[0143] 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.

[0144] "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.

[0145] 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.

[0146] 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".

[0147] 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.

[0148] "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.

[0149] "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.

[0150] "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.

[0151] 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.

[0152] "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.

[0153] I. Cells and compositions useful for adoptive cell therapy with enhanced properties

[0154] It is believed that a variety of HLA class I and class II proteins must be matched in allogeneic receptors to obtain tissue compatibility, thereby avoiding allogeneic rejection problems. In the absence of MHC matching, a method studied in allogeneic adoptive cell therapy is to eliminate or substantially reduce the expression of HLA class I and HLA class II proteins. HLA class I deficiency can be achieved by causing any region of HLA class I locus (chromosome 6p21) to have a functional loss or causing HLA class I related genes (including but not limited to beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene and TAP related protein) to lack or reduce expression levels. For example, the B2M gene encodes the common subunits necessary for the cell surface expression of all HLA class I heterodimers. B2M negative cells are HLA-I deficient. HLA class II deficiency can be achieved by causing HLA-II related genes (including but not limited to RFXANK, CIITA, RFX5 and RFXAP) to have a functional loss or reduction. CIITA is a transcriptional coactivator that works by the activation of the transcription factor RFX5 required for class II protein expression. CIITA negative cells are HLA-II deficient. Therefore, the application provides iPSC lines and derived cells thereof comprising HLA-I deficiency and / or HLA-II deficiency (e.g., lack of B2M and / or CIITA expression), wherein the derived effector cells obtained are achieved by eliminating the need for MHC (major histocompatibility complex) matching, avoiding host (allogeneic) T cells to identify and kill, to achieve allogeneic cell therapy.

[0155] However, lack of HLA class I expression increases sensitivity to NK cell lysis. In addition, the lack of both HLA-I and HLA-II still cannot prevent allogeneic rejection mediated by alloantigens other than the MHC of allogeneic adoptive cells. In addition, HLA-I dependent NK cell education processes (such as licensing, arming or disarming) are considered to have an impact on the innate immune response to allogeneic cells, which may lead to the reactivity or partial reactivity of recipient NK cells to allogeneic donor cells, even when those donor cells are HLA-I sufficient.

[0156] To address this "self-deficient" reaction, HLA-E, HLA-G or other non-classical HLA-I proteins can be optionally knocked in to avoid NK cell recognition and killing of effector cells derived from HLA-I deficient engineered iPSCs. In one embodiment, the provided HLA-I deficient iPSCs and their derivatives further comprise HLA-G knock-in. Alternatively, in one embodiment, the provided HLA-I deficient iPSCs and their derivatives further comprise one or both of CD58 knockout and CD54 knockout.

[0157] On the other hand, lymphocyte regulation chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine), which is usually administered with patient-specific CAR-T cell therapy, suppresses the patient's immune system to potentially create a suitable active window for allogeneic cell therapy to induce clinical response. However, long-term lymphocyte regulation is associated with poor immune reconstitution, increased susceptibility to opportunistic infections, and reduced contribution of host immunity to the development of durable anti-tumor efficacy. Therefore, it is necessary to study alternative methods that can be effectively treated with allogeneic cell therapy.

[0158] However, the present application provides a strategy that alternatively maintains HLA-I or HLA-II or both intact (i.e., comprising HLA-I wild type and HLA-II wild type) in allogeneic effector cells and does not require lymphoid regulation, while avoiding allogeneic rejection against allogeneic effector cells by eliminating or reducing activated T cells, B cells and NK cells in the recipient of adoptive cell therapy. The stealth strategy provided is comprehensive because it involves multiple immune recognition pathways to enable ready-made allogeneic effector cells to not only evade host immune cell alloreactivity, but also enhance their functional persistence, thereby making it possible to achieve effective cell-based adoptive cancer therapy.

[0159] 1. Allogeneic immune defense receptor (ADR) expression

[0160] This article provides a strategy that systematically engineers multiple regulatory circuits in cloned iPSCs without affecting differentiation potency and the cell developmental biology of iPSCs and their derivatives, while enhancing the therapeutic properties of derived cells differentiated from iPSCs. After the selective mode combination introduced into the cells by genetic engineering at the iPSC level, iPSC-derived cells are functionally improved and suitable for adoptive cell therapy. The engineering strategies and components are also suitable for modifying primary T cells or NK cells for similar purposes.

[0161] In order to control the pathological conditions caused by unwanted immune system activation, in various embodiments, the application provides immune cells, iPSC and iPSC-derived effector cells, except for other editing expected and described herein, it is genetically engineered to include allogeneic immune defense receptors (ADR) for effector cell enhancement and selective exhaustion of alloreactive host T cells or NK cells (including pathogenic T cells), while the resting cells in the remaining receptors. As provided in the application, the expression of alloreactive immune defense receptors (ADR) has the unique ability to eliminate alloreactive T cells and NK cells and escape host immune cells without the need for I HLA class deletion.

[0162] In some embodiments, the ADR is specific for 4-1BB (CD137, also referred to as "41BB"), and the ADR comprises an extracellular domain of 4-1BB that is targeted to be upregulated in host T cells or NK cells when the host T cells or NK cells are activated and a signaling domain that promotes effector cell activation. For example, the 41BB-ADR extracellular domain may comprise any suitable ligand of 4-1BB, including 4-1BBL, an antibody (or a functional fragment thereof) targeting 4-1BB, a fusion of Fc and 4-1BBL, or a functional derivative or fragment thereof. Additional upregulated surface proteins of host activated immune cells include, but are not limited to, CD38, CD25, CD69, CD44, OX40, and CD40L, on which ADRs may be constructed to include binding domains specific to CD38, CD25, CD69, CD44, OX40, and CD40L, respectively.

[0163] In some embodiments of the ADR specific for 4-1BB, the 41BB-ADR extracellular domain comprises 4-1BBL or a fragment thereof that effectively binds to 4-1BB. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 75. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 75. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 75. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 75. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence of SEQ ID NO: 75.

[0164] SEQ ID NO:75

[0165] GLLDLC

[0166] In one embodiment of the CD38-specific ADR, the CD38-ADR comprises an anti-CD38 antibody binding domain or a fragment thereof. In some embodiments, the anti-CD38 antibody comprises a mouse antibody, a human antibody, a humanized antibody, a camel Ig, a single variable new antigen receptor (VNAR), a shark antibody with only a heavy chain (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody binding domains or fragments thereof include Fab, Fab', F(ab')2, F(ab')3, Fv, single-chain antigen binding 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 a heavy chain (VHH), and other antibody fragments that maintain the binding specificity of the intact antibody.

[0167] In some embodiments, the anti-CD38 antibody binding domain or fragment thereof contained in the CD38-ADR comprises a heavy chain variable region and / or a light chain variable region consisting of SEQ ID NOs: 29 and 30, SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, SEQ ID NOs: 55 and 56, SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 72, SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 77, SEQ ID NOs: 78 and 78, SEQ ID NOs: 79 and 80, SEQ ID NOs: 81 and 82, SEQ ID NOs: 83 and 84, SEQ ID NOs: 85 and 86, SEQ ID NOs: 87 and 88, SEQ ID NOs: 89 and 90, SEQ ID NOs: SEQ ID NOs: 61 and 62, respectively, SEQ ID NOs: 63 and 64, respectively, SEQ ID NOs: 65 and 66, respectively, SEQ ID NOs: 67 and 68, respectively, SEQ ID NOs: 69 and 70, respectively, or SEQ ID NOs: 71 and 72, respectively, are represented by amino acid sequences having at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, and / or at least about 80% identity. Selected VH and VL sequences of exemplary CD38 antibodies are provided in Table 1 as numbered pairs 1-23. In some embodiments, the CD38-ADR extracellular domain comprises an amino acid sequence having at least about 90% sequence identity with a VH and / or VL sequence of any one of pairs 1-23 in Table 1. In some embodiments, the CD38-ADR extracellular domain comprises an amino acid sequence having at least about 95% sequence identity with a VH and / or VL sequence of any one of pairs 1-23 in Table 1. In some embodiments, the CD38-ADR extracellular domain comprises the amino acid sequence of the VH and / or VL sequences of any one of pairs 1-23 in Table 1.

[0168] Table 1.

[0169]

[0170]

[0171]

[0172]

[0173] In some embodiments, the extracellular domain of 41BB-ADR, CD38-ADR, CD25-ADR, CD69-ADR, CD44-ADR, OX40-ADR, or CD40L-ADR can be operably linked to one or more signaling domains that mediate downstream signaling upon effector cell activation following binding of 4-1BB, CD38, CD25, CD69, CD44, OX40, or CD40L (respectively) to an alloreactive host immune cell. In some embodiments, the ADR comprises CD3ζ, which is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 1, or a functional fragment thereof, or comprises a CD3ζ derivative (e.g., CD3ζ1XX, which is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 2, or a functional fragment thereof). In some embodiments, CD3ζ comprises an amino acid sequence having at least about 90% sequence identity with SEQID NO: 1 or 2. In some embodiments, CD3ζ comprises an amino acid sequence having at least about 95% sequence identity with SEQID NO: 1 or 2. In some embodiments, CD3ζ comprises the amino acid sequence of SEQID NO: 1. In some embodiments, CD3ζ comprises the amino acid sequence of SEQ ID NO: 2. CD3ζ mediates downstream ITAM-derived signaling during activation of effector T cells or NK cells. Other ITAM-containing signaling domains may include those derived from DAP12, Fc receptors, and other CD3 subunits. As used herein and throughout this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 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. The comparison of sequences and determination of the percent identity between two sequences can be achieved using a mathematical algorithm recognized in the art.

[0174] SEQ ID NO:1

[0175] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0176] (CD3ζ)

[0177] SEQ ID NO:2

[0178] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0179] (CD3ζ1XX- contains 2 mutations in ITAM1)

[0180] In some embodiments, the intracellular domain of the ADR comprising the signaling domain further comprises one, two, three or more costimulatory domains that enhance the production of cytokines from effector cells expressing the ADR. The costimulatory domain may be derived from the intracellular signaling domain of a costimulatory protein, including but not limited to CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, CD40, etc. In some embodiments, the ADR comprising CD3ζ further comprises a costimulatory domain derived from the intracellular domain of 4-1BB, and is represented by an amino acid sequence or a functional fragment thereof having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 73. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 73. In some embodiments, the 4-1BB intracellular domain comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO: 73. In some embodiments, the 4-1BB intracellular domain comprises the amino acid sequence of SEQ ID NO: 73. In one embodiment, when the ADR comprises 4-1BBL in its extracellular domain, the costimulatory domain of the ADR is not derived from 4-1BB.

[0181] SEQ ID NO:73

[0182] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0183] ( 41BB Intracellular-CD3ζ)

[0184] The intracellular domain of ADR can be non-covalently connected to the extracellular domain of ADR by a transmembrane domain.In some embodiments, ADR includes a transmembrane domain that can belong to any kind, as long as it allows the CD3ζ components of ADR to be located in the cell and the extracellular domain of targeting 4-1BB, CD38, CD25, CD69, CD44, OX40 or CD40L is located outside the cell.In other cases, ADR is a soluble protein, which can be bound to the corresponding ligand on activated T cells and promote cytotoxicity by cross-linking TCR (e.g., ADR-CD3 T cell adapter protein).In the case where the extracellular domain is from a surface protein (e.g., CD40) with a transmembrane domain, ADR may include a transmembrane domain from the corresponding endogenous molecule.In some embodiments where the ADR molecule includes one or more costimulatory domains, a transmembrane domain (TM) may be from the same endogenous molecule with a costimulatory domain. Non-limiting examples of TMs include those from CD3, CD8a, CD27, CD28, 4-1BB, OX40, and CD4.

[0185] In some embodiments, ADR is included in the spacer between the extracellular protein and the transmembrane domain. In some embodiments, the spacer may include a sequence that is inert or contributes substantially little or no contribution to any function that ADR may have; however, in other cases, the spacer includes a sequence that enhances the function of ADR and / or allows it to be detected and / or can be targeted for inhibition. In a specific embodiment, the spacer includes a coded protein sequence that promotes the detection of cells expressing ADR. For example, the spacer may encode an Fc region or a fragment thereof that will allow surface detection of cells expressing ADR (such as by using an anti-Fc antibody). In a specific embodiment, the spacer provides a separation between the ligand-binding extracellular domain and the membrane to avoid potential steric hindrance. As will be appreciated by those skilled in the art, the spacer may vary in sequence, length, regardless of whether functions other than physical separation are expected.

[0186] Exemplary spacers that may be included in the ADR are well known in the art, including but not limited to IgG4 spacers, CD28 spacers, CD8 spacers, or a combination of more than one spacers. The length of the spacer may also vary, from about 15 amino acids (aa) to about 300 amino acids or more amino acids. Non-limiting exemplary spacer peptides include those represented by an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with SEQ ID NO: 3 or 4. In some embodiments, the spacer peptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3 or 4. In some embodiments, the spacer peptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3 or 4. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO: 4.

[0187] SEQ ID NO:3

[0188] ESKYGPPCPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[0189] (88 amino acids)

[0190] SEQ ID NO:4

[0191] ESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKDPK

[0192] (123 amino acids IgG4 hinge - IgG1 CH3)

[0193] In one embodiment of the 4-1BB-specific ADR, 41BB-ADR is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with any one of SEQ ID NO: 5-8, wherein the signal peptide, spacer and transmembrane (TM) domain sequences are variable. In some embodiments, 41BB-ADR comprises an amino acid sequence having at least about 90% sequence identity with any one of SEQ ID NO: 5-8. In some embodiments, 41BB-ADR comprises an amino acid sequence having at least about 95% sequence identity with any one of SEQ ID NO: 5-8. In some embodiments, 41BB-ADR comprises an amino acid sequence having at least about 95% sequence identity with any one of SEQ ID NO: 5-8. In some embodiments, 41BB-ADR comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, 41BB-ADR comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, 41BB-ADR comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, 41BB-ADR comprises an amino acid sequence of SEQ ID NO: 8.

[0194] SEQ ID NO:5

[0195] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLL IDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPAS SEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPS PRSE ESKYGPPCPPCPGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNAYTQKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRS RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR

[0196] Signal peptide-41BBL- Spacer -CD28(TM)- CD3z (Signal peptide, spacer and TM / transmembrane domain may vary)

[0197] SEQ ID NO:6

[0198] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLL IDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPAS SEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPS PRSE ESKYGPPCPPCPGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNAYTQKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0199] Signal peptide-41BBL- Spacer -CD28(TM)-CD28(ICD)- CD3z (Signal peptide, spacer and TM / transmembrane domain may vary)

[0200] SEQ ID NO:7

[0201] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE ESKYGPPCPPCPGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYT QKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPP R

[0202] Signal peptide-41BBL- Spacer -CD28(TM)- CD3z1xx (Signal peptide, spacer and TM / transmembrane domain may vary)

[0203] SEQ ID NO:8

[0204] MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSE ESKYGPPCPPCPGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYT QKSLSLSPGKKDPK FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFS EIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0205] Signal peptide-41BBL- Spacer -CD28(TM)-CD28(ICD)- CD3z1xx (Signal peptide, spacer and TM / transmembrane domain may vary)

[0206] In one embodiment of a CD38-specific ADR, the CD38-ADR is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 74. In some embodiments, the CD38-ADR comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 74. In some embodiments, the CD38-ADR comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 74. In some embodiments, the CD38-ADR comprises the amino acid sequence of SEQ ID NO: 74.

[0207] SEQ ID NO:74

[0208] MDFQVQIFSFLLISASVIMSRDIQMTQSPSSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKPSGGTFRSYAISWVRQAPGQGLEWMGRIIVFLGKVNYAQRFQGRVTLTADKSTTTAYMELSSLRSEDTAVYYCTGEPGARDPDAFDIWGQGTM VTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0209] Signal peptide-anti-CD38VH- Linker - Anti-CD38VL-CD8(TM)-41BB(Intracellular)- CD3z(Intracellular) (Signal peptide, linker and TM / transmembrane domain may vary)

[0210] Therefore, in some embodiments, the present application provides immune cells, iPSCs, and iPSC-derived effector cells, which contain polynucleotides encoding ADRs specific to 4-1BB, CD38, CD25, CD69, CD44, OX40, or CD40L, and optionally other selected gene editing as described herein, so that the effector cells (including T cells and NK cells) are equipped with the ability to selectively deplete activated host immune cells, so that the genetically engineered effector cells are resistant to host immune alloreactivity, which is associated with the allogeneic use of effector cells for treating tumors and infectious diseases in patients. Further provided herein is a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one phenotype as provided herein, including ADRs specific for 4-1BB, CD38, CD25, CD69, CD44, OX40 or CD40L, 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, including but not limited to derived NK cells and T cells, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0211] 2. Endogenous TCR knockout

[0212] The α-β T cell receptor (TCRαβ) is an antigen-specific receptor generally considered essential for immune responses and is present on the cell surface of αβ T lymphocytes. Binding of TCRαβ to the peptide-major histocompatibility complex (pMHC) initiates TCR-CD3 intracellular activation, recruitment of a multitude of signaling molecules, and branching and integration of signaling pathways, leading to the mobilization of transcription factors that are critical for gene expression and T cell growth and acquisition of function. Disruption of the constant region (TRAC or TRBC) of endogenous TCRα or TCRβ, either by direct editing of T cells or by genomic iPSC editing and differentiation, is a promising approach to generate TCRs as a source for obtaining modified derivative T lineage cells. neg For example, inserting a 2A sequence at a preselected position in TRAC or TRBC, operably linked to an endogenous promoter or an exogenous promoter of TRAC or TRBC, can result in disruption (or truncation in this example) of TRAC or TRBC and TCR-negative cells (TCR neg In some embodiments, targeted truncation or disruption using a self-cleaving peptide such as 2A can optionally occur simultaneously with integration of one or more exogenous genes of interest at the truncation or disruption site, and expression of the integrated gene can be driven by an operably linked exogenous promoter or by an endogenous promoter of the integrated TCRα or TCRβ, resulting in TRAC or TRBC knockout, and thus TCR knockout (TCR KO ) simultaneously express one or more exogenous genes inserted at the TRAC or TRBC locus.

[0213] In certain embodiments, TCR neg The cell is an iPSC. In another embodiment, the TCR neg The cells are NK lineage cells. As used herein, the term "TCR negative" or "TCR neg ” refers to the lack of endogenous TCR expression, which is due to the disruption of TCR gene expression (TCR KO ) (such as in T lineage cells: primary or iPSC-derived T lineage cells) or due to the natural lack of TCR gene expression despite the presence of TCR loci in the genome (e.g., iPSC or NK lineage cells: primary or iPSC-derived NK lineage cells). The directed differentiation of engineered iPSCs to hematopoietic cells that are subsequently cloned makes it possible to produce iPSC-derived immune effector cells and / or their homologous groups without TCR expression.

[0214] iPSC-derived TCR-negative effector cells (with or without exogenous gene integration) obtained using this method do not require HLA matching, have reduced alloreactivity and are able to prevent GvHD (graft-versus-host disease) when used for allogeneic adoptive cell therapy.

[0215] Therefore, in some embodiments, the present invention provides iPSCs and cells derived therefrom, which comprise an exogenous polynucleotide encoding an ADR specific for 4-1BB and optionally an endogenous TCR knockout (TCR KO ), wherein the cells (including derived T cells and NK cells) can be used to overcome allogeneic reactivity in an immunocompetent host system. In some embodiments, iPSCs and their derivative cells contain exogenous polynucleotides encoding 41BB-ADR, and optionally TCR KO And 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 additionally provided in the present application, comprising a single cell sorting and amplified cloned engineered iPSC with at least one phenotype as provided herein, comprising an ADR specific to 4-1BB and an optional TCR KO , wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derived NK cells and T cells, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0216] 3. CD38 knockout

[0217] 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).

[0218] 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.

[0219] In the treatment of malignancies, systemic administration of CD38 antigen-binding receptor-transduced T cells has been shown to lyse 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 function of recipient immune effector cells. 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 decrease (Casneuf et al., "Hematological Research Progress (Blood Advances.)" 2017, Vol. 1, Issue 23, Pages 2105-2114).

[0220] Without being limited by theory, the application provides a strategy for making full use of the full potential of CD38 targeted cancer treatment by overcoming the effector cell exhaustion or reduction induced by CD38 specific antibodies and / or CD38 antigen binding domains via cannibalism. In addition, because CD38 is upregulated on activated lymphocytes (such as T cells or B cells) by using CD38 specific antibodies (such as daratumumab) to suppress the activation of these recipient lymphocytes in the recipient of allogeneic effector cells, the host allogeneic rejection for these effector cells will be reduced and / or prevented, thereby increasing 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.

[0221] In addition, the present application also discloses that when CD38 is used in the presence of a CD38 antagonist (including but not limited to an anti-CD38 antibody), a CD38-ADR or a CD38 inhibitor, - 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.

[0222] Furthermore, in various embodiments, the strategies provided herein for CD38 knockout are compatible with other components and processes contemplated herein, thereby generating a CD38 knockout (CD38 KO ) iPSC lines, master cell banks containing clonal CD38-negative iPSCs that were single-cell sorted and expanded, and CD38-negative (CD38) iPSCs that were derived by directed differentiation of engineered iPSC lines. neg ) derived effector cells, wherein when the CD38 targeting therapeutic moiety is used with the effector cells, these derived effector cells are protected from fratricide and allogeneic rejection, among other advantages. In addition, anti-CD38 monoclonal antibody therapy significantly depletes the patient's activated immune system without adversely affecting the patient's hematopoietic stem cell compartment, and when administered for purposes including but not limited to lymphodepletion is an example of a process known as "CD38 modulation". Thus, in some embodiments, CD38 is administered to the patient's immune system. KO Effector cells in combination with anti-CD38 monoclonal antibodies: (i) reduce or prevent host cell alloreactivity to allogeneic effector cells; (ii) eliminate or reduce the number of alloreactive host cells; (iii) prolong the survival and persistence of allogeneic effector cells; (iv) delay host immune reconstitution; and / or (v) prevent leakage protection of allogeneic effector cells to host cell alloreactivity via overexpression of HLA-G or HLA-E. CD38 neg The derived cells are resistant to CD38 antibody-mediated depletion and can be effectively administered in combination with anti-CD38 antibodies, CD38-CARs or CD38-ADRs without the use of toxic conditioning agents, thereby reducing and / or replacing chemotherapy-based lymphocyte depletion.

[0223] In one embodiment as provided herein, the CD38 knockout in the iPSC line is a biallelic knockout. As disclosed herein, in some embodiments, the CD38 negative iPSC line provided further comprises at least 41BB-ADR or CD38-ADR and optionally TCR KO, and may further comprise one or more additional engineering transformation modes described herein, and as shown in Table 2; and the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, including but not limited to immune effector cells, including but not limited to mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 + Hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional characteristics not present in primary NK, T and / or NKT cells. In some embodiments, when anti-CD38 antibodies are used to induce ADCC or anti-CD38 CAR or CD38-ADR is used for target cell killing, CD38 neg iPSC and / or its derived effector cells are not eliminated by anti-CD38 antibodies, anti-CD38CAR or CD38-ADR or receptor activated T cells or B cells, thereby increasing iPSC and its effector cells to remain and / or survive in the presence of such treatment parts and / or after being exposed to such treatment parts. In some embodiments, effector cells have increased retention and / or survival in vivo in the presence of such treatment parts and / or after being exposed to such treatment parts.

[0224] Further provided herein is a master cell bank comprising single cell sorted and expanded clonally engineered iPSCs having at least one phenotype as provided herein, comprising polynucleotides encoding an ADR specific for 4-1BB or CD38 and optionally a TCR KO and CD38 KO One or both of the above, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derived NK cells and T cells, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0225] 4. CD16 knock-in

[0226] 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 downregulation including 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 allele / 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, and the complete disclosure of the document 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).

[0227] 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 of the exemplary sequences SEQ ID NO: 9, 10 and 11, each of which comprises at least a portion of the CD64 extracellular domain.

[0228] SEQ ID NO:9

[0229] WFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFF HWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQ LYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFH YQVSFCLVMVLLFAVDTGLYFSV KTNIRSSTRDWKDHKFKWRKDPQDK

[0230] (340 amino acids CD64 domain-based construct ;CD16TM; CD16ICD )

[0231] SEQ ID NO:10

[0232] MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPS YRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKF FHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGL QLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGL FFPPGYQVSFCLVMVLLFAVDTGLYFSV KTNIRSSTRDWKDHKFKWRKDPQDK

[0233] (336 amino acids CD64 exon-based constructs ;CD16TM; CD16ICD )

[0234] SEQ ID NO:11

[0235] MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPS YRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKF FHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGL QLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLG FFPPGYQVSFCLVMVLLFAVDTGLYFSV KTNIRSSTRDWKDHKFKWRKDPQDK

[0236] (335 amino acids CD64 exon-based constructs ;CD16TM; CD16ICD )

[0237] Therefore, effector cells or iPSCs are provided herein, which are genetically engineered to include exogenous CD16 or variants thereof in other edits as covered and described herein, wherein these effector cells are cells derived from primary sources or derived from iPSC differentiation, or wherein these genetically engineered iPSCs are capable of differentiating into derived effector cells, and these derived effector cells include exogenous CD16 or variants thereof introduced into iPSCs. In some embodiments, the derived effector cells comprising hnCD16 are NK cells. In some embodiments, exogenous CD16 is a non-cleavable high-affinity CD16 receptor (hnCD16). In some embodiments, hnCD16 includes all or part of the length of the CD64 extracellular domain. In some embodiments, exogenous CD16 is in the form of a chimeric Fc receptor (CFcR) based on CD16, which includes a transmembrane domain, a stimulatory domain, and / or a signaling domain that is not derived from CD16.

[0238] In some embodiments, the primary source or derived effector cell including exogenous CD16 or its variant is NK lineage cell.In some embodiments, the primary source or derived effector cell including exogenous CD16 or its variant is T lineage cell.Exogenous CD16 or its functional variant contained in iPSC or effector cell has high affinity when combined with ligand, and the ligand triggers downstream signal transduction after such combination.Non-limiting examples of the ligand combined with exogenous CD16 or its functional variant include not only ADCC antibodies or fragments thereof, but also include bispecific, trispecific or multispecific adapters or binders of CD16 or CD64 extracellular binding domains that recognize the exogenous CD16.Examples of bispecific, trispecific or multispecific adapters or binders are further described in the following of the application. Therefore, at least one of the aspects of the present application provides a derived effector cell or a cell population thereof, which is pre-loaded with one or more preselected ADCC antibodies by expressing exogenous CD16 on the derived effector cells in an amount sufficient for therapeutic use in treating a condition, disease or infection as further detailed in the present application, wherein the exogenous CD16 comprises the extracellular binding domain of CD64 or comprises the extracellular binding domain of CD16 having F176V and S197P.

[0239] In some other embodiments, the exogenous CD16 comprises a CFcR based on CD16 or a variant thereof. 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 the transmembrane domain, the stimulatory domain or the signaling domain are derived from different receptors in addition to the receptor providing the extracellular domain. In the description herein, the CFcR based on CD16 or a variant thereof 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 one embodiment of a CD16-based CFcR, a chimeric Fc receptor is provided that includes a transmembrane domain and a signaling domain that are 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.

[0240] Various embodiments of the chimeric Fc receptor based on CD16 as described above can bind to the Fc region of an antibody or its fragment with high affinity; or bind to a bispecific, trispecific or multispecific adapter or binder. 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 the 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 a 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, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific 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.

[0241] 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 other 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, hnCD16 may comprise F176V and S197P in some embodiments, or may comprise an extracellular domain derived from all or part of the length of CD64 as exemplified in SEQ ID NO: 9, 10 or 11, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides a derivative NK cell or cell population thereof pre-loaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in treating a condition, disease or infection as further detailed herein.

[0242] 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 including expressed exogenous non-cleavable CD16 do not impair T cell developmental biology and are able to differentiate into functional derivative T lineage cells that not only express exogenous CD16, but also can 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 saving antigen escape that usually occurs with CAR-T cell therapy, wherein the tumor is reduced or lost with the expression of antigens targeting CAR-T or mutant antigens to avoid being recognized by CAR and recurring. When derived T lineage cells express acquired ADCC by exogenous CD16 (including functional variants and CD16-based CFcR), and when the antibody targets a tumor antigen different from the antigen targeted by CAR, the antibody can be used to save CAR-T antigen escape and reduce or prevent the recurrence or reappearance of common targeted tumors 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.

[0243] Thus, an embodiment of the present invention provides a derivative effector cell comprising, in addition to 41BB-ADR, exogenous CD16, and optionally CD38 as provided herein. KO and TCR KO In some embodiments, the CD16 contained in the derived effector cell is a hnCD16 comprising a CD16 extracellular domain, which CD16 extracellular domain comprises F176V and S197P. In some other embodiments, the hnCD16 contained in the derived effector cell comprises an extracellular domain derived from all or part of the length of CD64 as exemplified by SEQ ID NO: 9, 10 or 11; or may also comprise at least one of a non-native transmembrane domain, a stimulatory domain and a signaling domain. As explained herein, such derived effector cells have an acquired mechanism to target tumors with monoclonal antibodies mediated by ADCC, thereby enhancing the therapeutic effect of the antibody. As disclosed, the present application also provides a derived effector cell or a 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 below.

[0244] As disclosed herein, an iPSC line is provided, which comprises an exogenous polynucleotide encoding 41BB-ADR and optionally exogenous CD16, CD38 KO and endogenous TCR KO One or more of, wherein such cells may further comprise one or more additional engineering transformation patterns described herein and as shown in Table 2; and the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, including but not limited to immune effector cells, including but not limited to mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 + Hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, common myeloid progenitors, common lymphoid progenitors, erythrocytes, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional characteristics not present in primary NK, T and / or NKT cells.

[0245] Additionally provided herein is a master cell bank comprising single cell sorted and expanded clonally engineered iPSCs having at least one phenotype as provided herein, including but not limited to 41BB-ADR and optionally CD38 KO , endogenous TCR KOand one or more of exogenous CD16, 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, including but not limited to derived NK cells and T cells, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0246] 5. Chimeric Antigen Receptor (CAR) Expression

[0247] 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 region. 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 signal transduction domain, wherein the signal transduction 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-related antigen is a tumor antigen, wherein the tumor can 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, NK cells are derived from iPSCs comprising polynucleotides encoding CAR. In some embodiments, CAR has T cell specificity due to the inclusion of T cell-specific signaling components. In certain embodiments, T cells are derived from iPSCs comprising a polynucleotide encoding a CAR, and the derived T lineage cells may include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or a combination thereof.

[0248] In certain embodiments, the antigen recognition domain comprises a mouse antibody, a human antibody, a humanized antibody, a camel Ig, a single variable new antigen receptor (VNAR), a shark heavy chain antibody only (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. 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).

[0249] 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, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigens of cytomegalovirus (CMV) infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-3 (EGP-4), and leukocyte antigen (LEA). 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), MICA / B, MR1, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, NYESO1, 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. In still other embodiments, the CAR-T cell comprising an exogenous TCR complex further comprises a CAR specific for any one of MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Muc1, Muc16, CD19, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1.

[0250] In some embodiments, the present specification provides a CAR comprising an antigen recognition region targeting CD19 (referred to as "CD19-CAR"). Non-limiting examples of CAR are described in International Publication No. WO 2019 / 133969, which is incorporated herein by reference. In one embodiment, the CD19-CAR comprises a CD19 binding domain, which is represented by a heavy chain variable region with an amino acid sequence having at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85% or at least about 80% identity to SEQ ID NO: 24, and / or a light chain variable region represented by an amino acid sequence having at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85% or at least about 80% identity to SEQ ID NO: 25. In one embodiment, the CD19-CAR comprises a single chain variable fragment (scFV) having an amino acid sequence representation of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to SEQ ID NO: 26, wherein the linker and / or signal peptide are exemplary and are replaceable.

[0251] SEQ ID NO:24

[0252] EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS

[0253] (122 amino acids CD19 scFV heavy chain (HC))

[0254] SEQ ID NO:25

[0255] DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR

[0256] (108 amino acids CD19 scFV light chain (LC))

[0257] SEQ ID NO:26

[0258] MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSS GGGGSG GGGSGGGGS DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR

[0259] (263 amino acids CD19 scFv: signal peptide-HC- Connectors -LC)

[0260] In some embodiments, there is a spacer / hinge between the antigen recognition region and the transmembrane domain of CAR, although in some other embodiments, such a spacer / hinge is not required. Exemplary spacers that may be included in CAR are well known in the art, including but not limited to IgG4 spacers, CD28 spacers, CD8 spacers, or a combination of more than one spacer. The length of the spacer may also vary, from about 15 amino acids to about 300 amino acids or more. In the present application, for ease of description, a spacer of less than about 80 amino acids (e.g., 10-80 amino acids) is considered short; a spacer of about 80-180 amino acids is considered medium; and a spacer of more than 180 amino acids is considered long. Non-limiting exemplary spacer peptides include those spacer peptides represented by an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identity with any one of SEQ ID NO:3, 20, 21, 22 or 23.

[0261] SEQ ID NO:20

[0262] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0263] (39 amino acids)

[0264] SEQ ID NO:3

[0265] ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[0266] (88 amino acids)

[0267] SEQ ID NO:21

[0268] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLT

[0269] (89 amino acids)

[0270] SEQ ID NO:22

[0271] ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(129 amino acids)

[0272] SEQ ID NO:23

[0273] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0274] (229 amino acids)

[0275] 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.

[0276] In some embodiments, the signal transduction 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 2 containing transmembrane and immunoglobulin domains), CD3ζ (T cell surface glycoprotein CD3ζ chain; SEQ ID NO: 27), CD3ζ1XX (CD3ζ variant; SEQ ID NO: 30), CD3ζ2XX (CD3ζ variant; SEQ ID NO: 31), CD3ζ3XX (CD3ζ variant; SEQ ID NO: 32), CD3ζ4XX (CD3ζ variant; SEQ ID NO: 33), CD3ζ5XX (CD3ζ variant; SEQ ID NO: 34), CD3ζ6XX (CD3ζ variant; SEQ ID NO: 35), CD3ζ7XX (CD3ζ variant; SEQ ID NO: 36), CD3ζ9XX (CD3ζ variant; SEQ ID NO: 37), CD3ζ1XX (CD3ζ variant; SEQ ID NO: 38), CD3ζ1XX (CD3ζ variant; SEQ ID NO: 39), CD3ζ2XX (CD3ζ variant; SEQ ID NO: 40), CD3ζ1XX (CD3ζ variant; SEQ ID NO:28), DAP10 (hematopoietic cell signal 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-D type II integral membrane protein), NKp30 (natural cytotoxicity triggering receptor 3), NKp44 (natural cytotoxicity triggering receptor 2), NKp46 (natural cytotoxicity triggering receptor 1), CS1 (SLAM family member 7) and CD8 (T cell surface glycoprotein CD8α chain) full length or at least a portion of the polypeptide. Descriptions of exemplary signal transduction proteins, including transmembrane and cytoplasmic sequences of proteins suitable for incorporation into CARs, can be found in International Publication No. WO2021 / 077117, the entire disclosure of each of which is incorporated herein by reference).

[0277] SEQ ID NO:27

[0278] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0279] (113 amino acid CD3ζ cytoplasmic sequence)

[0280] SEQ ID NO:28

[0281] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0282] (112 amino acids CD3ζ1XX; contains 2 mutations in ITAM1)

[0283] In certain embodiments, the intracellular domain 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.

[0284] In one embodiment, a CAR suitable for cells provided herein comprises a costimulatory domain derived from CD28 and a signaling domain of an ITAM1 including a native or modified CD3ζ, the CD3ζ being 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 with SEQ ID NO: 12. In another embodiment, a CAR comprising a costimulatory 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 with SEQ ID NO: 13.

[0285] SEQ ID NO:12

[0286] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0287] (153 amino acids CD28 co-stimulatory + CD3ζ ITAM)

[0288] SEQ ID NO:13

[0289] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0290] (219 amino acids CD28 hinge + CD28 TM + CD28 co-stimulatory + CD3ζ ITAM)

[0291] In another embodiment, a CAR suitable for use in a cell provided herein comprises a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3 ζ, which CD3 ζ is represented by 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 SEQ ID NO: 14. The CAR comprising a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3 ζ may also comprise a CD8 hinge, wherein the amino acid sequence of such a structure is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 15.

[0292] SEQ ID NO:14

[0293] SNLFVASWIAVMIIFRIGMAVAIFCCFFFPSWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0294] (NKG2D of 263 amino acids TM +2B4+CD3ζ)

[0295] SEQ ID NO:15

[0296] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIF CCFFFPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0297] (308 amino acids, CD8 hinge + NKG2D TM +2B4+CD3ζ)

[0298] Non-limiting CAR strategies also include: heterodimers that conditionally activate CAR by dimerizing a pair of intracellular domains (see, e.g., U.S. Pat. No. 9,587,020); split CARs in which the antigen binding, hinge, and intracellular domains are homologously recombined to generate CARs (see, e.g., U.S. Publication No. 2017 / 0183407); multi-chain CARs that allow non-covalent attachment between two transmembrane domains that are respectively attached to an antigen binding domain and a signaling domain (see, e.g., U.S. Publication No. 2014 / 0134142); CARs that have bispecific antigen binding domains (see, e.g., U.S. Pat. No. 9,447,194), or a pair of antigen binding domains that recognize the same or different antigens or antigenic determinants (see, e.g., U.S. Pat. No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Pharmacol. 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.

[0299] In another embodiment, the invention comprises a CAR, a 41BB-ADR, and optionally a CD38 KO iPSCs and their derived effector cells have CAR inserted into the TCR constant region, resulting in endogenous TCR knockout (TCR KO ), thereby placing CAR expression under the control of the endogenous TCR promoter. Disruption of the constant region (TRAC or TRBC) of TCRα or TCRβ generates TCR negIn some other embodiments, the CAR inserted into the TCR constant region is specific for a tumor antigen comprising at least one of: MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Mucl, Mucl6, 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.

[0300] In addition, TCR expression was negative in NK lineage effector cells differentiated from iPSCs. neg The cells do not require HLA matching, have reduced alloreactivity, and can prevent GvHD (graft versus host disease) when used for allogeneic adoptive cell therapy. Additional insertion sites for CAR 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. In another embodiment, the immune cells, iPSCs and NK cells derived therefrom comprise a CAR, wherein the CAR is inserted into the NKG2A locus or the NKG2D locus, causing NKG2A or NKG2D knockout, thereby placing CAR expression under the control of the endogenously derived NKG2A or NKG2D promoter.

[0301] Thus, in addition to genetically engineered immune cells comprising functional patterns as provided herein, aspects of the present invention provide derived cells obtained from differentiated genome engineered iPSCs, wherein the iPSCs comprise an exogenous polynucleotide encoding 41BB-ADR and optionally exogenous CD16, CD38 KO and endogenous TCR KOOne or more of, and wherein the iPSC optionally further comprises one or more CARs and additional modified patterns, as discussed herein. The present application further provides a master cell bank comprising single cell sorted and expanded cloned engineered iPSCs, the iPSCs having at least an exogenous polynucleotide encoding 41BB-ADR and optionally exogenous CD16, CD38 KO , endogenous TCR KO and one or more of CAR, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products.

[0302] 6. Exogenously introduced cytokine signaling complexes

[0303] 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 achieved by retroviruses, Sendai viruses, adenoviruses, episomes, minicircles, or RNA (including mRNA)-borne expression constructs.

[0304] 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. For example, in embodiments where the signaling complex is used for IL15, the transmembrane (TM) domain may be native to the IL15 receptor, or may be modified or replaced by the transmembrane domain of any other membrane-bound protein. In various embodiments, the cytokine signaling complex comprises an IL15 receptor fusion (IL15RF), which comprises a full-length or partial-length IL15 and a full-length or partial-length IL15 receptor (IL15R). In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide to mimic 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 IL15's cis-presentation 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, simulating trans-presentation of IL15, maintaining IL15 membrane binding, and by additionally eliminating cis-presentation and / or any other potential signal transduction pathways mediated by normal IL15R through its intracellular domain. In other embodiments, IL15Rα is fused to IL15 without an intracellular domain (IL15Δ), as described in International Publication Nos. WO 2019 / 191495 and WO2019 / 126748, the entire disclosures of each of which are incorporated herein by reference.

[0305] In various embodiments, such truncated constructs comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 16. In one embodiment of the truncated IL15 / IL15Rα, the construct does not comprise the last 4 amino acid residues (KSRQ) of SEQ ID NO: 16, and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 17. In some embodiments, the construct comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct comprises the amino acid sequence of SEQ ID NO: 16.

[0306] SEQ ID NO:16

[0307] MDWTWILFLVAAATRVHSGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQ

[0308] (379 amino acids; signaling and linker peptides are underlined)

[0309] SEQ ID NO:17

[0310] MDWTWILFLVAAATRVHS GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYL

[0311] (375 amino acids; signaling and linker peptides are underlined)

[0312] 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 connector 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 by 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:18.

[0313] SEQ ID NO:18

[0314] MDWTWILFLVAAATRVHS GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0315] (242 amino acid signaling and linker peptides are underlined)

[0316] 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-representation. 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 referred to as common γ chain or CD132, also referred to 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.

[0317] 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. The transmembrane (TM) domain may be native to the IL7 receptor, or may be modified or replaced with the transmembrane domain of any other membrane-bound protein. In some embodiments, native (or wild-type) or modified IL7R may be fused to IL7 at the C-terminus via a linker (also referred to herein as "IL7RF") to achieve constitutive signaling and maintain membrane-bound IL7. In some embodiments, such constructs comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 19, wherein the transmembrane domain, signal peptide and linker are flexible and vary in length and / or sequence. In some embodiments, the construct comprises the amino acid sequence SEQ ID NO: 19.

[0318] SEQ ID NO:19

[0319] MDWTWILFLVAAATRVHSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNNKILMGTKEHS GGGSGGGGSGGGGSGGGGSGGGS LQESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKIDLTTIVK PEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMD PILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0320] (Signal peptide-IL7- Connectors -IL7R; transmembrane domain ( TM ), signal peptides and linkers may vary in length and sequence)

[0321] 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 another embodiment, an engineered IL7R that forms a homodimer in the absence of IL7 is also suitable for constitutive signaling of cytokines.

[0322] Those of ordinary skill in the art will appreciate that the above signal peptide and linker sequences are illustrative and in no way limit variants thereof useful as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those of ordinary skill in the art. Those of ordinary skill in the art understand that a signal peptide and / or linker sequence can replace another sequence without changing the activity of a functional peptide directed by a signal peptide or connected by a linker.

[0323] In iPSCs and their derivative cells comprising both CAR and exogenous cytokines and / or cytokine receptor signaling (cytokine signaling complex or "IL" in Table 2), CAR and IL can be expressed in separate constructs, or can be co-expressed in bicistronic constructs comprising both CAR and IL. In some other embodiments, the 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 IL signaling complex (e.g., IL7 signaling complex) and CAR can be in a single open reading frame (ORF). In one embodiment, the 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, where the transmembrane domain is anchored in the cell membrane. The CAR-2A-IL or IL-2A-CAR bicistronic design allows for coordinated expression of the CAR and IL signaling complexes 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 veined flat moth virus (TaV), and porcine Teschenovirus 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.

[0324] 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 and IL21.

[0325] In some embodiments, iPSCs and their derived effector cells comprising any of the genotypes in Table 2 may further comprise: disruption of at least one of TAP1, TAP2, TAP-related protein, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region; or HLA-E, 4-1BBL, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, TCR, Fc receptor, antibody and at least one of a surface triggering receptor for coupling to a bispecific, multispecific or universal adaptor.

[0326] Thus, in various embodiments, cytokines IL15 or IL7 and / or their receptors can be introduced into iPSCs using one or more of the above construct designs, and introduced into derived cells thereof after differentiation of the iPSCs. In addition, provided herein are induced pluripotent cells (iPSCs), cloned iPSCs, cloned iPS cell lines, or iPSC-derived cells comprising at least a polynucleotide encoding 41BB-ADR and optionally a CAR, exogenous CD16, a cytokine signaling complex, CD38 KO and endogenous TCR KO Also provided is a master cell bank comprising single cell sorted and expanded cloned engineered iPSCs having at least an exogenous polynucleotide encoding 41BB-ADR and optionally CAR, exogenous CD16 or its variant, CD38 KO , endogenous TCR KO and one or more of a cytokine signaling complex, wherein the cytokine signaling comprises a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface 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.

[0327] 6. CD58 and / or CD54 knockout

[0328] CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and promote cell (including immune cell) migration. CD58 knockout has been shown to be more efficient than CD54 knockout in reducing allogeneic NK cell activation; while double knockout of both CD58 and CD54 has the strongest reduction in NK cell activation. In some observations, double knockout of CD58 and CD54 is even more effective than HLA-G overexpression of HLA-I-deficient cells in overcoming the "self-deletion" effect.

[0329] As provided herein, in some embodiments, iPSCs and cells derived therefrom comprise 41BB-ADR and optionally CD38 KO and TCR KO In some other embodiments, the cell comprises an exogenous polynucleotide encoding 41BB-ADR and optionally CD38 KO and TCR KO One or both of the iPSCs and derived cells thereof are CD54 negative and CD58 negative, wherein the cells have improved resistance to host immune alloreactivity compared to cells without the exogenous polynucleotide. KO In some embodiments of iPSCs and cells derived therefrom containing exogenous CD16 or its variants, the cells are CD54 negative and / or CD58 negative. KO 、TCR KO In still other embodiments of iPSCs and cells derived therefrom containing exogenous CD16 or its variants, IL and CAR, the cells are CD58 negative and / or CD54 negative.

[0330] In some embodiments, the surface proteins upregulated in activated recipient immune cells include but are not limited to CD38, CD25, CD69, CD44, 4-1BB, OX40 or CD40L. When the cell expresses this inactivated CAR, it is preferred that the cell does not express or knock out the same surface protein targeted by CAR. In some embodiments, the inactivated CAR includes at least one of CD38-CAR, CD25-CAR, CD69-CAR, CD44-CAR, 41BB-CAR, OX40-CAR and CD40L-CAR.

[0331] The present application further provides a master cell bank comprising single cell sorted and expanded cloned engineered iPSCs having at least one phenotype as provided herein, including but not limited to 41BB-ADR and optionally exogenous CD16, CD38 KO , endogenous TCRKO , CAR and one or more of CD58 negative and / or CD54 negative, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derived NK cells and T cells, which are well-defined and uniform in composition and can be mass-produced in a cost-effective manner.

[0332] 7. Genetically engineered iPSC lines and derived cells provided herein

[0333] According to the above content, the present application provides iPSC, iPS cell line cells or a population thereof, and derived effector cells differentiated from iPSC, wherein each cell at least comprises a polynucleotide encoding 41BB-ADR and optionally CD38 KO 、TCR KO , exogenous CD16, CAR, cytokine signaling complex and CD58 negative and / or CD54 negative one or more, wherein the cell is a eukaryotic cell, an animal cell, a human cell, an induced pluripotent cell (iPSC), an iPSC-derived effector cell, an immune cell or a feeder cell. A master cell bank is also provided, which comprises a cloned engineered iPSC having a single cell sorting and amplification of a phenotype as described herein, wherein the cell bank provides a renewable source for making 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. In some embodiments, iPSC-derived cells are hematopoietic cells, including but not limited to mesoderm 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, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, bone marrow cells, neutrophil progenitor cells and / or with T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells and macrophages in common. In some embodiments, functional derived hematopoietic cells include effector cells, which have one or more functional features, and the one or more functional features are not present in corresponding primary T cells, NK cells, NKT cells and / or B cells.

[0334] iPSC-derived effector cells comprising 41BB-ADR can be used to overcome alloreactivity in an immunocompetent host system, because 4-1BB is upregulated on activated effector cells (including alloreactive NK and T cells). In some embodiments, targeting has activated T cells with upregulated expression of 4-1BB, which is opposite to unactivated T cells. In order to target these activated T cells, any suitable ligand of 4-1BB (including 4-1BBL, antibodies targeting 4-1BB (or their functional fragments), Fc and 4-1BBL fusions or their functional derivatives or fragments) can be used for ADR.

[0335] Also provided are iPSCs and effector cells derived therefrom comprising a polynucleotide encoding 41BB-ADR, wherein the cells further comprise a TCR KO In some embodiments, such cells have reduced alloreactivity and are able to prevent GvHD (graft versus host disease) when used in allogeneic adoptive cell therapy.

[0336] Also provided are iPSCs and effector cells derived therefrom comprising a polynucleotide encoding 41BB-ADR, wherein the cells further comprise CD38 KO And optional TCR KO and exogenous CD16. In the presence of an anti-CD38 therapeutic agent, which can be an anti-CD38 antibody, comprising CD38 KO The effector cells have increased persistence and / or survival in vivo. In some embodiments, the effector cells include T cells. KO and TCR KO iPSC-derived T cells undergo reduced cell depletion in the presence of anti-CD38 antibodies; ADCC is acquired, thereby providing an additional mechanism for T cell-mediated tumor killing. In some embodiments, the effector cells include NK cells. KO iPSC-derived NK cells with exogenous CD16 have enhanced cytotoxicity and reduced NK cell cannibalism in the presence of anti-CD38 antibodies. In some embodiments, when anti-CD38 antibodies are used to induce CD16-mediated enhanced ADCC, iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing effector cell elimination, i.e., reduction or depletion of effector cells expressing CD38, thereby increasing the retention and / or survival of iPSCs and their effector cells, and delaying host immune reconstitution.

[0337] Also provided are iPSCs comprising a polynucleotide encoding 41BB-ADR, and optionally CD38 knockout, TCR KOOne or more of and a polynucleotide encoding at least one cytokine signaling complex (IL) to achieve cytokine signaling that promotes cell survival, retention and / or amplification, wherein the iPSC line can be directed to differentiate to produce functional derived hematopoietic cells with improved survival, retention, amplification and effector cell function. In various embodiments, the exogenously introduced cytokine signaling includes any one, two or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18 and IL21. In some embodiments, the introduced cytokine signaling complex is expressed on the cell surface. In some embodiments, 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, the transient / temporary expression of cell surface cytokines / cytokine receptors is by retrovirus, Sendai virus, adenovirus, episome, small ring or RNA including mRNA. In some embodiments, the exogenous cell surface cytokine signaling complex contained in the iPSC or its derivative cells as described herein achieves IL7 signaling. In some embodiments, the exogenous cell surface cytokine signaling complex contained in the iPSC or its derivative cells as described herein achieves IL10 signaling. In some embodiments, the exogenous cell surface cytokine signaling complex contained in the iPSC or its derivative cells as described herein achieves IL15 signaling. Therefore, the iPSC and its derivative cells according to some embodiments are able to autonomously maintain or improve cell growth, proliferation, expansion and / or effector function without additional in vitro or in vivo contact with the supplied soluble cytokines. In some embodiments, the iPSC and its derivative cells comprising 41BB-ADR and optionally CD38 KO 、TCR KO iPSCs and their derived effector cells, and any other genetic forms described herein, are intact in HLA-I and / or HLA-II and have synergistically increased persistence and / or survival in the presence of activated recipient T, B and NK cells.

[0338] Also provided is an iPSC comprising an exogenous polynucleotide encoding 41BB-ADR and, optionally, CD38 KO 、TCR KO And any other gene form described herein, wherein the cell optionally further comprises a polynucleotide encoding HLA-G, but does not comprise a B2M knockout and / or a CIITA knockout, wherein the iPSC is capable of directed differentiation to produce functional derived hematopoietic cells. The CD38-CAR / CD38 - / - HLA-I WT or HLAWT iPSCs and their derived effector cells can be used with anti-CD38 antibodies to induce ADCC without causing effector cell elimination or allogeneic rejection by activated recipient T, B and NK cells, thereby increasing the persistence and / or survival of iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or survival in vivo.

[0339] Another aspect provided herein includes iPSCs or iPSC-derived cells comprising a polynucleotide encoding 41BB-ADR and optionally CD38 KO 、TCR KO And any other gene form described herein, including a truncated fusion protein of IL15 and IL15Rα, wherein the fusion protein does not comprise an intracellular domain. Also referred to as "IL15Rα (ΔICD) fusion" and "IL5 / mb-Sushi", these embodiments are further collectively referred to as "IL15Δ" in Table 2 and throughout this application. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 16, 17 or 18. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO:18. In yet other embodiments, the iPSCs or iPSC-derived cells comprising a truncated IL15 / IL15Rα fusion protein lacking the intracellular domain (IL15Δ) further comprise one or more of the following: 41BB-ADR, TCR knockout, CD38 knockout, exogenous CD16, an exogenous cytokine signaling complex, and CAR, wherein the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, and wherein the derived hematopoietic cells 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 (MPPs), T cell progenitors, NK cell progenitors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, bone marrow cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.

[0340] Therefore, the present application provides iPSC and its functional derivative effector cells, which include any of the following genotypes in Table 2. Unless specified as IL15Δ, which is described above as a truncated fusion protein of IL15 and IL15Rα but without an intracellular domain, it depends on which specific cytokine / receptor expression is selected, as provided in Table 2. "IL" means any one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18 and IL21. In addition, when iPSC and its functional derivative effector cells have a genotype comprising both CAR and IL, CAR and IL may be included in a bicistronic expression cassette comprising a 2A sequence. In contrast, in some other embodiments, CAR and IL are in separate expression cassettes contained in iPSC and its functional derivative hematopoietic cells. In a specific embodiment, the iPSCs and their functionally derived effector cells comprise a CAR and an IL, the IL being IL15, wherein the IL15 construct is contained in an expression cassette with the CAR or separate from the CAR.

[0341] Table 2: Suitable exemplary genotypes of provided cells :

[0342]

[0343]

[0344] 8. Additional Modifications

[0345] In some embodiments, the gene modification pattern further comprises one or more of the following: 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 iPSCs or their derivative cells. In some embodiments, the genetically modified iPSCs and their derivative cells comprise the genotypes listed in Table 2. In some embodiments, the iPSCs and their derived effector cells comprising any of the genotypes in Table 2 may additionally comprise: TAP1, TAP2, TAP-related proteins, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and at least one of the genes in the chromosome 6p21 region that are disrupted; or CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, Fc receptor, adapter and at least one of a surface triggering receptor for coupling with a bispecific, multispecific or universal adapter.

[0346] Adaptor is a fusion protein composed of two or more single-chain variable fragments (scFv) of different antibodies, 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. 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. Therefore, the 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.

[0347] In some embodiments, the adapter is used in combination with an effector cell population as described herein by simultaneous or continuous administration, wherein the effector cell comprises a surface molecule or surface triggering receptor recognized by the adapter. In some other embodiments, the adapter is a bispecific antibody expressed by a derived effector cell through genetic engineering iPSC as described herein, and then directed differentiation of engineered iPSC. Exemplary effector cell surface molecules or surface triggering receptors that can be used for bispecific or multispecific adapter recognition or coupling include but are not limited to CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C and chimeric Fc receptors as disclosed herein. As described herein, in some embodiments, the exogenous CD16 expressed on the surface of the derived effector cell for adapter recognition is hnCD16, which comprises CD16 (containing F176V and optional S197P) or CD64 extracellular domains as described herein and native or non-native transmembrane domains, stimulatory domains and / or signaling domains. In some embodiments, the exogenous CD16 expressed on the surface of the effector cell for adaptor recognition is a chimeric Fc receptor (CFcR) based on CD16. In some embodiments, the CFcR based on CD16 comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ; wherein the extracellular domain of the exogenous CD16 is derived from the full length or partial sequence of the extracellular domain of CD64 or CD16; wherein the extracellular domain of CD16 comprises F176V and optionally S197P.

[0348] 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, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. In one embodiment, the bispecific adapter is a bispecific antibody specific for CD3 and CD19 (CD3-CD19). In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In another embodiment, the bispecific antibody is CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody is CD3-CD33.

[0349] 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 effector cell expansion (referred to as TriKE or tri-specific killing adapter in some publications). In one embodiment, TriKE is CD16-IL15-EPCAM or CD64-IL15-EPCAM. In another embodiment, TriKE is CD16-IL15-CD33 or CD64-IL15-CD33. In another embodiment, TriKE is NKG2C-IL15-CD33 ("2C1533"). In addition to IL15, cytokines suitable for inclusion in TriKE include but are not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18 and IL21.

[0350] 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 effector cells, i.e., by additionally engineering iPSCs comprising the genotype listed in Table 2, and then directing the differentiation of the iPSCs toward T cells, NK cells, or any other effector cells comprising the same genotype and surface triggering receptor as the source iPSC.

[0351] 9. Antibodies for immunotherapy

[0352] In some embodiments, in addition to the effector cells of genome engineering as provided herein, other therapeutic agents comprising antibodies or antibody fragments targeting antigens associated with the patient's condition, disease or indication can be used together with these effector cells in combination therapy. In some embodiments, antibodies are used in combination with effector cell colonies as described herein by being administered to subjects simultaneously or continuously. In other embodiments, such antibodies or fragments thereof can be expressed by effector cells by genetically engineering iPSC using exogenous polynucleotide sequences encoding the antibodies or fragments thereof and guiding the differentiation of engineered iPSC. In some embodiments, effector cells express exogenous CD16 variants, wherein the cytotoxicity of effector cells is enhanced by antibodies via ADCC.

[0353] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, antibodies suitable for combination therapy as an additional therapeutic agent with the administered iPSC-derived effector cells include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, utoximab, okatuzumab, obtuzumab, isobetuzumab, ocrelizumab), anti-CD22 (inotuzumab, mosetuzumab, epratuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (cetuximab), anti-GD2 (detuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (etuzumab); and humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof. In some embodiments, antibodies suitable for combined treatment with iPSC-derived effector cells administered as additional therapeutic agents also include bispecific or multispecific antibodies, which target more than one antigen or epitope on the target cell, or recruit effector cells (e.g., T cells, NK cells, or macrophages) to the target cell while targeting the target cell. Such bispecific or multispecific antibodies serve as adapters that 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 antibody therapy.

[0354] In some embodiments, antibodies suitable for combined treatment with iPSC-derived effector cells administered as additional therapeutic agents also include bispecific or multispecific antibodies, which target more than one antigen or epitope on the target cell, or recruit effector cells (e.g., T cells, NK cells, or macrophages) to the target cell while targeting the target cell. Such bispecific or multispecific antibodies act as effector cells, whether adjacent immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils in therapy receptors) or engineered effector cells in therapeutic compositions, to tumor cells, and activate the adapter of immune effector cells when tumor antigens bind, and have shown great potential for maximizing the benefits of antibody therapy. The adapter is specific to at least one tumor antigen and specific to at least one surface triggering receptor of an immune cell, which can provide a multi-targeting method for the engineered effector cells disclosed herein to solve tumor antigen escape and tumor heterogeneity. Examples of adaptors include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs), or multispecific killer cell engagers, or universal adaptors that are compatible with multiple immune cell types.

[0355] 10. Checkpoint inhibitors

[0356] Checkpoints are cell molecules, usually cell surface molecules, that can inhibit or downregulate immune responses when not inhibited. It is now clear that tumors select certain immune checkpoint pathways as the primary mechanism of immune resistance, especially for T cells specific to tumor antigens. Checkpoint inhibitors (CI) 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, and these agents provide long-term relief for a variety of indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a major problem. Therefore, one aspect of the present application provides a method of overcoming CI resistance by including a functional iPSC-derived cell engineered as provided herein in a combination therapy with CI. In one embodiment of the combination therapy described herein, the iPSC-derived cell is a NK cell. In another embodiment of the combination therapy described herein, the iPSC-derived cell is a T cell. In addition to showing direct anti-tumor ability, the derived NK cells provided herein also show resistance to PDL1-PD1-mediated inhibition, and have the ability to enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at the tumor site. Therefore, the tumor infiltration of T cells promoted by functionally effective genome-engineered derived NK cells indicates that the NK cells can synergize with T cell-targeted immunotherapy (including checkpoint inhibitors) to relieve local immunosuppression and reduce tumor burden.

[0357] In some embodiments of the combination therapy, the check point inhibitor is used in combination with the effector cell population described herein by simultaneously or continuously administering the effector cell population to the subject. In some other embodiments, the check point inhibitor is expressed by effector cells by genetically engineering iPSC using an exogenous polynucleotide sequence encoding the check point inhibitor or a fragment or variant thereof and guiding the differentiation of the engineered iPSC. Some embodiments of the combination therapy with effector cells described herein include at least one check point inhibitor to target at least one check point molecule; wherein the effector cell has a genotype listed in Table 2.

[0358] In another embodiment, iPSC-derived effector cells for checkpoint inhibitor combination therapy comprise 41BB-ADR, and optionally TCR KO, CD38 knockout, exogenous CD16 expression, CAR expression and one or more of exogenous cell surface cytokine signaling complexes. In some embodiments, the derived T cells comprise any of the genotypes listed in Table 2. In some embodiments, the derived T cells further comprise at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3 and TIGIT, deletion, destruction or reduced expression; or CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptor, adapter, and at least one of a surface triggering receptor for coupling to a bispecific adapter, a multispecific adapter, or a universal adapter are introduced or the expression is increased.

[0359] In various embodiments, the derived effector cells are obtained from differentiated iPSC or iPS clonal cell line cells, which contain a polynucleotide encoding 41BB-ADR and optionally a TCR KO , CD38 knockout, exogenous CD16, CAR and one or more of exogenous cell surface cytokine signaling complexes. In some embodiments, the iPSC or iPS clonal cell line cells described above further comprise at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3 and TIGIT deletion, destruction or reduced expression; or CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, CAR, TCR, Fc receptor, adapter and at least one of a surface triggering receptor for coupling with a bispecific adapter, a multispecific adapter or a universal adapter, or the introduction or increased expression of the same.

[0360] Checkpoint inhibitors suitable for combination therapy with derived NK cells or T cells as provided herein include, but are not limited to, PD-1 (Pdcd1, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (CD223), CTLA-4 (CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A 2AR, 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).

[0361] 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 single variable new antigen receptor (VNAR), a shark antibody with only a heavy chain (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 (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR antibody), IPH43 (anti-MICA antibody), IPH33 (anti-TLR3 antibody), lirelumab (anti-KIR antibody), monalizumab (anti-NKG2A antibody), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and any derivatives, functional equivalents, or biosimilars thereof.

[0362] 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.

[0363] Some embodiments of combined therapy with the provided derived NK or T cells include at least one check point inhibitor to target at least one check point molecule; wherein the derived cells have the genotype listed in Table 2. Some other embodiments of the combined therapy with the provided derived NK cells or T cells include two, three or more check point inhibitors, so as to target two, three or more check point molecules. In some embodiments of the combined therapy comprising at least one check point inhibitor and a derived cell with a genotype listed in Table 2, the check point inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the check point inhibitor is produced by a derived cell by expressing an exogenous polynucleotide sequence encoding the antibody or its fragment or variant. In some embodiments, an exogenous polynucleotide sequence encoding an antibody or its fragment or variant that inhibits a check point is co-expressed with CAR in a separate construct or in a bicistronic construct comprising both CAR and a sequence encoding an antibody or its fragment. In some other embodiments, the sequence encoding the antibody or its fragment can be connected to the 5' end or 3' end of the CAR expression construct by a self-cleavage 2A coding sequence, which is described as, for example, CAR-2A-CI or CI-2A-CAR. Therefore, the coding sequences of the checkpoint inhibitor and CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered, expressed, and secreted as a payload by a derived effector cell capable of infiltrating the tumor microenvironment (TME), it counteracts the inhibitory checkpoint molecule after engaging the TME, allowing the effector cell to be activated by, for example, an activation mode of a CAR or an activating receptor. In some embodiments, the checkpoint inhibitor co-expressed with CAR inhibits at least one of the following checkpoint molecules: PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AR, 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 2 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.

[0364] In some other embodiments of the combination therapy comprising iPSC-derived cells provided herein and at least one antibody that inhibits a checkpoint molecule, the antibody is not produced by or in an iPSC-derived cell, and is administered before, simultaneously with, or after administering a derivative cell having a genotype listed in Table 2. In some embodiments, administration of one, two, three or more checkpoint inhibitors in the combination therapy with the provided derivative effector cells is simultaneous or sequential. In one embodiment of the combination therapy comprising a derivative NK cell or T cell having a genotype listed in Table 2, the checkpoint inhibitor included in the treatment is one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments of the combination therapy comprising derived NK cells or T cells having a genotype listed in Table 2, the checkpoint inhibitor included in the treatment is atezolizumab or a humanized or Fc-modified variant, fragment, or functional equivalent thereof, or biosimilar thereof. In some embodiments of the combination therapy comprising derived NK cells or T cells having a genotype listed in Table 2, the checkpoint inhibitor included in the treatment is nivolumab or a humanized or Fc-modified variant, fragment, or functional equivalent thereof, or biosimilar thereof. In some embodiments of the combination therapy comprising derived NK cells or T cells having a genotype listed in Table 2, the checkpoint inhibitor included in the treatment is pembrolizumab or a humanized or Fc-modified variant, fragment, or functional equivalent thereof, or biosimilar thereof.

[0365] II. Methods for Targeted Genome Editing at Selected Loci in iPSCs

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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 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.

[0370] 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, homing endonucleases and DICE (double integrase cassette exchange) systems utilizing phiC31 and Bxb1 integrases are also promising tools for targeted integration.

[0371] 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. A "selected" zinc finger domain is a domain not found in nature, and its production is mainly 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.

[0372] 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.

[0373] 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.).

[0374] 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.

[0375] 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.

[0376] 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.

[0377] 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 desired integration site in the genome. See, e.g., U.S. Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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 one embodiment, the endogenous gene comprising the targeted insertion / deletion is associated with immune response regulation and regulation. In some other embodiments, the endogenous gene comprising the targeted insertion / deletion is associated with the following: a targeting mode, a receptor, a signaling molecule, a transcription factor, a candidate drug target, immune response regulation and regulation, or a protein that inhibits transplantation, transport, homing, vitality, self-renewal, retention and / or survival of stem cells and / or progenitor cells and their derived cells.

[0382] Therefore, another aspect of the present invention provides a method for targeted integration in a selected locus, the selected locus includes a genomic safe harbor or a preselected locus known or confirmed to be safe and fully regulated to achieve continuous or temporary gene expression, such as B2M, TAP1, TAP2, Tap-related protein, TRAC or CD38 locus as provided herein. In one embodiment, the genomic safe harbor used in the targeted integration method comprises one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR or RUNX1, or other loci that meet the 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 the cell, and introducing a construct comprising a pair of homology arms specific to a desired integration site and one or more exogenous sequences to achieve site-directed homologous recombination by the cell host enzyme machinery, 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 β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0383] 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 β 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, 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 β 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, TCR α or β 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 β 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.

[0384] 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 antigenic determinants 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.

[0385] 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 disclosed herein include, but are not limited to, cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0386] 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.

[0387] 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 configuration. 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.

[0388] 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 the backbone of a viral vector such as an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a slow viral vector or a Sendai viral vector. In some embodiments, a plasmid vector is used to deliver exogenous polynucleotides to a target cell and / or to express exogenous polynucleotides in a target cell (e.g., pAl-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo) etc. In some other embodiments, an episomal vector is used to deliver exogenous polynucleotides to a target cell. In some embodiments, a recombinant adeno-associated virus (rAAV) can be used for genetic engineering to introduce insertions, deletions or substitutions by homologous recombination. Unlike slow viruses, rAAV is not integrated into the host genome. In addition, the episomal rAAV vector mediates homologous directional genes, which are targeted with a much higher ratio than 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 2.

[0389] III. Methods for obtaining and maintaining genome-engineered iPSCs

[0390] In one aspect, the present invention also provides a method for obtaining and maintaining genome engineering iPSC, the method includes one or more targeted editing (for example, multiple genome engineering) 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 derived cells into the genome (i.e., introducing targeted integration and / or insertion / deletion at selected sites). Compared with the primary effector cells of peripheral blood origin of direct engineering patient sources, many benefits of iPSC-derived effector cells of genome engineering 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 a variety of engineered 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 engineered iPSC as provided herein.

[0391] In some embodiments, iPSCs comprising genome engineering for one or more targeted editing at one or more selected sites are maintained, passaged and expanded for a long time in a cell maintenance medium (FMM) as a single cell, wherein iPSCs retain targeted editing and functional modification at selected sites. iPSCs cultured in FMM have been shown to continue to maintain their undifferentiated and basic or initial characteristics; provide genome stability without the need for culture cleaning or selection; and differentiate via in vitro embryoid bodies or monolayers (without forming embryoid bodies); and differentiation of teratoma formation in vivo easily produces all three somatic cell lineages. See, for example, International Publication No. WO2015 / 134652, the disclosure of which is incorporated herein by reference.

[0392] In some embodiments, genome-engineered iPSCs comprising one or more targeted integrations and / or insertions / deletions are maintained, passaged, and expanded in culture medium (FMM) 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.

[0393] 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.

[0394] 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 the 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).

[0395] In some embodiments, reprogramming is initiated by contacting a non-pluripotent cell with at least one reprogramming factor and an optional combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. In some embodiments, the genome-engineered iPSC produced by any of the above methods is further maintained and amplified using a mixture comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor.

[0396] In some embodiments of the method for generating genome-engineered iPSC, the method includes: genome engineering iPSC by introducing one or more targeted integrations and / or insertions / deletions into iPSC to obtain genome-engineered iPSC with genotype provided herein. Alternatively, the method for generating genome-engineered iPSC includes: (a) introducing one or more targeted edits into non-pluripotent cells to obtain genome-engineered non-pluripotent cells comprising targeted integration and / or insertions / deletions at selected sites, and (b) contacting genome-engineered non-pluripotent cells with one or more reprogramming factors and optional small molecule compositions to obtain genome-engineered iPSC comprising targeted integration and / or insertions / deletions at selected sites, the small molecule compositions comprising TGFβ receptor / ALK inhibitors, MEK inhibitors, GSK3 inhibitors and / or ROCK inhibitors. 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, and / or screening off-target editing and abnormal karyotypes in genome-engineered 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.

[0397] 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 disclosures of which are incorporated herein by reference. One or more reprogramming factors may be in the form of polypeptides. Reprogramming factors may also be in the form of polynucleotides 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.

[0398] In some embodiments, non-pluripotent cells are transferred by a variety of vectors for targeted integration at the same or different selected sites using a variety of constructs comprising different exogenous polynucleotides and / or different promoters. 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 polynucleotide is 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 transferred to non-pluripotent cells simultaneously or continuously. The non-pluripotent cells undergoing the targeted integration of multiple constructs can contact one or more reprogramming factors simultaneously to start reprogramming with genetic engineering, thereby obtaining the iPSC of genome engineering comprising multiple targeted integrations in the same cell pool. Therefore, this stable method realizes simultaneous reprogramming and engineering strategies, thereby obtaining the iPSC of genome engineering with multiple modes of cloning integrated into one or more selected targets.

[0399] IV. Methods for obtaining genetically engineered effector cells by differentiating genome-engineered iPSCs

[0400] 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, the insertion / deletion is included in one or more endogenous genes, which include but are not limited to at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tap-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β 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.

[0401] In some embodiments, the genome-engineered iPSCs comprising one or more genetic modifications as provided herein are used to derive hematopoietic cell lineages or any other specific cell type in vitro, wherein the derived non-pluripotent cells retain functional genetic modifications, including targeted editing at selected sites. In some embodiments, the genome-engineered iPSCs used to derive hematopoietic cell lineages or any other specific cell type in vitro are master cell bank cells that are cryopreserved and thawed just before their use. In one embodiment, the genome-engineered iPSC-derived cells include, but are not limited to, mesodermal cells with permanent hemogenic endothelial (HE) potential, permanent HE, CD34 + Hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells and macrophages, wherein cells derived from genome-engineered iPSCs retain functional genetic modifications, including targeted editing at desired sites.

[0402] 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.

[0403] 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.

[0404] 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.

[0405] 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 realizes the 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 carried out using the method provided herein produces functional hematopoietic lineage cells, which realizes 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, derived T lineage cells, derived NKT lineage cells, derived NK lineage cells, derived B lineage cells, T cells, NK cells, NKT cells, B cells, macrophages and neutrophils.

[0406] The method for directed differentiation of pluripotent stem cells into definitive hematopoietic lineage cells comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting the 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 the mesodermal cells, wherein the composition is optionally free of a TGFβ receptor / ALK inhibitor; (iii) contacting the 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.

[0407] 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.

[0408] 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 - .

[0409] 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 + .

[0410] 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.

[0411] In some embodiments, the genome-engineered iPSC-derived cells obtained by the above method contain 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 β 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 include polynucleotides encoding the following: safety switch proteins, targeting patterns, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or 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 iPSC-derived cells comprising one or more suicide genes also include one or more insertions / deletions contained in one or more endogenous genes, which are related to immune response regulation and mediation, including but not limited to checkpoint genes, endogenous T cell receptor genes and MHC class I inhibitory genes. In one embodiment, the genome-engineered iPSC-derived cells comprising one or more suicide genes also include insertions / deletions in the B2M gene, wherein B2M is knocked out.

[0412] In addition, methods and compositions applicable to dedifferentiation of genome-engineered hematopoietic cells of a first fate to genome-engineered hematopoietic cells of a 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 for partial reprogramming of starting non-pluripotent cells 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.

[0413] V. Therapeutic Use of Immune Cells Derived from Genetically Engineered iPSCs with Functional Patterns

[0414] In some embodiments, the present invention provides a composition comprising an isolated population or subpopulation of functionally enhanced derived immune cells differentiated from genome-engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSCs of the composition comprise one or more targeted gene edits as disclosed herein, which can be retained in iPSC-derived effector cells, wherein the genetically engineered iPSCs and their derived cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells of the composition comprises iPSC-derived CD34 + Cells. In one embodiment, the isolated population or subpopulation of the genetically engineered effector cells of the composition comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of the genetically engineered effector cells of the composition comprises iPSC-derived proT cells or T cells. In one embodiment, the isolated population or subpopulation of the genetically engineered effector cells of the composition comprises iPSC-derived proNK cells or NK cells. In one embodiment, the isolated population or subpopulation of the genetically engineered effector cells of the composition comprises iPSC-derived immunoregulatory cells or bone marrow-derived suppressor cells (MDSC).

[0415] In some embodiments of the composition, the genetically engineered effector cells derived from iPSC are further regulated in vitro to improve therapeutic potential. In one embodiment of the composition, the isolated population or subpopulation of the genetically engineered effector cells derived from iPSC comprises an increased number or ratio of initial T cells, stem cell memory T cells and / or central memory T cells. In one embodiment of the composition, the isolated population or subpopulation of the genetically engineered effector cells derived from iPSC comprises an increased number or ratio of type I NKT cells. In another embodiment of the composition, the isolated population or subpopulation of the genetically engineered effector cells derived from iPSC comprises an increased number or ratio of adaptive NK cells. In some embodiments of the composition, the CD34 derived from the genetically engineered iPSC + The isolated population or subpopulation of cells, HSC cells, T cells, NK cells or myeloid-derived suppressor cells is allogeneic. In some other embodiments of the composition, the genetically engineered CD34 derived from iPSCs + The isolated population or subpopulation of T cells, HSC cells, T cells, NK cells or MDSC cells is autologous.

[0416] In some embodiments of the composition, the iPSCs used for differentiation comprise a genetic imprint selected to convey a desired therapeutic property in the derived effector cells, with the proviso that the cell developmental biology is not disrupted during differentiation, and with the proviso that the genetic imprint is retained and functional in the differentiated hematopoietic cells derived from said iPSCs.

[0417] In some embodiments of the composition, the genetic imprint in the pluripotent stem cells comprises (i) one or more gene modification patterns acquired by genomic insertions, deletions, or substitutions in the pluripotent cell genome 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.

[0418] In some embodiments o...

Claims

1. A cell or a population thereof, wherein (i) the cell is (a) an immune cell; (b) an induced pluripotent cell (iPSC); or (c) a derivative cell obtained by differentiating the iPSC; and (ii) the cell comprises: (a) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); and Optional (b) One or both of CD38 knockout and endogenous TCR knockout.

2. The cell or population of claim 1, wherein the cell has improved resistance to host immune alloreactivity compared to cells not containing the exogenous polynucleotide.

3. The cell or population thereof according to claim 1, wherein (i) the iPSC is a clonal iPSC, a single-cell dissociated iPSC, an iPSC cell line cell, or an iPSC master cell bank (MCB) cell; (ii) the derived cells include 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 lineage cells, derived NKT lineage cells, derived NK lineage cells or derived B lineage cells; Or (iii) the derived cells include 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.

4. The cell or population thereof of claim 1, wherein the ADR is specific for 41BB or CD38.

5. The cell or population thereof according to claim 3, wherein the ADR comprises: (i) a 41BB-specific ligand operably linked to a signaling domain that promotes effector cell activation; or (ii) a CD38 binding domain that can be operably linked to a signaling domain that promotes effector cell activation.

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

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

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

9. The cell or population thereof of claim 8, wherein the one, two, three or more co-stimulatory domains are from the intracellular signaling domain of CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM or CD40.

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

8.

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

12. The cell or population thereof of claim 11, wherein the cell has therapeutic properties including one or more of the following compared to a corresponding primary cell of the cell obtained from peripheral blood, umbilical cord blood, or any other donor tissue without the same gene editing: (i) increased cytotoxicity; (ii) improved retention and / or survival; (iii) enhanced ability to mobilize neighboring immune cells to the tumor site and / or activate or recruit neighboring immune cells; (iv) improved tumor penetration; (v) enhanced ability to reduce tumor immunosuppression; (vi) improved ability to rescue tumor antigen escape; (vii) controlled apoptosis; (viii) enhanced or acquired ADCC; and (ix) The ability to avoid cannibalism.

13. The cell or population thereof according to claim 11, wherein the exogenous CD16 or variant thereof comprises at least one of the following: (a) Non-cleavable high-affinity CD16 (hnCD16); (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.

14. The cell or population thereof according to any one of claims 11 to 13, 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 another CAR; (ix) 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 a separate construct or in a bicistronic construct; (x) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct; (xi) is specific for at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1; and / or (xii) 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, CLEC12A, an antigen of cytomegalovirus (CMV) infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase erb B2,3,4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit 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, melanin Tumor antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGFR2), Wilms tumor protein (WT-1) and pathogen antigens; and optionally, wherein the CAR of any one of (i) to (xii) is inserted at the TCR locus and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by the insertion of the CAR.

15. The cell or population thereof according to claim 11, wherein the cytokine signaling complex: (a) a partial or complete peptide comprising at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and their corresponding receptors; or (b) includes at least one of the following: (i) Co-expression of IL15 and IL15Rα by using a self-cleaving peptide; (ii) fusion protein of IL15 and IL15Rα; (iii) IL15 / IL15Rα fusion protein with truncated intracellular domain of IL15Rα; (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) includes 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) a homodimer of IL7Rβ, wherein any of (c)(i)-(iii) is optionally co-expressed with CAR in a separate construct or in a bicistronic expression cassette; and optionally, (d) Transient expression.

16. The cell or population thereof according to claim 1, wherein the cell is a NK lineage cell or a T lineage cell, wherein: (i) the NK lineage cells or the T lineage cells have improved infiltration and / or retention at the tumor site; (ii) the NK lineage cells are capable of recruiting T cells and / or migrating the T cells to the tumor site; or (iii) the NK lineage cells or the T lineage cells are capable of reducing tumor immunosuppression in the presence of one or more checkpoint inhibitors.

17. The cell or population thereof according to claim 14 or 16, wherein the checkpoint inhibitor is an antagonist against one or more checkpoint molecules, wherein the one or more checkpoint molecules include 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, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR.

18. The cell or population thereof according to claim 17, wherein the checkpoint inhibitor comprises: (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.

19. The cell or population thereof according to any one of claims 1 to 18, wherein the cell comprises: (i) one or more exogenous polynucleotides integrated into a safe harbor locus or a selected locus; or (ii) more than two exogenous polynucleotides integrated in different safe harbor loci or two or more selected loci.

20. The cell or population thereof of claim 19, wherein the safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1; or wherein the selected locus is one of B2M, TAP1, TAP2, TAP-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT; and / or wherein integration of the exogenous polynucleotide knocks out expression of the gene in the locus.

21. The cell or population thereof according to claim 20, wherein the TCR locus is the constant region of TCRα and / or TCRβ.

22. The cell or population thereof of claim 1, wherein the cell comprises: (i) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); (ii) CD38 knockout and exogenous CD16 or its variants; and (iii) TCR knockout; and wherein the cell or population thereof has improved resistance to host immune alloreactivity compared to a cell not possessing all of (i), (ii) and (iii).

23. The cell or population thereof of claim 1, wherein the cell comprises: (i) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); (ii) CD38 knockout; (iii) exogenous CD16 or its variants; (iv) a partial or complete peptide of IL15 and a partial or complete peptide of IL15 receptor; and (v) a CAR; wherein the cell or population thereof has improved resistance to host immune alloreactivity compared to a cell not possessing all of (i), (ii) and (iii).

24. The cell or population thereof of claim 23, wherein the CAR is specific for at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1.

25. The cell or population thereof of claim 23, wherein the CAR is specific for CD19.

26. A method for improving effector cell resistance to host immune alloreactivity, wherein the method comprises: (i) obtaining engineered iPSCs comprising an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR), and optionally one or both of CD38 knockout and endogenous TCR knockout, and (ii) differentiating the iPSCs into effector cells, thereby generating effector cells having improved resistance to host immune alloreactivity compared to corresponding effector cells without the exogenous polynucleotide.

27. The method of claim 26, wherein the obtaining step comprises: (i) engineering induced pluripotent cells (iPSCs) to generate genome-edited iPSCs comprising one or more exogenous polynucleotides encoding allogeneic immune defense receptors (ADRs) and optionally knocking out one or both of CD38 and endogenous TCRs; or (ii) engineering immune cells by introducing a polynucleotide encoding an allogeneic immune defense receptor (ADR) and optionally knocking out one or both of CD38 and endogenous TCR to produce genome-edited effector cells comprising said ADR and optionally one or both of CD38 knockout and endogenous TCR knockout.

28. The method of claim 26, wherein the ADR is specific for 41BB or CD38.

29. The method of claim 28, wherein the ADR comprises (i) an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 8; or (ii) a CD38 binding domain.

30. The method of claim 26, wherein the engineered iPSC further comprises one or more edits compared to a corresponding primary cell of the engineered iPSC obtained from peripheral blood, umbilical cord blood, or any other donor tissue without the same genome edits, the one or more edits producing: (i) Chimeric antigen receptor (CAR); (ii) introduction of exogenous CD16 or its variants; (iii) a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface; (iv) at least one of the genotypes listed in Table 2; (v) disruption of at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (vi)CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, Fc receptor, antibody or its functional variant or fragment, check point inhibitor, adapter and at least one of surface triggering receptor for coupling with agonist.

31. The method of any one of claims 26 to 30, wherein the improved effector cell resistance to host immune alloreactivity is in vivo.

32. A method for improving in vivo resistance of CAR-T cells to host immune alloreactivity according to any one of claims 26 to 31.

33. A composition comprising the cell or population thereof according to any one of claims 1 to 25.

34. The composition of claim 33, further comprising one or more therapeutic agents.

35. The composition of claim 34, wherein the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, an effector, an antibody or a functional variant or fragment thereof, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector comprising one or more polynucleic acids of interest, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

36. The composition of claim 35, wherein: (a) The checkpoint inhibitors include: (i) 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 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, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR; (ii) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (iii) at least one of atezolizumab, nivolumab, and pembrolizumab; or (b) The therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide.

37. The composition of claim 35, wherein the antibody or functional variant or fragment thereof comprises: (a) anti-CD20 antibody, anti-HER2 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-CD123 antibody, anti-GD2 antibody, anti-PDL1 antibody and / or anti-CD38 antibody; (b) one or more of rituximab, veltuzumab, ofatumumab, utuuximab, okatuzumab, obtuzumab, isobemomab, ocrelizumab, inotuzumab, mosetuzumab, epratuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dietuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, etozuzumab and humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof; or (c) daratumumab, and wherein the derived effector cells comprise CD38 knockout, and optionally expression of CD16 or a variant thereof.

38. The composition of claim 33, further comprising a sensitizer.

39. The composition of claim 38, wherein the sensitizer comprises at least one of a chemotherapeutic agent, external beam radiation, brachytherapy, and a radiopharmaceutical.

40. The composition of claim 38 or 39, wherein the sensitizer increases chemokine secretion and / or surface expression of tumor cells when in contact with the tumor cells.

41. The composition of claim 38, wherein the sensitizer comprises: (i) at least one of calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90; or (ii) Paclitaxel.

42. The therapeutic use of a composition according to any one of claims 33 to 41 by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject suffers from: an autoimmune disorder, a hematological malignancy, a solid tumor, a cancer or a viral infection.

43. A master cell bank (MCB) comprising the clonal iPSCs according to any one of claims 1 to 25.

44. A method for making derived effector cells, the derived effector cells comprising an allogeneic immune defense receptor (ADR), and optionally one or both of CD38 knockout and endogenous TCR knockout, wherein the method comprises differentiating genetically engineered iPSCs into the derived effector cells, wherein the genetically engineered iPSCs comprise an exogenous polynucleotide encoding the ADR, and optionally one or both of CD38 knockout and endogenous TCR knockout.

45. The method of claim 44, wherein the genome engineered iPSC further comprises one or more of the following: (i) CAR; (ii) exogenous CD16 or its variants; (iii) a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface; (iv) at least one of the genotypes listed in Table 2; (v) disruption of at least one of TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (vi)CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, Fc receptor, antibody or its functional variant or fragment, check point inhibitor, adapter and at least one of surface triggering receptor for coupling with agonist.

46. ​​The method of claim 44, wherein (i) the iPSC is a clonal iPSC, a single-cell dissociated iPSC, an iPSC line cell, or an iPSC master cell bank (MCB) cell; (ii) the derived cells include 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 lineage cells, derived NKT lineage cells, derived NK lineage cells or derived B lineage cells; Or (iii) the derived cells include 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.

47. The method of claim 45, wherein the exogenous CD16 or variant thereof comprises at least one of: (a) Non-cleavable high-affinity CD16 (hnCD16); (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.

48. The method of claim 45, 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 another CAR; (ix) 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 a separate construct or in a bicistronic construct; (x) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct; (xi) is specific for at least one of CD19, B7H3, BCMA, CD20, CD22, CD38, CD79b, CD123, CD52, EGFR, EpCAM, GD2, GPRC5D, HER2, KLK2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1; and / or (xii) 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, CLEC12A, an antigen of cytomegalovirus (CMV) infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase erb B2,3,4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit 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, melanin Tumor antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGFR2), Wilms tumor protein (WT-1) and pathogen antigens; and optionally, The CAR of any one of (i) to (xii) is inserted at the TCR locus and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by the insertion of the CAR.

49. The method of claim 44, further comprising genome engineering the clonal iPSC to knock in a polynucleotide encoding the ADR; and optionally: (i) knocking out one or both of CD38 and endogenous TCR, and / or (ii) introducing one or more of exogenous CD16 or its variants, CAR and / or a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface.

50. The method of claim 49, wherein the genome engineering comprises targeted editing.

51. The method of claim 50, 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.

52. A method for generating a clonal master engineered iPSC line using CRISPR, ZFN or TALEN-mediated editing of clonal iPSCs, wherein the editing comprises knocking in a polynucleotide encoding an allogeneic immune defense receptor (ADR), and optionally one or both of CD38 knockout and endogenous TCR knockout, thereby generating the engineered iPSCs.

53. The method of claim 52, wherein the ADR is inserted at one of the loci comprising: B2M, TAP1, TAP2, TAP-associated protein, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT; and wherein the insertion knocks out expression of the gene in the locus.

54. The method of claim 52, further comprising sorting the engineered iPSCs to obtain single cell dissociated iPSCs comprising the polynucleotide encoding the ADR and optionally one or both of CD38 knockout and endogenous TCR knockout.

55. The method of claim 54, further comprising expanding the single cell dissociated iPSCs to generate a clonal master engineered iPSC population.

56. The method of claim 55, further comprising cryopreserving the resulting clonal master engineered iPSC line.

57. The method of any one of claims 53 to 56, further comprising analyzing the engineered iPSCs for off-target editing and / or karyotype.

58. A clonal master engineered iPSC line generated using the method of claims 52 to 57.

59. A method of treating a disease or condition comprising administering to a subject in need thereof a composition according to any one of claims 33 to 41.

60. The method of claim 59, wherein the method comprises administering to the subject in need thereof the cell or population thereof of any one of claims 1 to 25.

61. The method of claim 60, wherein the method further comprises administering a sensitizer to the subject, thereby pre-treating tumor cells in the subject.

62. The method of claim 60 or 61, wherein the method further comprises administering one or more therapeutic agents to the subject.

63. A method of treating a subject, the method comprising: (a) administering a sensitizer to the subject to pre-treat tumor cells in the subject; as well as (b) administering the cell or population thereof according to any one of claims 1 to 25 to the subject after administering the sensitizer, wherein the subject suffers from an autoimmune disorder; a hematological malignancy; a solid tumor; a cancer or a viral infection.

64. The method of claim 63, wherein the sensitizing agent comprises at least one of a chemotherapeutic agent, external beam radiation, brachytherapy, and a radiopharmaceutical.

65. The method of claim 63, wherein the sensitizer increases the secretion and / or surface expression of 4-1BB and / or CD38 of the tumor cells when in contact with the tumor cells.

66. The method of claim 64, wherein the sensitizer comprises: (i) at least one of x-ray radiation, gamma radiation, photon radiation, proton radiation, and neutron radiation; or (ii) at least one of calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen-3, indium-111, iodine-123, iodine-125, iodine-131, iron-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90; or (iii) Paclitaxel.

67. The method of claim 63, further comprising administering one or more therapeutic agents.

68. The method of claim 67, wherein the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, an effector, an antibody or a functional variant or fragment thereof, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector comprising one or more polynucleic acids of interest, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

69. The method of claim 68, wherein: (a) The checkpoint inhibitors include: (i) 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 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, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR; (ii) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (iii) at least one of atezolizumab, nivolumab, and pembrolizumab; or (b) The therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide.

70. The method of claim 68, wherein the antibody or functional variant or fragment thereof comprises: (a) anti-CD20 antibody, anti-HER2 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-CD123 antibody, anti-GD2 antibody, anti-PDL1 antibody and / or anti-CD38 antibody; (b) one or more of rituximab, veltuzumab, ofatumumab, utuuximab, okatuzumab, obtuzumab, isobemomab, ocrelizumab, inotuzumab, mosetuzumab, epratuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dietuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, etozuzumab and humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof; or (c) daratumumab, and wherein the derived effector cells comprise CD38 knockout, and optionally expression of CD16 or a variant thereof.

71. A method of reducing or preventing host cell alloreactivity against allogeneic effector cells in adoptive cell therapy provided to a subject in need thereof, wherein the allogeneic effector cells comprise cells or populations thereof according to any one of claims 1 to 25, and wherein the method optionally comprises CD38 modulation.

72. The method of claim 71, wherein the host cells comprise alloreactive immune cells comprising primary T cells, B cells and / or NK cells.

73. The method of claim 71, wherein the CD38 modulation comprises: (i) administering a CD38 antagonist to the subject before, during or after infusion of the allogeneic effector cells into the subject; or (ii) pre-loading the allogeneic effector cells with a CD38 antagonist in vitro prior to infusing the allogeneic effector cells into the subject.

74. The method of claim 73, wherein the CD38 antagonist comprises: (i) anti-CD38 antibody or CD38-CAR; (ii) daratumumab, isatuximab or MOR202; and / or (iii) Daratumumab.

75. The method of claim 71, wherein the method: (i) reducing or preventing the alloreactivity of host cells to said allogeneic effector cells; (ii) eliminating or reducing the number of alloreactive host cells; (iii) prolonging the survival and persistence of the allogeneic effector cells; (iv) delaying host immune reconstitution; and / or (v) Preventing leaky protection of the allogeneic effector cells against alloreactivity of host cells via overexpression of HLA-G or HLA-E.

76. A method of treating a subject in need of adoptive cell therapy according to any one of claims 71 to 75.

77. A cell or a population thereof, wherein (i) the cell is (a) an immune cell; (b) an induced pluripotent cell (iPSC); or (c) an effector cell derived from differentiating the iPSC; and (ii) the cell comprises: (a) an exogenous polynucleotide encoding an allogeneic immune defense receptor (ADR); (b) an exogenous polynucleotide encoding exogenous CD16 or a variant thereof; (c) an exogenous polynucleotide encoding a cytokine signaling complex, wherein the cytokine signaling complex comprises a part or all of a peptide of an exogenous cytokine and / or its receptor expressed on the cell surface; and (d) CD38 knockout, And wherein the cell has improved resistance to host immune alloreactivity compared to a cell without the exogenous polynucleotide.

78. The cell or population thereof of claim 77, wherein the cell further comprises a CAR.

79. The cell or population thereof of claim 77, wherein the cell further comprises a double knockout of CD58 and CD54.

80. The cell or population thereof of claim 77, wherein the ADR comprises a 41BB-specific ligand operably linked to a signaling domain that promotes effector cell activation.

81. The cell or population thereof of claim 80, wherein the 41BB-specific ligand is 4-1BBL, an antibody or fragment thereof targeting 4-1BB, or a 4-1BBL-Fc fusion.

82. The cell or population thereof of claim 80, wherein the signaling domain comprises CD3ζ or a functional fragment thereof from DAP12, an Fc receptor, or a combination thereof.

83. The cell or colony thereof of any one of claims 77 to 82, wherein the ADR further comprises one, two, three or more co-stimulatory domains.

84. The cell or population thereof of claim 83, wherein the one, two, three or more co-stimulatory domains are from the intracellular signaling domain of CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM or CD40.

85. The cell or population thereof of claim 77, wherein the ADR comprises (i) an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 8; or (ii) a CD38 binding domain.

86. The cell or population thereof according to claim 77, wherein the cytokine signaling complex comprises a fusion protein of IL7 and IL7Rα, or an IL15 / IL15Rα fusion protein with a truncated intracellular domain of IL15Rα.

87. The cell or population thereof of claim 77, wherein the exogenous CD16 or variant thereof is high affinity uncleavable CD16 (hnCD16).

88. The cell or population thereof of claim 77, wherein the high affinity non-cleavable CD16 (hnCD16) comprises an extracellular domain of CD16 having F176V and S197P.

89. The cell or population thereof of claim 77, wherein at least one of (i) the exogenous polynucleotide encoding exogenous CD16 or a variant thereof; and (ii) the exogenous polynucleotide encoding a cytokine signaling complex is inserted into the CD38 locus, thereby resulting in CD38 knockout.

90. A method of treating a subject, the method comprising administering to a subject in need thereof a cell or population thereof according to any one of claims 77 to 89, wherein the subject has an autoimmune disorder; a hematological malignancy; a solid tumor; a cancer or a viral infection.

91. The method of claim 90, wherein the method does not comprise administering Cy / Flu-based lymphodepletion therapy.

92. A method of reducing or preventing host cell alloreactivity against allogeneic effector cells in adoptive cell therapy provided to a subject in need thereof, wherein the allogeneic effector cells comprise cells or populations thereof according to any one of claims 77 to 89, wherein the method optionally comprises CD38 modulation.

93. The method of claim 92, wherein the method does not comprise administering Cy / Flu-based lymphodepletion therapy.

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