Enhanced chimeric antigen receptor effector cell engineering for immunity and use thereof

Abstract

To provide methods and compositions for obtaining functionally enhanced derivative effector cells obtained from the differentiation of genomically engineered iPSCs.SOLUTION: Derivative cells provided herein have stable and functional genome editing that delivers improved or enhanced therapeutic effects. Also there are provided therapeutic compositions and the use thereof comprising the functionally enhanced derivative effector cells alone, or with antibodies or checkpoint inhibitors in combination therapies.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 916,468, filed October 17, 2019, and International Application PCT / US20 / 54601, filed October 7, 2020, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Reference to Electronically Submitted Sequence Listing This application incorporates by reference a computer-readable format (CRF) of the sequence listing in ASCII text format, submitted together with this application, created on October 19, 2020, titled 056932-530001WO_SEQUENCE_LISTING_ST25.txt, and having a size of 92,883 bytes.

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

Background Art

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

Summary of the Invention

[0005] There is a need for effector cells that are functionally improved to address issues related to the effectiveness against solid tumors, namely, tumor microenvironment and associated immunosuppression, recruitment, trafficking, and infiltration, from response rate, cell depletion, loss of transfused cells (survival and / or persistence), tumor escape due to target loss or lineage conversion, accuracy of tumor targeting, off-target toxicity, and off-tumor effects.

[0006] The object of the present invention is to provide a method and composition for generating derivative non-pluripotent cells differentiated from a single-cell-derived iPSC (induced pluripotent stem cell) clone strain, wherein the iPSC contains one or several gene modifications in its genome. The one or several gene modifications include DNA insertion, deletion, and substitution, and these modifications are retained and continue to function in the subsequent derived cells after differentiation, expansion, passage, and / or transplantation.

[0007] The iPSC-derived non-pluripotent cells of the present application include, but are not limited to, CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, B cells, and immune effector cells having one or more functional characteristics present in primary NK, T, and / or NKT cells. The iPSC-derived non-pluripotent cells of the present application contain one or several genetic modifications in their genome through differentiation from iPSCs containing the same genetic modification. In the engineered clone iPSC differentiation strategy for obtaining genetically engineered derivative cells, it is also necessary that the possibility of iPSC generation in differentiation is not adversely affected by the engineered modality of the iPSC and that the engineered modality functions as intended in the derivative cells. Furthermore, this strategy overcomes the current barriers in engineering primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely, that such cells often result in cells that lack reproducibility and uniformity and exhibit insufficient cell persistence with high cell death and low cell proliferation, making it difficult to engineer such cells. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations obtained in another way using initially heterogeneous primary cell sources.

[0008] Some aspects of the invention provide genome-engineered iPSCs obtained using a method comprising (I), (II), or (III), each reflecting a strategy of genome engineering after, simultaneously with, and prior to the reprogramming process. (I): Genetically engineer iPSCs by performing one or both of (i) and (ii) in any order: (i) introduce one or more constructs into the iPSCs to enable targeted integration at a selected site; (ii) (a) introduce one or more double-strand breaks into the iPSCs at the selected site using one or more endonucleases capable of recognizing the selected site; (b) culture the iPSCs of step (I)(ii)(a) to allow endogenous DNA repair to generate indels targeted at the selected site, thereby obtaining genomically engineered iPSCs capable of differentiating into partially or fully differentiated cells. (II): Genetically engineer reprogramming non-pluripotent cells to obtain genomically engineered iPSCs, which includes: (i) contacting the non-pluripotent cells with one or more reprogramming factors and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells; (ii) introducing into the reprogramming non-pluripotent cells of step (II)(i) either or both of (a) and (b) in any order: (a) one or more constructs enabling targeted integration at a selected site; (b) one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, and then culturing the cells of step (II)(ii)(b) to allow endogenous DNA repair to generate indels targeted at the selected site, such that the resulting genomically engineered iPSCs contain at least one functional targeted genomic edit and are capable of differentiating into partially or fully differentiated cells. (III): Genetically engineering non-pluripotent cells to obtain genome-edited iPSCs for reprogramming: This involves (i) introducing into the non-pluripotent cells, in either any order or both, (a) and (b): (a) one or more constructs that enable targeted integration at a selected site, (b) at least one endonuclease capable of recognizing the selected site, to introduce one or more double-strand breaks at the selected site (culturing the cells of step (III)(i)(b) to allow for the generation of indels targeted to the selected site by endogenous DNA repair). And (ii) contacting the cells of step (III)(i) with one or more reprogramming factors and optionally a small molecule composition containing a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to obtain genome-edited iPSCs containing edits targeted to the selected site, thereby obtaining genome-edited iPSCs containing at least one functional targeted genome edit, and the genome-edited iPSCs can differentiate into partially differentiated cells or fully differentiated cells.

[0009] In one embodiment of the above method, at least one targeted genomic editing at one or more selected sites comprises the insertion of one or more exogenous polynucleotides encoding a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of genomically engineered iPSCs or their derivative cells. In some embodiments, the exogenous polynucleotide for insertion is (1) one or more exogenous promoters comprising CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, transient, tissue-specific, or cell-type specific promoters; or (2) operably linked to one or more endogenous promoters contained in a selected site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting the criteria of genomic safe harbors. In some embodiments, the genomically engineered iPSCs generated using the above method comprise one or more different exogenous polynucleotides encoding a protein comprising caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, wherein when the genomically engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated into different safe harbor loci comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotide encodes a partial or complete peptide of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors. In some embodiments, the partial or complete peptide of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors encoded by the exogenous polynucleotide is in the form of a fusion protein.

[0010] In some other embodiments, the genome-edited iPSCs generated using the methods provided herein include indels in one or more endogenous genes related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins that suppress engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derivative cells. In some embodiments, the endogenous genes for disruption include at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.

[0011] In yet some other embodiments, the genome-edited iPSCs generated using the methods provided herein include caspase encoding an exogenous polynucleotide at the AAVS1 locus and thymidine kinase encoding an exogenous polynucleotide at the H11 locus.

[0012] In still some other embodiments, approaches (I), (II), and / or (III) maintain the pluripotency of genome-edited iPSCs by contacting the genome-edited iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. In one embodiment, the resulting genome-edited iPSCs, including at least one targeted genome edit, are functional and pluripotent and can differentiate into non-pluripotent cells containing the same functional genome edit.

[0013] One aspect of the present application provides a chimeric antigen receptor comprising an extracellular domain comprising at least one antigen recognition domain, a transmembrane domain, and an endodomain comprising at least one signaling domain, wherein the at least one signaling domain can be derived from the cytoplasmic domain of a signaling protein specific for the activation or function of T and / or NK cells, and when including induced pluripotent stem cells (iPSCs), the chimeric antigen receptor promotes the differentiation of iPSCs towards desired effector cells, and the iPSC-derived effector cells differentiated from iPSCs have (i) improved persistence and / or survival, (ii) improved cell proliferation, (iii) increased cytotoxicity, (iv) increased resistance to allograft rejection, (v) improved tumor infiltration, (vi) enhanced ability to migrate, activate and / or mobilize bystander immune cells to the tumor site, and / or (vii) enhanced ability to reduce tumor immunosuppression, compared to primary immune cells obtained from peripheral blood, cord blood, or any other donor tissue, including but not limited to at least one of these characteristics.In various embodiments, (a) the signaling protein comprises any one of 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell-specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain-containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain), DAP10 (hematopoietic cell signal transducer), DAP12 (TYRO protein tyrosine kinase-binding protein), DNAM1 (CD226 antigen), FcERIγ (high-affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta), IL-2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D type II integral membrane protein), NKp30 (natural cytotoxicity triggering receptor 3), NKp44 (natural cytotoxicity triggering receptor 2), NKp46 (natural cytotoxicity triggering receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain), and / or (b) at least one signaling domain comprises, respectively, (a) amino acid sequences represented by SEQ ID NOs: 21-41, 54, and 56 for 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8; and / or (b) amino acid sequences having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a portion thereof of 2B4, CD28H, CD3ζ, DAP10, FcERIγ, KIR2DS2, NKG2D, CD3ζ, CD3ζ1XX, DNAM1, CS1.In certain embodiments, each of the at least one signaling domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8, wherein the portion of the cytoplasmic domain comprises an ITAM (immunoreceptor tyrosine-based activation motif), YxxM motif, TxYxxV / I motif, FcRγ, hemi-ITAM, and / or ITT-like motif.

[0014] In various embodiments, the end domain comprises a first signaling domain, a second signaling domain, and optionally a third signaling domain, wherein the first, second, and third signaling domains are different. In some of these embodiments where the end domain comprises the second signaling domain and optionally the third signaling domain, the second or third signaling domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8, as represented by SEQ ID NOs: 21-41, 54, and 56, respectively. In various embodiments, the end domain comprises two different signaling domains, and the end domain comprises a fusion cytoplasmic domain or a portion thereof in any one of the forms including, but not limited to, 2B4-CD3ζ / 1XX, 2B4-DNAM1, 2B4-FcERIγ, 2B4-DAP10, CD16-DNAM1, CD16-DAP10, CD16-DAP12, CD2-CD3ζ / 1XX, CD2-DNAM1, CD2-FcERIγ, CD2-DAP10, CD28-DNAM1, CD28-FcERIγ, CD28-DAP10, CD28-DAP12, CD28H-CD3ζ / 1XX, DAP10-CD3ζ / 1XX, DAP10-DAP12, DAP12-CD3ζ / 1XX, DAP12-DAP10, DNAM1-CD3ζ / 1XX, KIR2DS2-CD3ζ / 1XX, KIR2DS2-DAP10, KIR2DS2-2B4, and NKp46-2B4.In various embodiments, the end domain includes three different signaling domains, and the end domain further includes a fusion cytoplasmic domain or a part thereof in any one of the forms selected from 2B4-DAP10-CD3ζ / 1XX, 2B4-IL21R-DAP10, 2B4-IL2RB-DAP10, 2B4-IL2RB-CD3ζ / 1XX, 2B4-41BB-DAP10, CD16-2B4-DAP10, and KIR2DS2-2B4-CD3ζ / 1XX.

[0015] In some embodiments, the end domain includes only one signaling domain, and the end domain includes an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain of DNAM1, CD28H, KIR2DS2, DAP12, or DAP10 or a part thereof.

[0016] In various embodiments of the chimeric antigen receptor, the transmembrane domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the transmembrane region or a portion thereof of CD2, CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide. In various embodiments, the transmembrane domain comprises, respectively, (a) 2B4, CD2, CD16, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, represented by SEQ ID NOs: 1-20, 53, and 55; or (b) an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the transmembrane region or a portion thereof of DAP10, KIR2DS2, 2B4, NKG2D, CD28H, and DNAM1. In various embodiments, the transmembrane domain and its directly linked signaling domain are derived from the same protein or different proteins.

[0017] In various embodiments of the chimeric antigen receptor, the chimeric antigen receptor comprises a transmembrane domain and an endodomain (TM-(endodomain)), and the chimeric antigen receptor (i) one of the forms of NKG2D-(2B4-IL2RB-CD3ζ), CD8-(41BB-CD3ζ1XX), CD28-(CD28-2B4-CD3ζ), CD28H-(CD28H-CD3ζ), CD28H-(CD28H-2B4), CD28H-(CD28H-2B4-CD3ζ), DNAM1-(DNAM1-CD3ζ), DNAM1-(DNAM1-CS1), DAP10-(DAP10-CD3ζ), KIR2DS2-(KIR2DS2-CD3ζ), KIR2DS2-(KIR2DS2-DAP10), KIR2DS2-(KIR2DS2-DAP10-CD3ζ), KIR2DS2-(KIR2DS2-2B4), CD16-(CD16-2B4-DAP10), CD16-(CD16-DNAM1), NKp46-(NKp46-2B4), NKp46-(NKp46-2B4-CD3ζ), NKp46-(NKp46-CD2-DAP10), CD2-(CD2-CD3ζ), 2B4-(2B4-CD3ζ), 2B4-(2B4-FcERIg), CS1-(CS1-CD3ζ), NKG2D-(CS1), NKG2D-(2B4-CS1), and NKG2D-(2B4-CS1-CD3ζ), or (ii) one of the forms of DAP10-(DAP10-CD3ζ), KIR2DS2-(KIR2DS2-CD3ζ), KIR2DS2-(KIR2DS2-DAP10), KIR2DS2-(KIR2DS2-2B4), 2B4-(2B4-CD3ζ), 2B4-(2B4-FcERIg), NKG2D-(2B4-CS1), CD28H-(CD28H-2B4), CD28H-(CD28H-2B4-CD3ζ), and DNAM1-(DNAM1-CS1), or (iii) comprising an amino acid sequence having about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the sequences represented by each of SEQ ID NOs: 57 to 74.

[0018] In various embodiments of the chimeric antigen receptor, the antigen recognition domain specifically binds to an antigen associated with a disease, pathogen, liquid tumor, or solid tumor. In various embodiments, the antigen recognition domain is (i) any one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1, or (ii) ADGRE2, carbonic anhydrase IX (CAlX), CCRI, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family 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, tumor fetal 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 may be specific to any one of the pathogen antigens.

[0019] In various embodiments of the chimeric antigen receptor, the ectodomain comprises one or more of (i) two antigen recognition domains, (ii) a signal peptide, and / or (iii) a spacer / hinge. In some embodiments, the chimeric antigen receptor can be included in a bicistronic construct that co-expresses a cell surface-expressed exogenous cytokine or a partial or full-length peptide of its receptor, and the exogenous cytokine or its receptor is (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (b) (i) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein having an intracellular domain of cleaved or deleted 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 common receptor γC, wherein the common receptor γC is natural or modified, and (vii) at least one of homodimers of IL15Rβ.

[0020] In some of these embodiments where the chimeric antigen receptor is included in induced pluripotent stem cells (iPSCs) and promotes the differentiation of iPSCs towards desired derived effector cells, the derived effector cells from iPSC differentiation include one or more of derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived immune effector cells. In various embodiments, the iPSC-derived immune effector cells express the chimeric antigen receptor, and the iPSC-derived immune effector cells include at least one functional property that is not present in primary T, NK, and / or NKT cells.

[0021] In another aspect, the present invention provides a cell or a population thereof, wherein (i) the cell can be an immune cell, an induced pluripotent stem cell (iPSC), a cloned iPSC, or an iPS cell line cell, or the cell can be a derived effector cell obtained by differentiating an iPSC, and (ii) the cell comprises at least one chimeric antigen receptor (CAR) provided herein. In various embodiments, the cell is (i) CD38 knockout, (ii) B2M null or low, optionally CIITA null or low, compared to its corresponding cell, (iii) introduced expression of HLA-G or non-cleavable HLA-G, or knockout of one or both of CD58 and CD54, (iv) CD16 or a variant thereof, (v) a second CAR having different targeting specificities, (vi) a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, (vii) at least one of the genotypes listed in Table 2, (viii) deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT compared to its corresponding primary cell, or (ix) introduced or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, Fc receptor, engager, and surface trigger receptor for binding an agonist, compared to its corresponding cell.

[0022] In various embodiments, the cell further comprises high-affinity non-cleavable CD16 (hnCD16) or a variant thereof. Some embodiments of high-affinity non-cleavable CD16 or a variant thereof include at least one of: (a) F176V and S197P in the ectodomain domain of CD16, (b) a complete or partial ectodomain derived from CD64, (c) a non-natural (or non-CD16) transmembrane domain, (d) a non-natural (or non-CD16) intracellular domain, (e) a non-natural (or non-CD16) signaling domain, (f) a non-natural stimulatory domain, and (g) transmembrane, signaling, and stimulatory domains that are not derived from CD16 and are derived from the same or different polypeptides. In certain embodiments, (a) the non-natural transmembrane domain can be derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor (TCR) polypeptide; (b) the non-natural stimulatory domain can be derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or an NKG2D polypeptide; (c) the non-natural signaling domain can be derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or an NKG2D polypeptide; or (d) the non-natural transmembrane domain can be derived from NKG2D, the non-natural stimulatory domain can be derived from 2B4, and the non-natural signaling domain can be derived from CD3ζ.

[0023] In various embodiments, the cell further comprises a second CAR, and the second CAR is (i) T cell-specific or NK cell-specific, (ii) a bispecific antigen-binding CAR, (iii) a switchable CAR, (iv) a dimerized CAR, (v) a split CAR, (vi) a multi-chain CAR, (vii) an inducible CAR, (viii) a partial or complete peptide of an extracellular cytokine and / or its receptor expressed on the cell surface, optionally co-expressed in a separate construct or a bicistronic construct, (xi) a checkpoint inhibitor, optionally co-expressed in a separate construct or a bicistronic construct, (xii) specific for at least one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1, and / or (xiii) ADGRE2, carbonic anhydrase IX (CAlX), CCRI, 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 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9) Specific to any one of 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, tumor fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigens.

[0024] In various embodiments, the cell comprises a partial or complete peptide of an exogenous cytokine and / or its receptor for cell surface expression, and the exogenous cytokine or its receptor comprises (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (b) (i) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein having an intracellular domain of cleaved or excluded IL15Rα, (iv) a fusion protein of the membrane-bound Sushi domain of IL15 and IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and common receptor γC, wherein the common receptor γC is natural or modified, and (vii) a homodimer of IL15Rβ, and any one of (i)-(vii) can be co-expressed with the CAR in a separate construct or in a bicistronic construct, and optionally, (c) transiently expressed.

[0025] In some embodiments where the cell or population of cells is a derived effector cell, the derived effector cell can be a hematopoietic cell and can contain longer telomeres compared to the corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, or the CAR has the following characteristics: (i) being specific for T or NK cells, (ii) being bispecific in antigen binding, (iii) being a switchable CAR, (iv) being a dimerized CAR, (v) being a split CAR, (vi) being a multi-chain CAR, (vii) being an inducible CAR, and (viii) being inserted into one of the gene loci of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, wherein the insertion knocks out or reduces the expression of the gene at the locus, having at least one of being inserted. In various embodiments, the derived effector cell can be capable of mobilizing and / or migrating T cells to the tumor site, and the derived effector cell can be capable of reducing tumor immunosuppression in the presence of one or more checkpoint inhibitors. In various embodiments, the derived effector 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 cells, derived NKT cells, derived NK cells, derived B cells, or derived immune effector cells.

[0026] In some of these embodiments, the cell further comprises a second CAR co-expressed with a checkpoint inhibitor, or the derived effector cell can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. The checkpoint inhibitor can be an antagonist to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR. In various embodiments, the checkpoint inhibitor comprises (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0027] In some of these embodiments of the cell or population thereof, the derived effector cell has at least one of the following characteristics compared to its corresponding primary cell obtained from peripheral blood, cord blood, or any other donor tissue: (i) improved persistence and / or survival, (ii) increased resistance to alloreactive recipient immune cells, (iii) increased cytotoxicity, (iv) improved tumor infiltration, (v) enhanced or acquired ADCC, (vi) enhanced ability to migrate, activate, or mobilize bystander immune cells to the tumor site, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability in rescuing tumor antigen escape, (ix) ability to stabilize tumor antigens, and (x) ability to avoid fratricide.

[0028] In some embodiments of the cell or population thereof, the cell comprises (i) one or more exogenous polynucleotides integrated into a safe harbor locus or a selected locus, or (ii) two or more exogenous polynucleotides integrated into different safe harbor loci or more than two selected loci. In certain embodiments, the safe harbor locus can comprise at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, and the selected locus can be one of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and the integration of the exogenous polynucleotide knocks out the expression of the gene at the locus. In embodiments where the locus is TCR, the TCR locus can be the constant region of TCR alpha or TCR beta.

[0029] In another aspect, the present invention provides a composition comprising the cells or a population thereof described herein. In related aspects, the present invention provides a composition for therapeutic use comprising the derived effector cells provided herein and one or more therapeutic agents. In various embodiments, the one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacements thereof, vectors comprising one or more polynucleotides of interest, antibodies or functional variants or fragments thereof, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). In some of these embodiments where the composition comprises a checkpoint inhibitor, the checkpoint inhibitor is (a) one or more antagonists to a checkpoint molecule including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR, (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (c) may comprise at least one of atezolizumab, nivolumab, and pembrolizumab, or the one or more therapeutic agents include one or more of venetoclax, azacitidine, and pomalidomide.In some of these embodiments where the composition comprises an antibody, the antibody is (a) an anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibody, (b) rituximab, belzutifan, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, certuximab, dinutuximab, abemaciclib, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and one or more of their humanized or Fc-modified variants or fragments, and their functional equivalents and biosimilars, or (c) daratumumab, and derivative effector cells comprising a CD38 knockout, and optionally may comprise the expression of CD16 or a variant thereof.

[0030] In another aspect, the present invention provides a therapeutic use of the compositions provided herein by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection.

[0031] In yet another aspect, the present invention provides a method for producing derivative effector cells comprising the CARs described herein, the method comprising differentiating genetically engineered iPSCs, the iPSCs comprising a polynucleotide encoding a CAR, and optionally, by one or more edits, (i) CD38 knockout, (ii) B2M null or low, optionally CIITA null or low, compared to their corresponding cells, (iii) introduction of HLA-G or non-cleavable HLA-G expression, or knockout of one or both of CD58 and CD54, (iv) CD16 or a variant thereof, (v) a chimeric antigen receptor (CAR) having different targeting specificities, (vi) a partial or full peptide of an exogenous cytokine or its receptor expressed on the cell surface, (vii) at least one of the genotypes listed in Table 2, (viii) deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, compared to their corresponding primary cells, and / or (ix) introduction or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, Fc receptor, engager, and surface trigger receptor for binding to a bispecific or multispecific or universal engager, compared to their corresponding primary cells.In various embodiments, the method further includes genome engineering of the cloned iPSCs to knock in a polynucleotide encoding a CAR, and optionally, (i) knocking out CD38, (ii) knocking out B2M and CIITA, (iii) knocking out one or both of CD58 and CD54, and / or (iv) introducing the expression of HLA-G or non-cleavable HLA-G, high-affinity non-cleavable CD16 or a variant thereof, a second CAR, and / or a partial or full peptide of a cell surface-expressed exogenous cytokine or its receptor. In some embodiments, the genome engineering includes targeted editing. In certain embodiments, the targeted editing includes deletions, insertions, or indels and can be performed by CRISPR, ZFNs, TALENs, homing nucleases, homologous recombination, or any other functional variation of these methods.

[0032] In yet another aspect, the invention provides CRISPR-mediated editing of cloned iPSCs, the editing including knocking in a polynucleotide encoding a CAR as described herein. In various embodiments, the editing of the cloned iPSCs further includes knocking out CD38, and the CAR can be inserted into one of the gene loci including B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and the insertion knocks out the expression of the gene at the locus.

[0033] In yet another aspect, the present invention provides a method of treating a disease or condition comprising administering to a subject in need thereof a cell comprising a CAR as described herein. In various embodiments, the cell comprises a CD38 knockout, a derivative effector cell comprising CD16 or a variant thereof, and optionally (i) a B2M and CIITA knockout, (ii) an introduced expression of HLA-G or non-cleavable HLA-G, or a knockout of one or both of CD58 and CD54, (iii) an introduced expression of a second CAR, and / or a cell surface expressed exogenous cytokine or a partial or full peptide of its receptor, and / or (iii) at least one of the genotypes listed in Table 2. In various embodiments, administration of the cell results in one or more of (i) reducing shedding of MICA / B antigen on the surface of tumor cells, (ii) increasing MICA / B density on the surface of tumor cells, (iii) preventing tumor antigen escape, (iv) overcoming tumor microenvironment suppression, (v) enhancing activation and killing functions of effector cells, and (vi) controlling in vivo tumor progression, reducing tumor cell burden, tumor clearance, and / or improved survival rate, as compared to treatment using effector cells without a CAR as described herein.

[0034] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description when taken in conjunction with the accompanying drawings which illustrate certain embodiments of the invention by way of example and not limitation.

Brief Description of the Drawings

[0035]

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Figure 3F

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Modes for Carrying Out the Invention

[0036] Genome modification of iPSCs (induced pluripotent stem cells) includes insertion, deletion, and substitution of polynucleotides. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types from cells derived from genome-engineered iPSCs. On the other hand, it is difficult to directly manipulate primary immune cells such as T cells or NK cells, which poses an obstacle to the preparation and delivery of engineered immune cells for adoptive cell therapy. The present invention provides an efficient and reliable targeting approach for stably integrating one or more exogenous genes including suicide genes and other functional modalities, which confers improved therapeutic properties related to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, lifespan, self-renewal, persistence, and / or survival to iPSC-derived cells including, but not limited to, HSCs (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, and NK cells.

[0037] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular forms shall include pluralities and plural forms shall include singulars.

[0038] It is to be understood that the present invention is not limited to the specific methodologies, protocols, and reagents, etc. described herein and may, therefore, vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined only by the claims.

[0039] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0040] The use of an alternative (e.g., "or") should be understood to mean one, both, or any combination of them.

[0041] The term "and / or" should be understood to mean either one or both of the alternatives.

[0042] As used herein, the terms "about" or "approximately" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the terms "about" or "approximately" refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0043] As used herein, the terms "substantially" or "essentially" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the terms "substantially the same" or "essentially the same" refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is approximately the same as the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0044] As used herein, the terms "substantially free of" and "essentially free of" are used interchangeably and, when used to describe a composition such as a cell population or a culture medium, mean that the composition is free of a particular substance or source thereof, e.g., a composition that is 95%, 96%, 97%, 98%, 99% free of a particular substance or source thereof, or undetectable as measured by conventional means. The terms "free of" or "essentially free of" a particular component or substance in a composition also mean that such component or substance is either (1) not present in the composition at any concentration or (2) is functionally inert and present in the composition at low concentration. A similar meaning can be applied to the term "absent", which refers to the absence of a particular substance or source thereof in a composition.

[0045] Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises" and "comprising" are to be construed as including the stated step or element or group of steps or elements but not excluding any other step or element or group of steps or elements. In certain embodiments, the terms "include", "have", "contain" and "comprise" are used as synonyms.

[0046] "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are necessary or essential and that no other elements can be present.

[0047] "Consisting essentially of" means including any elements listed after the clause, and is limited to other elements that do not interfere with or contribute to the activities or operations specified in the disclosure of the listed elements. Thus, the clause "consisting essentially of" indicates that the listed elements are necessary or essential, but other elements are not optional and may or may not be present depending on whether they affect the activity or operation of the listed elements.

[0048] Throughout this specification, references to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "a certain particular embodiment", "additional embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in connection with the embodiments are included in at least one embodiment of the invention. Thus, the appearances of the foregoing clause at various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0049] The term "ex vivo" generally refers to activities performed outside the body, such as experiments or measurements performed within or on living tissue in an artificial environment outside the body, preferably with minimal modification of natural conditions. In certain embodiments, "ex vivo" procedures involve living cells or tissues that are harvested from an organism and cultured in an experimental apparatus, typically under aseptic conditions, for typically several hours or up to about 24 hours (but including up to 48 or 72 hours or more depending on the circumstances). In certain particular embodiments, such tissues or cells can be collected and frozen and later thawed for ex vivo processing. Tissue culture experiments or procedures that continue for longer than several days using living cells or tissues are typically considered to be "in vitro", but in certain embodiments, this term may be used interchangeably with ex vivo.

[0050] The term "in vivo" generally refers to activities performed within a living organism.

[0051] As used herein, the terms "reprogramming", "dedifferentiation", "increased differentiation potential", or "increased developmental potential" refer to methods of increasing the differentiation potential of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased differentiation potential has greater developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0052] As used herein, the term "differentiation" is the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells such as, for example, blood cells or muscle cells. Differentiated or induced-differentiated cells are cells that have taken a more specialized ("committed") position within the cell lineage. The term "committed", when applied to the process of differentiation, refers to a cell that has progressed along a differentiation pathway to the point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, cannot differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential that ranges from less primitive and less pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0053] As used herein, the term "induced pluripotent stem cell" or "iPSC" means that the stem cell has been derived from an induced or modified differentiated adult, neonatal, or fetal cell, i.e., reprogrammed into a cell capable of differentiating into all tissues of the three germ layers or dermal layer: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells found in nature.

[0054] As used herein, the term "embryonic stem cell" refers to pluripotent stem cells that naturally exist in the inner cell mass of the blastocyst. Embryonic stem cells are pluripotent and give rise to all derivatives of the three major germ layers, ectoderm, endoderm, and mesoderm, during development. They do not contribute to the extraembryonic membranes or placenta, i.e., they are not totipotent.

[0055] As used herein, the term "multipotent stem cell" refers to a cell that has the potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Thus, multipotent cells are also referred to as "partially differentiated cells". Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells such as, for example, hematopoietic stem cells and neural stem cells. "Multipotent" indicates that a cell has the potential to form many cell types within a given lineage, but may not be able to form cells of other lineages. For example, multipotent hematopoietic cells can form many different blood cell types (red, white, platelets, etc.), but cannot form neurons. Thus, the term "multipotent" refers to a state of a cell with a lower degree of developmental potential than totipotency and pluripotency.

[0056] Pluripotency can be determined, in part, by assessing the characteristics of pluripotent cells. Characteristics of pluripotency include, but are not limited to, (i) the morphology of pluripotent stem cells, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50, (iv) the ability to differentiate into all three somatic lineages (ectoderm, mesoderm, endoderm), (v) the formation of teratomas consisting of the three somatic lineages, and (vi) the formation of embryoid bodies consisting of cells from the three somatic lineages, among others.

[0057] Two types of pluripotency have been previously described: a “primed” or “quasi-stable” state of pluripotency similar to epiblast stem cells (EpiSCs) of late blastocysts, and a “naïve” or “ground” state of pluripotency similar to the cell mass of early / pre-implantation blastocysts. Both pluripotent states exhibit the characteristics as described above, but the naïve or ground state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single-cell culture, (iii) overall reduction of DNA methylation, (iv) reduction of deposition of the H3K27me3 repressive chromatin mark to the developmental control gene promoter, and (v) reduced expression of differentiation markers compared to pluripotent cells in the primed state. The standard methodology of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, is generally considered to have the characteristics of the primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in the primed state unless the expression of exogenous introduced genes, in which the characteristics of the ground state are observed, is maintained.

[0058] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological features of embryonic stem cells. The morphology of normal embryonic stem cells is characterized by a high nucleus-to-cytoplasm ratio, prominent nucleoli, and typical intercellular spacing, with a round and small shape.

[0059] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0060] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0061] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (in each case, the singular "medium"), "supplemental component", and "medium supplement component" refer to the nutrient composition for culturing cell cultures.

[0062] "Culturing" or "maintaining" refers to maintaining, proliferating (growing), and / or differentiating cells outside of a tissue or outside of the body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culture" or "maintenance" can utilize a culture medium as a source of nutrients, hormones, and / or other factors useful for growing and / or maintaining cells.

[0063] As used herein, the term "mesoderm" refers to one of three germ layers that appear during early embryonic development and give rise to various specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0064] As used herein, the terms "secondary hematopoietic endothelial cells" (HE) or "induced hematopoietic endothelial cells from pluripotent stem cells" (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial-hematopoietic transition. Hematopoietic cell development in the embryo proceeds sequentially from the lateral plate mesoderm through angioblasts to secondary hematopoietic endothelial cells and hematopoietic progenitor cells.

[0065] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic progenitors" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation and include multipotent hematopoietic stem cells (hemocytoblasts), myeloid progenitors, megakaryocyte progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid (T cells, B cells, NK cells). The term "secondary hematopoietic stem cells" as used herein refers to CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types, including T-lineage cells, NK-lineage cells, and B-lineage cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0066] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to a major type of white blood cell that matures in the thymus and has various roles in the immune system, including the activation and inactivation of other immune cells in a specific and MHC class I-restricted manner in response to specific foreign antigens in the body. T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), etc., at any stage of development. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also be differentiated from stem cells or progenitor cells. T cell-like derived effector cells can have a T cell lineage in some respects but simultaneously have one or more functional characteristics that are not present in primary T cells.

[0067] "CD4+ T cells" refers to a subset of T cells that express CD4 on their surface and are involved in cell-mediated immune responses. They are characterized by their secretion profile after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55kD glycoprotein initially defined as a differentiation antigen on T lymphocytes, but is also found on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as an associated recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, they define the helper / inducer subset.

[0068] "CD8+ T cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes, as well as cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is an associated recognition element in major histocompatibility complex class I-restricted interactions.

[0069] As used herein, the terms "NK cells" or "natural killer cells" refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and are phenotypically CD3- and CD56+, express at least one of NKG2C and CD57, and optionally CD16, but PLZF, SYK,

[0070]

Number

[0071] As used herein, the term "NKT cell" or "natural killer T cell" refers to CD1d-restricted T cells that express a T cell receptor (TCR). Different from 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 have been recognized. Invariant or type I NKT cells express a very limited TCR repertoire, i.e., a standard α-chain (Vα24-Jα18 in humans) associated with a limited spectrum of β-chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, shows a more heterogeneous use of TCRαβ. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0072] As used herein, terms such as "isolated" refer to a cell or population of cells that has been separated from its original environment, i.e., the environment of an isolated cell is substantially free of at least one component found in the environment in which "non-isolated" reference cells are found. This term includes cells that have been removed from some or all of the components found in their natural environment, e.g., cells isolated from tissue or a biopsy sample. This term also includes cells that are found in an environment not found in nature, e.g., cells isolated from cell culture or a cell suspension and thus removed from at least one, some, or all of their components. Thus, an isolated cell, when found in nature or when grown, stored, or surviving in an environment not found in nature, is partially or completely separated from at least one component that includes other substances, cells, or populations of cells. Specific examples of isolated cells include a partially pure cell composition, a substantially pure cell composition, and cells cultured in a medium not found in nature. An isolated cell can be obtained by separating the desired cell or population thereof from other substances or cells in the environment or by removing one or more other cell populations or subpopulations from the environment.

[0073] As used herein, terms such as "purify" refer to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0074] As used herein, the term "encoding" refers to the unique property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that serves as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produce the protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is typically the nucleotide sequence provided in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0075] "Construct" refers to a macromolecule or molecular complex that includes a polynucleotide delivered to a host cell either 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 and capable of replicating and / or expressing in the target cell. As used herein, the term "vector" includes the construct being delivered. A vector can be a linear or circular molecule. A vector can be integrative or non-integrative. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, etc.

[0076] "Integration" means that one or more nucleotides of a construct are stably inserted into the cell genome, i.e., covalently bound to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of a construct are inserted into the chromosomal or mitochondrial DNA of a cell at a preselected site or "integration site". As used herein, the term "integration" further refers to a process involving the insertion of one or more exogenous sequences or nucleotides of a construct, regardless of the presence or absence of a deletion of an endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" may further include replacing the deleted endogenous sequence or nucleotide with one or more inserted nucleotides.

[0077] As used herein, the term "exogenous" is intended to mean that the referenced molecule or activity is introduced into the host cell or is not native to the host cell. A molecule can be introduced, for example, by introducing a coding nucleic acid into the genetic material of the host, such as by integrating it into the host's chromosome or by introducing it as non-chromosomal genetic material such as a plasmid. Thus, the term when used with respect to the expression of a coding nucleic acid refers to introducing the coding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity present in the 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 within the cell and not introduced exogenously.

[0078] As used herein, "gene of interest" or "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and optionally translated into a polypeptide in vivo. The gene or polynucleotide of interest can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, the gene of interest can encode miRNA, shRNA, a native polypeptide (i.e., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (i.e., a variant of a native polypeptide having less than 100% sequence identity to the native polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0079] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, in the case of polynucleotides that are RNA, uracil (U) in place of thymine. Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to both double-stranded and single-stranded molecules.

[0080] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, which are generally also referred to as peptides, oligopeptides, and oligomers in the art, and longer chains, which are generally referred to as polypeptides or proteins in the art. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0081] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it can affect the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation.

[0082] As used herein, the term "gene imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic attributes in the source cell or iPSC and can be retained in iPSC derived from the source cell and / or hematopoietic cells derived from iPSC. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSC through reprogramming, and iPSC derived from the source cell can be further differentiated into a specific cell type including any hematopoietic cell line. The iPSC derived from the source cell, and the cells differentiated therefrom, are sometimes collectively referred to as "derived" or "descendant" cells depending on the context. For example, the derived effector cells, or derived NK lineage cells or derived T lineage cells used throughout this specification are cells differentiated from iPSC as compared to their corresponding primary cells obtained from natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, the gene imprint conferring preferential therapeutic attributes is incorporated into the iPSC either by reprogramming selected source cells that are donor-specific, disease-specific, or treatment response-specific, or by introducing a genetically modified modality into the iPSC using genome editing. In the context of source cell aspects obtained from a specifically selected donor, disease, or treatment situation, the genetic imprint contributing to the preferential therapeutic attributes may include a heritable phenotype, i.e., a situation-specific genetic or epigenetic modification representing the preferential therapeutic attribute, passed on to the descendant cells of the selected source cell, regardless of whether the underlying molecular events have been identified. Donor-specific, disease-specific, or treatment response-specific source cells may contain gene imprints that can be retained in the iPSC and the derived hematopoietic cells, and these gene imprints include, for example, pre-arranged single-specificity TCRs from virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable gene polymorphisms, e.g., homozygosity for a point mutation encoding the high-affinity CD16 receptor of a selected donor; predetermined HLA requirements, i.e., selected HLA-matched donor cells presenting an increased haplotype, but are not limited thereto.As used herein, preferred therapeutic attributes include improved engraftment, transport, homing, viability, self-renewal, persistence, control and regulation of the immune response, survival, and cytotoxicity of the source cells. Preferred therapeutic attributes may also relate to the expression of antigen-targeting receptors, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modulation, improved target specificity by reduction of off-tumor effects, and resistance to treatments such as chemotherapy.

[0083] As used herein, the term "enhanced therapeutic properties" refers to enhanced therapeutic properties of cells as compared to typical immune cells of the same general cell type. For example, NK cells having "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties as compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells can include, but are not limited to, cell engraftment, transport, homing, viability, self-renewal, persistence, control and regulation of the immune response, survival, and cytotoxicity. Therapeutic properties of immune cells are also indicated by the expression of antigen-targeting receptors, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modulation, improved target specificity by reduction of off-tumor effects, and resistance to treatments such as chemotherapy.

[0084] As used herein, the term "engager" refers to a molecule, such as a fusion polypeptide, that forms a linkage between immune cells, such as T cells, NK cells, NKT cells, B cells, macrophages, neutrophils, and tumor cells and can activate the immune cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTE), bispecific killer cell engagers (BiKE), trispecific killer cell engagers, or multispecific killer cell engagers, or universal engagers that are compatible with multiple immune cell types.

[0085] As used herein, the term "surface trigger receptor" refers to a receptor that can induce or initiate an immune response, such as a cytotoxic response. The surface trigger receptor can be engineered and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, neutrophils. In some embodiments, the surface trigger receptor facilitates the binding of a bispecific or multispecific antibody between an effector cell and a specific target cell, such as a tumor cell, regardless of the natural receptor and cell type of the effector cell. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and then differentiated into populations of various effector cell types that express the universal surface trigger receptor. "Universal" means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind or ligate to an engager having the same epitope recognizable by the surface trigger receptor regardless of the tumor-binding specificity of the engager. In some embodiments, engagers having the same tumor targeting specificity are used to bind to the universal surface trigger receptor. In some embodiments, engagers having different tumor targeting specificities are used to bind to the universal surface trigger receptor. Thus, one or more effector cell types may be used to kill one particular type of tumor cell or two or more types of tumors. The surface trigger receptor generally includes a co-stimulatory domain for effector cell activation and an epitope-binding region specific for the epitope of the engager. The bispecific engager is specific for the epitope-binding region of the surface trigger receptor at one end and specific for a tumor antigen at the other end.

[0086] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent potential toxicity or otherwise harmful effects of cell therapy. In some cases, the expression of the safety switch protein is conditionally controlled to address concerns about the safety of transplanted engineered cells that have permanently integrated the gene encoding the safety switch protein into their genome. This conditional control may be variable and may include post-translational activation via small molecules as well as control by tissue-specific and / or temporal transcriptional regulation. The safety switch may mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene control, and / or depletion via antibodies. In some cases, the safety switch protein is activated by an exogenous molecule, such as a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, the prodrug administered in the event of a harmful event is activated by the suicide gene product to kill the transduced cells.

[0087] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. A pharmaceutically active protein has curative, radical, or palliative properties against a disease and can be administered to restore, relieve, reduce, reverse, or alleviate the severity of the disease. A pharmaceutically active protein also has preventive properties and is used to prevent the onset of a disease or to alleviate the severity of such a disease or pathological condition when it appears. Pharmaceutically active proteins include whole proteins or peptides, or pharmaceutically active fragments thereof. It 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 multiple proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0088] As used herein, the term "signaling molecule" refers to any molecule that regulates, is involved in, inhibits, activates, reduces, or increases cell signaling. Cell signaling refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along a pathway that ultimately causes biochemical events within the cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Toll-like receptor signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0089] As used herein, the term "targeting modality" refers to a molecule, e.g., a polypeptide, that is genetically incorporated into a cell and promotes antigen and / or epitope specificity, including, but not limited to, antigen specificity when associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR), engager specificity when associated with a monoclonal antibody or bispecific engager, targeting of transformed cells, targeting of cancer stem cells, and other targeting strategies in the absence of a specific antigen or surface molecule.

[0090] As used herein, the terms "specific" or "specificity" can be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as contrasted with non-specific or non-selective binding.

[0091] As used herein, the term "adoptive cell therapy" refers to a cell-based immunotherapy associated with the injection of autologous or allogeneic lymphocytes, identified as T cells or B cells expanded ex vivo prior to injection, whether or not genetically modified.

[0092] As used herein, a "therapeutically sufficient amount" includes, within the meaning thereof, a non-toxic but sufficient and / or effective amount of the particular treatment and / or pharmaceutical composition being referred to, and provides the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the general health of the patient, the patient's age, and the stage and severity of the condition. In certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to restore, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0093] The differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium or changes in the physical state of the cells. The most common strategy is to utilize the formation of embryoid bodies (EBs) as a common and important intermediate for initiating lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryonic development in order to generate multiple lineages within a three-dimensional area. Typically, through a differentiation process that lasts from several hours to several days, simple EBs (e.g., aggregated pluripotent stem cells that induce differentiation) continue to mature and develop into cystic EBs, at which point (typically from several days to several weeks), they are further processed to continue differentiating. EB formation is initiated by bringing pluripotent stem cells into close proximity with each other within a three-dimensional multi-layer cell cluster, and typically this is achieved by one of several methods, including sedimenting the pluripotent cells into droplets, sedimenting the cells into "U" bottom well plates, or by mechanical agitation. Aggregates maintained in pluripotent culture maintenance medium do not form appropriate EBs, so in order to promote EB development, pluripotent stem cell aggregates require cues for further differentiation. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cues for induction towards the selected lineage. EB-based culture of pluripotent stem cells typically results in the generation of a differentiated cell population (ectoderm, mesoderm, and endoderm germ layers) with moderate proliferation within the EB cell cluster. Although proven to promote cell differentiation, EBs generate heterogeneous cells in different differentiation states because the cells in the three-dimensional structure are not consistently exposed to cues for differentiation from the environment. In addition, EBs are difficult to produce and maintain. Furthermore, cell differentiation via EBs is accompanied by moderate cell expansion, which also contributes to a decrease in differentiation efficiency.

[0094] In contrast, "aggregate formation", which is different from "EB formation", can be used to expand populations of pluripotent stem cell-derived cells. For example, during the expansion of aggregate-based pluripotent stem cells, a culture medium for maintaining proliferation and pluripotency is selected. Cell proliferation generally increases the size of the aggregates that form larger aggregates, and these aggregates can be routinely dissociated mechanically or enzymatically into smaller aggregates in order to maintain cell proliferation in culture and increase the number of cells. Unlike EB culture, cells cultured within aggregates under maintenance culture maintain pluripotency markers. Pluripotent stem cell aggregates require additional differentiation cues to induce differentiation.

[0095] As used herein, "monolayer differentiation" is a term that refers to a differentiation method different from differentiation by three-dimensional multi-layer clusters of cells, i.e., "EB formation". Among other advantages disclosed herein, monolayer differentiation avoids the need for EB formation for the initiation of differentiation. Since monolayer culture does not mimic embryogenesis such as EB formation, differentiation into a specific lineage is considered minimal compared to the differentiation of all three germ layers of the EB.

[0096] As used herein, "dissociated" cells refer to cells that are substantially separated or purified from other cells or from a surface (e.g., the surface of a culture plate). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be dissociated from each other enzymatically or mechanically, such as by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture plate or other surface. Thus, dissociation can involve disruption of cell interactions with the extracellular matrix (ECM) and substrate (e.g., the culture surface), or disruption of the ECM between cells.

[0097] As used herein, "feeder cells" or "feeders" are terms that describe a type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, expand, or differentiate, because the feeder cells provide stimuli, growth factors, and nutrients for supporting the second cell type. Feeder cells are optionally from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. Feeder cells can typically be inactivated by treatment with a mitotic antagonist such as irradiation or mitomycin when co-cultured with other cells, to prevent them from growing more than the cells they support. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type can be a human feeder such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation to a particular lineage, enhance proliferation capacity, and promote maturation to specialized cell types such as effector cells.

[0098] As used herein, a "feeder-free" (FF) environment refers to an environment such as a culture condition, cell culture, or culture medium that is essentially free of feeder cells or stromal cells and / or not pre-conditioned by the culture of feeder cells. A "pre-conditioned" medium refers to a medium taken after feeder cells have been cultured in the medium for a period such as at least one day. Pre-conditioned media contain many mediator substances, including growth factors and cytokines secreted by feeder cells cultured in the medium. In some embodiments, a feeder-free environment contains neither feeder cells nor stromal cells and is not pre-conditioned by the culture of feeder cells.

[0099] When used in the context of genome editing or modification of iPSCs and their differentiated non-pluripotent derivative cells, or genome editing or modification of non-pluripotent cells and their reprogrammed iPSC derivatives, "functional" means (1) good knock-in, knockout, knockdown gene expression, transgenic or regulated gene expression at the genetic level achieved by direct genome editing or modification, or by "transmission" through differentiation or reprogramming from the starting cells initially genome-manipulated, e.g., inducible or transient expression at the desired cell developmental stage, or (2) (i) modification of gene expression obtained in the cell by direct genome editing, (ii) modification of gene expression maintained in the cell by "transmission" through differentiation or reprogramming from the starting cells initially genome-manipulated, (iii) downstream gene regulation in the cell as a result of modification of gene expression that appears only at the early developmental stage of the cell, or that appears only in the starting cells that give rise to the cell through differentiation or reprogramming, or (iv) removal, addition, or modification of good cell functions / characteristics at the cell level by enhanced or newly acquired cell functions or attributes presented within mature cell products, which are derived from genome editing or modification initially performed on iPSCs, progenitor cells, or dedifferentiated cells.

[0100] "HLA deficiency", including HLA class I deficiency, HLA class II deficiency, or both, refers to any cell in which the surface expression of the complete MHC complex containing the HLA class I protein heterodimer and / or the HLA class II heterodimer is absent, or is no longer maintained, or has a reduction in level such that the reduction is lower than the level naturally detectable by other cells or synthetic methods.

[0101] As used herein, "modified HLA-deficient iPSC" refers to HLA-deficient iPSCs that are further modified by introducing genes that express proteins related to, but not limited to, improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, 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, 41BB, DAP10, DAP12, CD24, CD3ζ, 41BBL, CD47, CD113, and PDL1. Cells with "modified HLA deficiency" include cells other than iPSCs.

[0102] "Fc receptor," abbreviated as FcR, is classified based on the type of antibody it recognizes. For example, those that bind to IgG, the most common class of antibody, are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). The classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells, and B cells) and the signal transduction properties of each receptor. Fc-gamma receptors (FcγR) include several members such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different antibody affinities due to different molecular structures.

[0103] The term "chimeric Fc receptor," abbreviated as CFcR, is used to describe engineered Fc receptors in which the native transmembrane and / or intracellular signaling domains have been modified or replaced with non-native transmembrane and / or intracellular signaling domains. In some embodiments of the chimeric Fc receptor, in addition to one or both of the transmembrane domain and the signaling domain being non-native, one or more stimulatory domains are introduced into the intracellular portion of the engineered Fc receptor to enhance cell activation, expansion, and function upon receptor engagement. Unlike chimeric antigen receptors (CARs) that contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to the Fc fragment, or the Fc region of an antibody, or the Fc region contained in an engager or binding molecule, and activate cells regardless of whether they bring the targeted cells into proximity. For example, Fcγ receptors can be engineered to include a selected transmembrane domain, stimulatory domain, and / or signaling domain within the intracellular region that responds to the binding of IgG at the extracellular domain, thereby generating a CFcR. In one example, a CFcR is generated by engineering CD16, which is an Fcγ receptor, by replacing its transmembrane domain and / or intracellular domain. To further improve the binding affinity of the CD16-based CFcR, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of the CFcR with the high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or replaced such that the extracellular domain of the receptor is non-cleavable, i.e., not affected by shedding, thereby yielding an hnCD16-based CFcR.

[0104] CD16, an Fcγ receptor, has been identified to have two isoforms, the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activates the NK cells, and facilitates antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high affinity CD16", "non-cleavable CD16", or "high affinity non-cleavable CD16 (hnCD16)" refers to a natural or non-natural variant of CD16. Wild-type CD16 has low affinity and, upon activation of NK cells, is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane-proximal region (positions 189-212) has been modified or eliminated do not undergo shedding. The cleavage site and the membrane-proximal region are described in detail in WO2015 / 148926, the complete disclosure of which is incorporated herein by reference. The CD16 S197P variant is a designed non-cleavable version of CD16. CD16 variants containing both F158V and S197P have high affinity and are non-cleavable. Another exemplary high affinity non-cleavable CD16 (hnCD16) variant is an engineered CD16 that contains an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0105] I. Cells and Compositions Useful for Adoptive Cell Therapies with Enhanced Properties Provided herein is a strategy for systematically manipulating the regulatory circuitry of clonal iPSCs while enhancing the therapeutic properties of their derivative cells without affecting the differentiation potential of the iPSCs and the cell developmental biology of the iPSCs and their derivative cells. The derivative cells are functionally improved, and a combination of select modalities is suitable for adoptive cell therapy after being introduced into the cells at the iPSC level through genome engineering. Prior to the present invention, it was unclear whether modified iPSCs containing one or more provided gene edits still had the ability to enter cell development and / or the ability to mature and generate functional differentiated cells while retaining regulated activity. Unexpected failures during directed cell differentiation from iPSCs are due to, but not limited to, aspects including stage-specific gene expression or lack thereof during development, requirements for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure differentiation protocols that allow for changes in cell phenotype and / or function. This application shows that one or more selected genome modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from the engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from the individual or combined genome modifications that are retained in the effector cells after iPSC differentiation.

[0106] 1. CAR with a novel endodomain In embodiments, a chimeric antigen receptor (CAR) is a fusion protein generally comprising an antigen recognition domain, an extracellular domain including a transmembrane domain, and an intracellular domain including one or more signaling domains. In embodiments, the CARs described herein are designed to be expressed and functional in induced pluripotent stem cells (iPSCs) and in effector cells derived from iPSCs engineered to contain the CAR. In embodiments, the CARs described herein are designed not to interfere with iPSC differentiation and / or to promote the differentiation of iPSCs toward a desired effector cell type. In embodiments, the CAR enhances effector cell proliferation, persistence, survival, cytotoxicity, resistance to allograft rejection, tumor infiltration, migration, the ability to activate and / or mobilize bystander immune cells, and / or the ability to overcome tumor suppression. In embodiments, the CARs provided herein can also be directly expressed in cell line cells and in cells from primary sources (primary cells), i.e., natural / native sources such as peripheral blood, cord blood, or other donor tissues.

[0107] In some embodiments, the CAR is suitable for activating T cells, NK cells, or NKT cells that express the CAR. In certain embodiments, the T cells are derived from CAR-expressing iPSCs, and the derived T cells can include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof. In certain embodiments, the NK cells are derived from CAR-expressing iPSCs. In certain embodiments, the NKT cells are derived from CAR-expressing iPSCs. In some embodiments, a CAR comprising NK cell-specific signaling components is specific for NK cells. In some embodiments, a CAR comprising NK cell-specific signaling components is also suitable for T cells or other cell types. In some embodiments, a CAR comprising T cell-specific signaling components is specific for T cells. In some embodiments, a CAR comprising T cell-specific signaling components is also suitable for NK cells or other cell types. In some embodiments, a CAR comprising NKT cell-specific signaling components is specific for NKT cells. In some embodiments, a CAR comprising NKT cell-specific signaling components is also suitable for NK or T cells, or other cell types.

[0108] In embodiments, the CARs described herein include at least an ectodomain, a transmembrane domain, and an endodomain. The endodomain of the CAR includes at least one signaling domain that affects the growth and function of cells expressing the CAR and activates effector cells expressing the CAR upon antigen binding. In some embodiments of the CAR endodomain, one or more costimulatory domains (often also referred to as additional signaling domains) are further included to affect cell lifespan, memory differentiation, and metabolic properties. Here, signaling proteins specific for T cells and / or NK cells are used to provide one or more signaling domains included in components of the CAR fusion protein, such as the transmembrane domain and the endodomain of the CAR. Exemplary signaling proteins suitable for CAR design include, but are not limited to, 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, and CD8. Descriptions of exemplary signaling proteins including the transmembrane and cytoplasmic sequences of the proteins are provided below and further in Table 1A.

[0109] 2B4 (natural killer cell receptor 2B4) is a receptor for CD48, a signaling lymphocyte activation molecule (SLAM). Upon ligand binding, 2B4 is involved in the regulation and interconnectedness of both innate and adaptive immune responses to regulate the activation and differentiation of a wide variety of immune cells. Acting as an activating NK cell receptor, 2B4 stimulates NK cell cytotoxicity, IFN-γ production, and granule exocytosis. Optimal growth and activation of NK cells appears to depend on the engagement of CD48 and 2B4 expressed on adjacent NK cells. 2B4 is also involved in the regulation of CD8+ T cell proliferation. Expression of 2B4 and its binding to CD48 on activated T cells provide a function such as costimulation to adjacent T cells. Additionally, 2B4 is involved in leukocyte migration.

[0110] 4-1BB (tumor necrosis factor receptor superfamily member 9) is a receptor for TNFSF9 / 4-1BBL and is involved in the activation of T cells through the tumor necrosis factor-mediated signaling pathway.

[0111] CD16 (IgG Fc region receptor III-A) is a receptor for the Fc region of IgG. It mediates antibody-dependent cell cytotoxicity (ADCC) and other antibody-dependent responses and is involved in the regulation of the immune response.

[0112] CD2 (T cell surface antigen CD2) interacts with lymphocyte function-associated antigen CD58 (LFA-3) and CD48 / BCM1 to mediate adhesion between T cells and other cell types. The cytoplasmic domain of CD2 is involved in the signaling function that triggers the activation of T cells. CD2 is also involved in leukocyte migration, the activation of NK cells, the differentiation of T cells, and the regulation of IFN-γ and IL8 secretion.

[0113] CD28 (T cell-specific surface glycoprotein CD28) is involved in the T cell receptor signaling pathway and affects the activation and co-stimulation of T cells, the induction of cell proliferation, cytokine production, and the promotion of T cell survival. CD28 also regulates the differentiation of regulatory T cells and enhances the production of IL4 and IL10 in T cells in combination with TCR / CD3 ligation and CD40L co-stimulation.

[0114] CD28H (transmembrane and immunoglobulin domain-containing protein 2) plays a role in immune responses, cell-cell interactions, cell migration, and angiogenesis. Through its interaction with HHLA2, CD28H co-stimulates T cells in the context of TCR-mediated activation. Furthermore, CD28H enhances T cell proliferation and cytokine production through the AKT-dependent signaling cascade.

[0115] CD3ζ (or CD3Z; T cell surface glycoprotein CD3 zeta chain) is part of the TCR-CD3 complex presented on the surface of T lymphocyte cells, which plays an essential role in the adaptive immune response. When an antigen-presenting cell (APC) activates the T cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains CD3D, CD3E, CD3G, and CD3Z. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains. When the TCR is involved, these motifs are phosphorylated, leading to the activation of downstream signaling pathways. CD3Z plays an important role in the differentiation of thymic T cells.

[0116] DAP10 (hematopoietic cell signal transducer) is a transmembrane adapter protein that associates with KLRK1 to form the activating receptor KLRK1-HCST in lymphoid and myeloid cells. The KLRK1-HCST receptor plays a role in the immunological surveillance against tumors and is usually required for the cell lysis of tumor cells that express cell surface ligands such as MHC class I chain-related MICA and MICB, as well as UL16-binding protein (ULBP), and these ligands are upregulated by stress conditions and pathological states such as viral infection and tumor transformation. In NK cells, KLRK1-HCST signaling directly induces cytotoxicity and enhances cytokine production. In T cells, it provides co-stimulation of TCR-induced signals.

[0117] DAP12 (TYRO protein tyrosine kinase-binding protein) is an adapter protein associated with the activation of receptors found on the surface of various immune cells and mediates signal transduction and cell activation after ligand binding by the receptor. DAP12 is associated with natural killer (NK) cell receptors such as KIR2DS2 and the KLRD1 / KLRC2 heterodimer and mediates the activation of NK cells. DAP12 also enhances the transport and cell surface expression of the NK cell receptors KIR2DS1, KIR2DS2, and KIR2DS4 and ensures their stability on the cell surface. Furthermore, DAP12 negatively regulates the proliferation of B cells.

[0118] DNAM1 (CD226 antigen) is involved in immune response, cell adhesion, lymphocyte signaling, cytotoxicity mediated by cytotoxic T lymphocytes (CTLs) and NK cells, and lymphokine secretion. DNAM1 also regulates T cell receptor signaling and stimulates T cell proliferation and cytokine production, including that of IL2, IL5, IL10, IL13, and IFNγ.

[0119] FcERIγ (high-affinity immunoglobulin epsilon receptor subunit gamma) is an adapter protein that transmits activation signals from various immune receptors. It is involved in antigen processing and presentation of exogenous peptide antigens through positive regulation of MHC class I and II, immunoglobulin-mediated immune responses, innate immune responses, leukocyte migration, IL-10, IL-6, TNF, and T cell differentiation.

[0120] IL21R (interleukin-21 receptor) is involved in the IL21-mediated signaling pathway and plays a role in the activation of natural killer cells.

[0121] IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta) is the beta subunit of the interleukin 2 receptor, is associated with IL15RA, is involved in receptor-mediated endocytosis, and transduces the signal of IL2. IL-2Rβ can affect cell persistence through negative regulation of the apoptosis process.

[0122] IL-2Rγ (cytokine receptor common subunit gamma) is a subunit common to the receptors of various interleukins and is involved in the signaling pathways mediated by IL15, IL21, IL2, IL4, IL7, and IL9.

[0123] IL-7R (Interleukin-7 receptor subunit alpha) is a receptor for interleukin 7 and is involved in IL7-mediated signaling pathways, cell morphogenesis, T cell differentiation, cell number homeostasis, cell proliferation, immune responses, and immunoglobulin production.

[0124] KIR2DS2 (Killer cell immunoglobulin-like receptor 2DS2) is a receptor for natural killer (NK) cells of HLA-C alleles. KIR2DS2 does not inhibit the activity of NK cells and is involved in the regulation of innate immune responses and immune responses.

[0125] NKG2D (NKG2-D type II integral membrane protein) functions as an activating and costimulatory receptor involved in immunological surveillance when it binds to various cell stress-inducible ligands presented on the surface of autologous tumor cells and virus-infected cells. For example, NKG2D binds to ligands belonging to various subfamilies of MHC class I-related glycoproteins including MICA, MICB, RAET1E, RAET1G, RAET1L / ULBP6, ULBP1, ULBP2, ULBP3 (ULBP2 > ULBP1 > ULBP3), and ULBP4. NKG2D activates NK cells, provides both stimulatory and costimulatory innate immune responses to activated killer (NK) cells, and results in cytotoxic activity. NKG2D functions as a costimulatory receptor for the T cell receptor (TCR) in CD8+ T cell-mediated adaptive immune responses by amplifying T cell activation. NKG2D stimulates the perforin-mediated elimination of ligand-expressing tumor cells. NKG2D is also involved in signal transduction involving calcium influx, reaches a peak in TNF-α expression, and is involved in NK cell-mediated bone marrow allograft rejection. NKG2D can also play a regulatory role in NK cell differentiation and survival.

[0126] NKp30 (Natural Cytotoxicity Triggering Receptor 3) is a cell membrane receptor of natural killer cells and is activated by the binding of extracellular ligands including BAG6 and NCR3LG1. NKp30 is involved in cell recognition, immune response, and regulation of immune response. Furthermore, NKp30 stimulates the cytotoxicity of NK cells against adjacent cells including tumor cells and produces these ligands. For example, it controls the cytotoxicity of NK cells against tumor cells.

[0127] NKp44 (Natural Cytotoxicity Triggering Receptor 2) and NKp46 (Natural Cytotoxicity Triggering Receptor 1) are receptors that activate cytotoxicity and can contribute to the improvement of the efficiency of activated natural killer (NK) cells that mediate the lysis of tumor cells. Both NKp44 and NKp46 are involved in cell defense responses, innate immune responses, and their regulation.

[0128] CS1 (SLAM Family Member 7) is an autoregulatory ligand receptor of the signaling lymphocyte activation molecule (SLAM) family. Since SLAM receptors regulate the activation of a wide variety of immune cells and function in their differentiation, they are involved in the regulation and interconnection of both innate and adaptive immune responses. The activity of SLAM receptors is controlled by the presence or absence of small cytoplasmic adapter proteins, SH2D1A / SAP, and / or SH2D1B / EAT-2. SLAM receptors positively regulate the activation and cytotoxicity of NK cells by a mechanism dependent on phosphorylated SH2D1B. SLAM receptors are also involved in cell adhesion.

[0129] CD8 (T cell surface glycoprotein CD8 alpha chain) is an integral membrane glycoprotein that plays an essential role in the immune response and performs multiple functions in response to both external and internal attacks. In T cells, CD8 mainly functions as a coreceptor for MHC class I molecule:peptide complexes. In NK cells, the presence of CD8A homodimers on the cell surface provides a survival mechanism that enables the engagement and lysis of multiple target cells. CD8A homodimer molecules also promote the survival of activated lymphocytes and their differentiation into memory CD8 T cells.

[0130]

Table 1-1

[0131]

Table 1-2

[0132]

Table 1-3

[0133] In some embodiments of the CARs provided, the end domains of the CARs each comprise at least a first signaling domain having 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 the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8, as represented by SEQ ID NOs: 21-41, 54, and 56, respectively. In some embodiments, the signaling domain of the CARs disclosed herein comprises only a portion of the cytoplasmic domain of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8. In some embodiments, a portion of the cytoplasmic domain selected for the CAR signaling domain is 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 an ITAM (immunoreceptor tyrosine-based activation motif), YxxM motif, TxYxxV / I motif, FcRγ, hemi-ITAM, and / or ITT-like motif.

[0134] In some embodiments of the provided CARs, the end domain of the CAR comprising the first signaling domain further comprises a second signaling domain comprising 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 the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ / 1XX (i.e., CD3ζ or CD3ζ1XX), CS1, or CD8, respectively, as represented by SEQ ID NOs: 21-41, 54, and 56, and the second signaling domain is different from the first signaling domain.

[0135] In some embodiments of the provided CARs, the end domain of the CAR comprising the first and second signaling domains further comprises a third signaling domain comprising 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 the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ / 1XX (i.e., CD3ζ or CD3ζ1XX), CS1, or CD8, respectively, as represented by SEQ ID NOs: 21-41, 54, and 56, and the third signaling domain is different from the first and second signaling domains.

[0136] In some exemplary embodiments of a CAR having an end domain consisting of only one signaling domain, the end domain comprises, but is not limited to, the cytoplasmic domain of a protein or a portion thereof that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to DNAM1, CD28H, KIR2DS2, DAP12, or DAP10.

[0137] In some exemplary embodiments of a CAR having an end domain consisting of two different signaling domains, the end domain comprises, but is not limited to, a chimeric cytoplasmic domain or a portion thereof, including 2B4-CD3ζ / 1XX, 2B4-DNAM1, 2B4-FcERIγ, 2B4-DAP10, CD16-DNAM1, CD16-DAP10, CD16-DAP12, CD2-CD3ζ / 1XX, CD2-DNAM1, CD2-FcERIγ, CD2-DAP10, CD28-DNAM1, CD28-FcERIγ, CD28-DAP10, CD28-DAP12, CD28H-CD3ζ / 1XX, DAP10-CD3ζ / 1XX, or DAP10-DAP12, DAP12-CD3ζ / 1XX, DAP12-DAP10, DNAM1-CD3ζ / 1XX, KIR2DS2-CD3ζ / 1XX, KIR2DS2-DAP10, KIR2DS2-2B4, or NKp46-2B4.

[0138] In some exemplary embodiments of a CAR having an end domain consisting of three different signaling domains, the end domain comprises, but is not limited to, a chimeric cytoplasmic domain or a portion thereof, including 2B4-DAP10-CD3ζ / 1XX, 2B4-IL21R-DAP10, 2B4-IL2RB-DAP10, 2B4-IL2RB-CD3ζ / 1XX, 2B4-41BB-DAP10, CD16-2B4-DAP10, or KIR2DS2-2B4-CD3ζ / 1XX.

[0139] In some embodiments, the transmembrane domain of the CAR comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the full-length or a portion of the transmembrane region of CD2, CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide. In some other embodiments, the transmembrane domain of the CAR comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the full-length or a portion of the transmembrane region of 2B4, CD2, CD16, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, respectively, as represented by SEQ ID NOs: 1-20, 53, and 55. In some embodiments of the CAR, the transmembrane domain and its directly linked signaling domain are derived from the same protein. In some other embodiments of the CAR, the transmembrane domain and the directly linked signaling domain are derived from different proteins.

[0140] Table 1B provides non-limiting examples of CAR constructs that include transmembrane domains and endodomains (designated as TM-(endodomain)). Generally, each of the exemplified CAR constructs includes a transmembrane domain and an endodomain that includes one or more signaling domains derived from the cytoplasmic region of one or more signaling proteins. In embodiments, the one or more signaling domains included in the CAR endodomain are derived from the same or a different protein from which the TM is derived. As shown in Table 1B, the portion presenting the transmembrane domain (TM) of the CAR is underlined, the domains included in the endodomain are indicated by parentheses “()”, and each of the TM and the signaling domains is represented by the name of the signaling protein from which the domain sequence is derived. In embodiments, the amino acid sequence of each TM or signaling domain can be about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the full length or a portion of the corresponding transmembrane region or cytoplasmic region of the designated signaling protein. Exemplary CAR constructs that include transmembrane domains and endodomains provided herein include, [Sequence Listing 2] TIFF2025108510000006.tif48165, but are not limited thereto. In some embodiments, each of the above exemplary CAR constructs that include transmembrane domains and endodomains includes an amino acid sequence that is about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence represented by each of SEQ ID NOs: 57-74 in Table 1B. Exemplary sequences for each construct provided in Table 1B have text formatted to match the format (i.e., underlined, normal, or bold text) of the corresponding region of the figure on the left side of the sequence. In most of the exemplary constructs in Table 1B, the TM is the first sequence region, but the construct can include an extracellular domain that precedes the TM (see, e.g., Construct 6), and can be derived from the same or a different protein as the TM. In some embodiments, two or more signaling domains included in the CAR endodomain can be separated by one or more additional sequences such as a spacer or a linker.

[0141]

Table 2-1

[0142]

Table 2-2

[0143]

Table 2-3

[0144] A CAR comprising any of the TM-(end domains) provided above can be constructed to specifically target at least one antigen determined by an antigen-binding domain contained in the ectodomain of the CAR. In some embodiments, the CAR can specifically target an antigen associated with a disease or pathogen. In some embodiments, the CAR can specifically target a tumor antigen, and the tumor can be a liquid or solid tumor. The ectodomain of the CAR contains one or more antigen recognition domains for antigen-specific binding. In some embodiments, the external domain can further contain a signal peptide or leader sequence, and / or a spacer.

[0145] In certain embodiments, the ectodomain of the provided CAR contains an antigen recognition domain comprising a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark variable new antigen receptor (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, single-chain variable fragment (scFv) for antigen binding, (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant variable heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody.

[0146] Non-limiting examples of antigens that can be targeted by CARs contained in genetically engineered iPSCs and derived effector cells include ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD269 (BCMA), CDS, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCS1, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0147] In some embodiments, the provided CAR ectodomain further includes a signal peptide. The signal peptide directs the CAR polypeptide to the endoplasmic reticulum (ER) for proper glycosylation and anchoring to the plasma membrane. Generally, any eukaryotic signal sequence that targets a secreted protein to the ER pathway can be used. Exemplary suitable signal peptides include, but are not limited to, the IL-2 signal sequence, kappa leader sequence, CD8α leader sequence, albumin signal sequence, prolactin signal sequence, and IgG signal peptide, and GM-CSF signal peptide.

[0148] In some embodiments, the provided CAR ectodomain may optionally include a hinge (also referred to as a spacer) region to provide flexibility between the antigen recognition domain and the transmembrane domain of the CAR. In some exemplary and non-limiting embodiments, the CAR hinge has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the hinge region of a known polypeptide such as CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8α, CD8β, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, IgG1, or the CH2 / CH3 domain of an immunoglobulin, or a combination thereof. In some embodiments, the provided CAR hinge region has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the CH2 / CH3 domain of an immunoglobulin and includes an amino acid sequence.

[0149] In some embodiments, effector cells comprising one or more CARs provided can be used to treat autoimmune disorders, hematological malignancies, solid tumors, or infectious diseases associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes). Examples of solid cancers include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpesvirus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella-zoster virus), adenovirus-, lentivirus-, BK polyomavirus-related disorders).

[0150] One aspect of the present invention provides iPSCs and derivative effector cells differentiated therefrom, comprising a polynucleotide encoding a CAR comprising one of the endodomains provided herein. In one embodiment of the CAR, the CAR is specific for CD19. In another embodiment, the CAR is specific for MICA / B. In another embodiment, the CAR is specific for BCMA. In yet another embodiment, the CAR is specific for CD38. In yet another embodiment, the CAR is specific for HER2. In another embodiment, the CAR is specific for MSLN. Further, in another embodiment, the CAR is specific for PSMA. In yet another embodiment, the CAR is specific for VEGF-R2.

[0151] In another aspect of the present invention, iPSCs and derivative effector cells differentiated therefrom, comprising a polynucleotide encoding a first CAR comprising one of the endodomains provided herein, may further comprise a second CAR having a different antigen specificity. The endodomain of the second CAR may or may not be the same as the endodomain of the first CAR. In some embodiments, the second CAR comprises an endodomain different from the endodomain of the first CAR and is one of the endodomains provided herein. In some other embodiments, the second CAR comprises an endodomain different from the endodomain of the first CAR and is not one of the endodomains provided herein.

[0152] Non-limiting CAR strategies include conditional activation CARs of heterodimers via dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9,587,020); split CARs that are homologous recombination of antigen-binding, hinge, and end domains to generate a CAR (see, e.g., U.S. Publication No. 2017 / 0183407); multi-chain CARs that allow non-covalent binding between two transmembrane domains each connected to an antigen-binding domain and a signaling domain (see, e.g., U.S. Publication No. 2014 / 0134142); CARs having a bispecific antigen-binding domain (see, e.g., U.S. Patent No. 9,447,194), or a pair of antigen-binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Patent No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Invest. 2016;126(8):3036-3052); inducible CARs (see, e.g., U.S. Publication Nos. 2016 / 0046700, 2016 / 0058857, 2017 / 0166877); switchable CARs (see, e.g., U.S. Publication No. 2014 / 0219975); and any other designs known in the art.

[0153] Genomic loci suitable for insertion of one or more of the CARs provided herein include loci that meet the criteria of genomic safe harbors and / or loci where knockdown or knockout of a gene is desired as a result of the insertion. In some embodiments, suitable genomic loci for CAR insertion include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant regions, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0154] In one embodiment, iPSCs and their derivative cells containing a CAR are inserted into the TCR constant region (TRAC or TRBC), resulting in a TCR knockout, and optionally placing CAR expression under the control of the endogenous TCR promoter. In one particular embodiment of an iPSC-derived cell containing a TCR null and a CAR containing one of the provided endodomains, the derivative cell is a T cell. In another embodiment, iPSCs and their derivative cells containing a CAR have a CAR inserted into the NKG2A locus or the NKG2D locus, resulting in an NKG2A or NKG2D knockout, and optionally placing CAR expression under the control of the endogenous NKG2A or NKG2D promoter. In one particular embodiment of an iPSC-derived cell containing an NKG2A or NKG2D null and a CAR, the derivative cell is an NK cell. In yet another embodiment, iPSCs and their derivative cells containing a CAR have a CAR inserted into the CD38 coding region, resulting in a CD38 knockout, and optionally placing CAR expression under the control of the endogenous CD38 promoter. In one embodiment of a cell containing a CD38 null and a CAR containing one of the provided endodomains, the CAR is specific for CD38. In one embodiment, iPSCs and their derivative cells containing a CAR containing one of the provided endodomains have a CAR inserted into the CD58 coding region, resulting in a CD58 knockout. In one embodiment, iPSCs and their derivative cells containing a CAR containing one of the endodomains have a CAR inserted into the CD54 coding region, resulting in a CD54 knockout. In one embodiment, iPSCs and their derivative cells containing a CAR containing one of the endodomains have a CAR inserted into the CIS (cytokine-inducible SH2-containing protein) coding region, resulting in a CIS knockout. In one embodiment, iPSCs and their derivative cells containing a CAR containing one of the endodomains have a CAR inserted into the CBL-B (E3 ubiquitin protein ligase CBL-B) coding region, resulting in a CBL-B knockout. In one embodiment, iPSCs and their derivative cells containing the provided CAR have a CAR inserted into the SOCS2 coding region, resulting in a SOCS2 knockout.In one embodiment, the iPSCs and their derivative cells comprising the provided CAR have the CAR inserted into the CD56 (NCAM1) coding region. In another embodiment, the iPSCs and their derivative cells comprising the provided CAR have the CAR inserted into the coding region of any one of PD1, CTLA4, LAG3, and TIM3, resulting in knockout or knockdown of the checkpoint receptor at the insertion site. In a further embodiment, the iPSCs and their derivative cells comprising the provided CAR have the CAR inserted into the coding region of TIGIT, resulting in TIGIT knockout.

[0155] As described in more detail herein, further provided embodiments include effector cells derived from differentiating genomically engineered iPSCs, wherein the iPSCs and derivative cells comprise the CARs described herein, and the iPSCs and derivative cells further comprise one or more additional modified modalities including, but not limited to, CD38 knockout, CD38-CAR, hnCD16, exogenous cytokines and / or their signaling components, HLA-I and / or HLA-II deficiency, overexpression of HLA-G, and knockout of one or both of CD58 and CD54, TCR null, surface presenting CD3, antigen-specific TCR, NKG2C, DAP10 / 12, NKG2C-IL15-CD33 ("2C1533").

[0156] 2. CD38 Knockout The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies, including multiple myeloma and CD20-negative B-cell malignancies, both of lymphoid and myeloid origin, and is an attractive target for antibody therapies that deplete cancer cells. Antibody-mediated depletion of cancer cells typically results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cell-mediated cytotoxicity). In addition to ADCC, immune effector mechanisms that cooperate with therapeutic antibodies may also include phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

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

[0158] In the treatment of malignant tumors, when T cells transduced with a CD38 antigen-binding receptor are used systemically, the CD38+ fraction of CD34+ hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells is lysed, and due to recipient immune effector cell dysfunction, the treatment response is incomplete and the efficacy is reduced or eliminated. In addition, in multiple myeloma patients treated with daratumumab, a CD38-specific antibody, a reduction in NK cells was observed in both the bone marrow and peripheral blood, while other immune cell types such as T cells and B cells were not affected despite the expression of CD38 (Casneuf et al., Blood Advances. 2017;1(23):2105-2114). Without being limited by theory, the provided CD38-null effector cells containing MICA / B-CAR overcome fratricide mediated by CD38 and can avoid depletion or reduction of effector cells induced by specific antibodies and / or CD38 antigen-binding domains. Furthermore, since CD38 is upregulated in activated lymphocytes such as T cells or B cells, CD38-specific antibodies such as daratumumab can be used to eliminate activated lymphocytes in the recipient of the provided CD38-null allogeneic effector cells or suppress the activation of these lymphocytes, and as a result, allogeneic rejection by host lymphocytes against these effector cells can be reduced and / or prevented, and the survival and persistence of these effector cells can be increased despite the presence of CD38 antibodies used for lymphocyte depletion. Thus, the present application also provides a strategy for reducing or preventing allogeneic rejection reactions by using CD38-specific antibodies, secreted CD38-specific engagers, or CD38 CAR (chimeric antigen receptor) while enhancing the persistence and / or survival of effector cells and against activation of recipient T cells and B cells and / or eliminating activated recipient T cells and B cells.

[0159] In one embodiment provided herein, the CD38 knockout in iPSCs is a biallelic knockout. As disclosed herein, the provided CD38 null iPSCs can differentiate into mesodermal cells with the potential to become definitive hemogenic endothelium (HE), definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), 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 derivative effector cells having one or more functional properties not present in primary NK, T, and / or NKT cells, including but not limited to, functional derivative effector cells that can differentiate to generate. In some embodiments, when using a CD38 antibody to induce ADCC or using CD38-CAR for targeted cell killing, CD38 - / - iPSCs and / or their derivative effector cells are not eliminated by the CD38 antibody or CD38 CAR, thereby increasing the persistence and / or survival of iPSCs and their effector cells in the presence and / or after exposure to such therapeutic agents. In some embodiments, the effector cells increase their persistence and / or survival in vivo in the presence of such therapeutic agents and / or after exposure. In some embodiments, the CD38 null effector cells are NK cells derived from iPSCs. In some embodiments, the CD38 null effector cells are T cells derived from iPSCs. In some embodiments, the CD38 null iPSCs and derivative cells include one or more additional genome edits described herein, including but not limited to hnCD16 expression, CAR expression, cytokine / cytokine receptor expression, HLA I and / or HLAII knockout, and additional modalities provided herein.

[0160] In another embodiment, inserting one or more transgenes provided herein at a selected position of CD38 while knocking out CD38 can be achieved, for example, by a knock-in / knock-out (CD38-KI / KO) construct targeting CD38. In some embodiments of the construct, the construct includes a pair of CD38-targeting homology arms for site-selective insertion within the CD38 locus. In some embodiments, the pre-selected targeting site is within an exon of CD38. The CD38-KI / KO constructs provided herein allow the transgene to be expressed either under the CD38 endogenous promoter or under an exogenous promoter included in the construct. When two or more transgenes are inserted at a selected position of the CD38 locus, a linker sequence, such as a 2A linker or an IRES, is placed between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-I, and TaV (also referred to as "F2A", "E2A", "P2A", and "T2A", respectively), enabling the production of separate proteins from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter included in the CD38-KI / KO construct can be a constitutive, inducible, temporal-specific, tissue-specific, or cell-type-specific promoter such as CAG, or other promoters including but not limited to CMV, EF1α, PGK, and UBC.

[0161] 3. CD16 Knock-In CD16 has been identified as two isoforms of the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activates NK cells, and facilitates antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high affinity CD16", "non-cleavable CD16", or "high affinity non-cleavable CD16" refers to various CD16 variants. Wild-type CD16 has low affinity and undergoes ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes upon activation of NK cells. F176V (also called F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is an exemplary genetically engineered non-cleavable version of CD16. Engineered CD16 variants containing both F176V and S197P have high affinity and are non-cleavable, which is described in more detail in WO2015 / 148926, the complete disclosure of which is incorporated herein by reference. In addition, chimeric CD16 receptors in which the ectodomain of CD16 is essentially replaced by at least a portion of the CD64 ectodomain can also achieve the desired high affinity and non-cleavable characteristics of CD16 receptors capable of performing ADCC. In some embodiments, the replaced ectodomain of the chimeric CD16 comprises one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoforms or polymorphic variants).

[0162] Thus, in some embodiments, the high-affinity non-cleavable CD16 receptor (hnCD16) includes both F176V and S197P, and in some embodiments, includes F176V and the cleavage region is excluded. In some other embodiments, hnCD16 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between identity with any of SEQ ID NOs: 42, 43, and 44 of the exemplary sequences, each of which includes at least a portion of the CD64 ectodomain. SEQ ID NOs: 42, 43 and 44 are each encoded by exemplifying SEQ ID NOs: 45 - 47. As used throughout this specification and this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % identity = number of identical positions / total number of positions × 100). Comparison of sequences and determination of the percent identity between two sequences can be accomplished using mathematical algorithms recognized in the art. [Sequence Listing 3] TIFF2025108510000010.tif46167[Sequence Listing 4] TIFF2025108510000011.tif46169[Sequence Listing 5] TIFF2025108510000012.tif51169[Sequence Listing 6] TIFF2025108510000013.tif92169[Sequence Listing 7] TIFF2025108510000014.tif97169[Sequence Listing 8] TIFF2025108510000015.tif92169

[0163] Accordingly, among other edits contemplated and described herein, cloned iPSCs genetically engineered to contain a high-affinity non-cleavable CD16 receptor (hnCD16) are provided herein, and the genetically engineered iPSCs can differentiate into effector cells containing the hnCD16 introduced into the iPSCs. In some embodiments, the effector cells derived to contain hnCD16 are NK cells. In some embodiments, the effector cells derived to contain hnCD16 are T cells. The exogenous hnCD16 expressed in the iPSCs or their derivative cells exhibits high affinity not only for ADCC antibodies or fragments thereof, but also for binding to bispecific, trispecific, or multispecific engagers or binders that recognize the extracellular binding domain of CD16 or CD64 of the hnCD16. Bispecific, trispecific, or multispecific engagers or binders are further described below in this application (see below). Thus, this application provides a derivative effector cell or a population of its cells pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed in the following sections, via high-affinity binding to the extracellular domain of hnCD16 expressed on the derivative effector cells, wherein the hnCD16 comprises the extracellular binding domain of CD64 or CD16 having F176V and S197P.

[0164] In some other embodiments, the native CD16 transmembrane domain and / or intracellular domain of hnCD16 are further modified or replaced such that a chimeric Fc receptor (CFcR) is produced to include a non-native transmembrane domain, a non-native stimulatory domain, and / or a non-native signaling domain. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor other than the receptor that provides the extracellular domain. In the figures herein, the CFcR based on CD16 or its variant does not have a transmembrane domain, stimulatory domain, or signaling domain derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native transmembrane domain derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T cell receptor polypeptide. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native stimulatory / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD1, LAG3, 2B4, BTLA, DAP10, DAP12, CTLA4, or NKG2D polypeptide. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of hnCD16, the chimeric receptor provided includes a transmembrane domain and a signaling domain, both of which are derived from one of IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides.One particular embodiment of the hnCD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the extracellular domain of the full-length or partial sequence of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P. Another embodiment of the hnCD16-based chimeric Fc receptor comprises the transmembrane domain and the signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the extracellular domain of the full-length or partial sequence of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P.

[0165] The various embodiments of the hnCD16-based chimeric Fc receptors described above can bind to the Fc region of an antibody or its fragment with high affinity, or to the Fc region of a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory and / or signaling domains of the chimeric receptor enable effector cell activation and cytokine secretion, as well as killing of tumor cells targeted by the antibody, or the tumor antigen-binding component and the Fc region of the bispecific, trispecific, or multispecific engager or binder. Without being limited by theory, through the non-native transmembrane, stimulatory and / or signaling domains of the hnCD16-based chimeric Fc receptor, or through binding of the engager to the external domain, the CFcR contributes to the killing capacity of effector cells and increases the proliferation and / or the potential for proliferation of effector cells. Antibodies and engagers can bring tumor cells expressing an antigen into proximity with effector cells expressing CFcR, which also contributes to enhanced killing of tumor cells. Exemplary tumor antigens for bispecific, trispecific, multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding effector cells expressing the hnCD16-based CFcR when attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0166] Unlike the endogenous CD16 receptor expressed by primary NK cells that is cleaved from the cell surface after NK cell activation, the various uncleavable versions of CD16 in derived NK avoid shedding of CD16 and maintain a certain level of expression. In derived NK cells, uncleavable CD16 increases the expression of TNFα and CD107a, which exhibit improved cell function. Uncleavable CD16 also enhances antibody-dependent cell-mediated cytotoxicity (ADCC), as well as the binding of bispecific, trispecific, or multispecific engagers. ADCC is an NK cell-mediated lysis mechanism via the binding of CD16 to antibody-coated target cells. The additional high-affinity characteristics of the introduced hnCD16 in derived NK cells also enable in vitro loading of ADCC antibodies onto NK cells via hnCD16 prior to administering the cells to a subject in need of cell therapy. As provided, hnCD16 can, in some embodiments, include F176V and S197P, or can include a full or partial ectodomain derived from CD64 as exemplified by SEQ ID NO: 42, 43, or 44, or can further include at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides derived NK or a cell population thereof pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as detailed in the following sections.

[0167] Unlike primary NK cells, mature T cells from a primary source (i.e., natural / natural sources such as peripheral blood, cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing exogenous non-cleavable CD16 could differentiate into functional derivative T cells that not only express exogenous CD16 but also can perform functions through the acquired ADCC mechanism without impairing the developmental biology of T cells. This acquired ADCC in the derivative T cells can additionally be used as an approach for dual targeting and / or as an approach to rescue the antigen escape that often occurs in CAR-T cell therapy, where tumors relapse with a reduced or lost expression of the CAR-T-targeted antigen or with mutated antigens that avoid recognition by the CAR (chimeric antigen receptor). When the derivative T cells contain acquired ADCC via exogenous CD16 expression and the antibody targets a tumor antigen different from that targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent the recurrence or relapse of the targeted tumor, which is common in CAR-T therapy. Such strategies that reduce and / or prevent antigen escape while achieving dual targeting are similarly applicable to NK cells expressing one or more CARs. The various CARs that can be used in this antigen escape reduction and prevention strategy include the CARs described in this application.

[0168] Thus, in embodiments, the present invention provides derived T cells that include exogenous CD16 in addition to at least one CAR provided. In further provided embodiments, the derived T cells obtained herein include CD38 knockout in addition to the expression of hnCD16 and CAR. In some embodiments, the hnCD16 included in the derived T cells includes F176V and S197P. In some other embodiments, the hnCD16 included in the derived T cells includes a full or partial ectodomain derived from CD64 as exemplified by SEQ ID NO: 42, 43, or 44, or may further include at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As explained, such derived T cells have an acquired mechanism of targeting tumors with monoclonal antibodies contemplated by ADCC to enhance the therapeutic effect of the antibodies. As disclosed, this application also provides derived T cells or a cell population thereof pre-filled with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed in the following sections. In some other embodiments, the derived T cells expressing the provided hnCD16 and CAR are also CD38 null, such that the cells can be avoided from being eliminated when in the presence of a therapeutic agent that targets the tumor antigen CD38. In one embodiment, the therapeutic agent that targets the tumor antigen CD38 is a CD38 antibody. In another embodiment, the therapeutic agent that targets the tumor antigen CD38 is a CD38-CAR that includes an endodomain as described herein.

[0169] 4. Exogenously introduced cytokines and / or cytokine signaling By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicity from such practice is reduced while cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, one or more partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors are introduced into cells to enable cytokine signaling, with or without the expression of the cytokine itself, thereby reducing the risk of cytokine toxicity and maintaining or improving cell growth, proliferation, expansion, and / or effector function. In some embodiments, the introduced cytokines and / or their respective native or modified receptors for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary.

[0170] Figure 1 shows several construct designs using IL15 as an example. Any transmembrane (TM) domain of the designs in Figure 1 can be native to the IL15 receptor or modified or substituted with the transmembrane domain of any other membrane-bound protein.

[0171] Design 1: IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics trans-presentation of IL15 without eliminating cis-presentation of IL15.

[0172] Design 2: IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without eliminating cis-presentation of IL15 and ensuring membrane binding of IL15.

[0173] Design 3: IL15Rα with a truncated intracellular domain is fused to IL15 at the C-terminus via a linker, mimicking the trans-presentation of IL15, maintaining the membrane binding of IL15, and eliminating cis-presentation and / or any other potential signaling pathways mediated by normal IL15R via its intracellular domain. The intracellular domain of IL15Rα is thought to be important for the receptor to be expressed in IL15-responsive cells and for the responsive cells to expand and function. Such a truncated construct includes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 48, which can be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 49. In one embodiment of the truncated IL15 / IL15Rα, the construct does not include the last four amino acids "KSRQ" of SEQ ID NO: 48 and includes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 50. [Sequence Listing 9] TIFF2025108510000016.tif50169[Sequence Listing 10] TIFF2025108510000017.tif109167[Sequence Listing 11] TIFF2025108510000018.tif52169

[0174] One of ordinary skill in the art will understand that the signal peptide and linker sequences above are exemplary and in no way limit those variations suitable for use as a signal peptide or linker. There are many suitable signal peptide or linker sequences known to and available to one of ordinary skill in the art. One of ordinary skill in the art will understand that the signal peptide and / or linker sequence may be replaced with another sequence without modifying the activity of the functional peptide provided by the signal peptide or linked by the linker.

[0175] Design 4: Since the construct of Design 3 has been shown to function in promoting the survival and proliferation of effector cells, the cytoplasmic domain of IL15Rα has been shown to be dispensable in such a design without adversely affecting the autonomous function of effector cells equipped with IL15. Design 4 is a construct that provides another functional alternative to Design 3. Except for the Sushi domain, essentially the entire IL15Rα is deleted, and IL15 is fused to one transmembrane domain at one end (mb-Sushi), and, optionally, includes a linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of any membrane-bound protein. In constructs such as Design 4, when only the desirable trans-presentation of IL15 is retained, unwanted signaling via IL15Rα, including cis-presentation, is excluded. 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 to SEQ ID NO: 51, which may be encoded by the exemplary nucleic acid sequence represented by SEQ ID NO: 52. [Sequence Listing 12] TIFF2025108510000019.tif46169[Sequence Listing 13] TIFF2025108510000020.tif82169

[0176] One of ordinary skill in the art will understand that the signal peptide and linker sequences described above are exemplary and in no way limit those modifications suitable for use as a signal peptide or linker. There are many suitable signal peptide or linker sequences known to and available to those of ordinary skill in the art. One of ordinary skill in the art will understand that the signal peptide and / or linker sequence may be replaced with another sequence without modifying the activity of the functional peptide provided by the signal peptide or linked by the linker.

[0177] Design 5: A native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to enable constitutive signaling and maintain IL15 membrane binding and trans-presentation.

[0178] Design 6: A native or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling of the cytokine and membrane-bound trans-presentation. The common receptor γC, also called the common gamma chain or CD132, is also known as the interleukin 2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukins, including but not limited to the IL2, IL4, IL7, IL9, IL15, and IL21 receptors.

[0179] Design 7: An engineered IL15Rβ that forms homodimers in the absence of IL15 is useful for generating constitutive signaling of the cytokine.

[0180] In some embodiments, one or more of the cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or their receptors can be introduced into iPSCs and their derived cells upon iPSC differentiation using one or more of the designs of FIG. 1. In some embodiments, the expression and signaling of IL2 or IL15 on the cell surface is through the constructs depicted in any one of Designs 1-7. In some embodiments, the expression and signaling of IL4, IL7, IL9, or IL21 on the cell surface is through the constructs depicted in Design 5, 6, or 7 by using either a common receptor or a cytokine-specific receptor. In some embodiments, the IL7 surface expression and signaling is through the constructs depicted in Design 5, 6, or 7 by using either a common receptor or a cytokine-specific receptor (such as the IL4 receptor). Any transmembrane (TM) domain of any of the designs of FIG. 1 can be native to the corresponding cytokine receptor or modified or replaced with the transmembrane domain of any other membrane-bound protein.

[0181] In iPSCs and derivative cells therefrom that include both a CAR and exogenous cytokine and / or cytokine receptor signaling, the CAR and IL can be expressed in separate constructs or co-expressed in a bicistronic construct that includes both the CAR and IL. In some further embodiments, IL15 in the form represented by any of the construct designs of FIG. 1 can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence exemplified, for example, as CAR-2A-IL15 or IL15-2A-CAR. Thus, IL15 and the CAR are in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct includes the IL15 of Design 3 of FIG. 1. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct includes the IL15 of Design 3 of FIG. 1. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct includes the IL15 of Design 7 of FIG. 1. When CAR-2A-IL15 or IL15-2A-CAR is expressed, the expressed CAR and IL15 dissociate by the self-cleaving 2A peptide, and the dissociated IL15 is presented on the cell surface. The CAR-2A-IL15 or IL15-2A-CAR bicistronic design enables coordinated CAR and IL15 expression, both in terms of timing and amount, and under the same regulatory mechanisms that can be selected, for example, to incorporate an inducible promoter for expression of a single ORF. Self-cleaving peptides are found in members of the picornaviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-I) (Donnelly, ML, et al, J. Gen. Virol, 82, 1027-101(2001), Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732(2001)), as well as in members of the cardioviruses such as Theiler's virus (e.g., Theiler's murine encephalomyelitis virus) and encephalomyocarditis virus.The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV may also be referred to as "F2A", "E2A", "P2A", and "T2A", respectively.

[0182] The bicistronic CAR-2A-IL15 or IL15-2A-CAR embodiments disclosed herein for IL15 also contemplate the expression of any other cytokine provided herein, such as IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, IL2 cell surface expression and signaling are via constructs illustrated in any of Designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling are via constructs illustrated in Designs 5, 6, or 7 using either a common receptor and / or a cytokine-specific receptor.

[0183] 5. HLA-I and HLA-II deficiency Often, to avoid the problem of allogeneic rejection, it is necessary to match multiple HLA class I and class II proteins for the tissue compatibility of an allogeneic recipient. iPSC cell lines with both HLA class I and HLA class II protein expression eliminated or substantially reduced and their derived cells differentiated therefrom are provided herein. HLA class I deficiency can be achieved by a functional deletion of any region of the HLA class I locus (chromosome 6p21), or a deletion or reduced expression level of HLA class I-related genes including, but not limited to, the beta2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for the cell surface expression of all HLA class I heterodimers. B2M null cells are HLA-I deficient. HLA class II deficiency can be achieved by a functional deletion or reduction of HLA-II-related genes including, but not limited to, RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through the activation of the transcription factor RFX5 required for the expression of class II proteins. CIITA null cells are HLA-II deficient. For example, iPSCs and their derived cells having both HLA-I and HLA-II deficiencies for the lack of both B2M and CIITA expression are provided herein, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoid recognition and killing by host (allogeneic) T cells.

[0184] However, in some cell types, the lack of class I expression results in lysis by NK cells. To overcome this "self-loss" response, HLA-G can be optionally knocked in to avoid recognition and killing of NK cells by HLA-I-deficient effector cells derived from engineered iPSCs. In one embodiment, the provided HLA-I-deficient iPSCs and their derived cells further comprise an HLA-G knock-in. Alternatively, in one embodiment, the provided HLA-I-deficient iPSCs and their derived cells further comprise one or both of a CD58 knockout and a CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and facilitate the migration of cells, including immune cells. Whether and how disruption of CD58 and / or CD54 in iPSCs affects pluripotent cells and developmental biology in the differentiation of iPSCs into functional immune effector cells, including T cells and NK cells, was previously unknown. Also unknown was whether CD58 and / or CD54 knockout could effectively and / or sufficiently reduce the susceptibility of effector cells derived from HLA-I-deficient iPSCs to allogeneic NK cell killing. Here, it was shown that CD58 knockout is more efficient than CD54 knockout in reducing allogeneic NK cell activation, while double knockout of both CD58 and CD54 results in the most enhanced reduction of NK cell activation. In some observations, CD58 and CD54 double knockout is more effective than HLA-G overexpression in HLA-I-deficient cells in overcoming the "self-loss" effect.

[0185] As provided above, in some embodiments, HLA-I and HLA-II deficient iPSCs and their derivative cells have an exogenous polynucleotide encoding HLA-G. In some embodiments, HLA-I and HLA-II deficient iPSCs and their derivative cells are CD58 null. In some other embodiments, HLA-I and HLA-II deficient iPSCs and their derivative cells are CD54 null. In yet some other embodiments, HLA-I and HLA-II deficient iPSCs and their derivative cells are CD58 null and CD54 null.

[0186] In some embodiments, engineering for HLA-I and / or HLA-II deficiency can be bypassed or kept intact by expressing an inactivated CAR targeting surface proteins upregulated in activated recipient immune cells to avoid allograft rejection. In some embodiments, such upregulated surface proteins in activated recipient immune cells include, but are not limited to, CD38, CD25, CD69, or CD44. When a cell expresses such an inactivated CAR, it is preferred that the cell does not express or has not knocked out the same surface protein targeted by the CAR.

[0187] 6. Genetically engineered iPSCs and derivative cells provided herein In light of the above, the present application provides iPSCs, iPSC line cells, or populations thereof, and derived effector cells obtained from differentiating the iPSCs, each cell comprising at least a CAR having an endodomain as described herein. In some embodiments, the derived effector cells include, but are not limited to, mesodermal cells with the potential to become definitive hemogenic endothelium (HE), definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), 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 properties not present in primary NK, T, and / or NKT cells.

[0188] Also provided herein is a CAR further comprising CD38− / − (also referred to herein as "CD38 null" or CD38 knockout), and the cells are iPSCs, iPSC line cells, or derived functional effector cells comprising the CAR and CD38 knockout obtained from iPSC differentiation. In some embodiments, the derived effector cells include, but are not limited to, mesodermal cells with the potential to become definitive hemogenic endothelium (HE), definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), 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 properties not present in primary NK, T, and / or NKT cells.

[0189] iPSCs are further provided herein that include a polynucleotide encoding a CAR and a polynucleotide encoding high affinity non-cleavable CD16 (hnCD16), and the iPSCs are capable of differentiating to generate functional derived hematopoietic cells. Cells containing both CAR and hnCD16 are suitable for dual targeting by CAR binding and CD16-mediated ADCC, enhancing the precision of tumor targeting, promoting tumor killing, and minimizing the impact of tumor antigen escape. Further, in some embodiments, the iPSCs and / or their derived effector cells containing CD38-CAR and hnCD16 having the endodomains provided herein are also CD38 null, such that when using a CD38 antibody to induce hnCD16-mediated enhanced ADCC, the iPSCs and / or their derived effector cells containing CD38 knockout, CD38-CAR, and hnCD16-CD38 can target CD38-expressing (tumor) cells and / or alloreactivated recipient cells without causing effector cell elimination, thereby increasing the persistence and / or survival of the iPSCs and their effector cells. In some embodiments, the effector cells include T cells. In some embodiments, the effector cells include NK cells. T cells or NK cells derived from iPSCs containing CAR, CD38 null, and hnCD16 experience reduced cell depletion in the presence of a CD38 antibody or CD38 CAR, have ADCC (acquired ADCC in the case of T cells), and provide additional multiple mechanisms of tumor killing while improving cell persistence.

[0190] The iPSCs comprising the first CAR provided herein may include a polynucleotide encoding a second chimeric antigen receptor (CAR) having target specificity other than the first CAR, and the iPSCs can differentiate to generate functional effector cells having two CARs targeting two different tumor antigens. In one embodiment, the two different antigens targeted by the CARs included in the iPSCs and their derived effector cells include, but are not limited to, MICA / B, CD19, BCMA, CD20, CD22, CD38, CD123, CD25, CD69, CD44, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1. In one embodiment, the iPSCs and / or their derived effector cells have a CAR targeting CD38, CD25, CD69, or CD44, and the cells are also null in the targeted protein.

[0191] Also provided are iPSCs comprising a polynucleotide encoding a CAR provided herein, as well as a polynucleotide encoding at least one exogenous cytokine and / or its receptor (IL) that enables cytokine signaling contributing to cell survival, persistence, and / or proliferation. The iPSCs can differentiate to generate functional derivative effector cells with improved survival, persistence, proliferation, and effector cell function. Exogenously introduced cytokine signaling includes any one or two or more of the signaling of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or complete peptide of the cytokine and / or its respective receptor for cytokine signaling is expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary. In some embodiments, the transient / temporary expression of cell surface cytokine / cytokine receptor is via RNA including retrovirus, Sendai virus, adenovirus, episome, minicircle, or mRNA. In some embodiments, the exogenous cell surface cytokine and / or receptor contained in the iPSC or its derivative cells included in the CAR enables the signaling of IL7. In some embodiments, the exogenous cell surface cytokine and / or receptor contained in the iPSC or its derivative cells included in the CAR enables the signaling of IL10. In some embodiments, the exogenous cell surface cytokine and / or receptor contained in the iPSC or its derivative cells included in the CAR enables the signaling of IL15. In some embodiments of the CAR IL iPSC, the IL15 expression is by construct 3 in FIG. 1. In some embodiments of the CAR IL iPSC, the IL15 expression is by construct 4 in FIG. 1.The CAR IL iPSC and its derivative cells of the above embodiments can autonomously maintain or improve cell growth, proliferation, expansion, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo. In some embodiments of the CAR IL iPSC and its derivative effector cells, the cells are CD38 null and can be used with a CD38 antibody to induce ADCC without causing effector cell elimination, thereby synergistically increasing the persistence and / or survival of the iPSC and its effector cells.

[0192] Also provided are iPSCs comprising a CAR, B2M knockout, and CIITA knockout, and optionally one of HLA-G overexpression, CD58 knockout, and CD54 knockout, which can differentiate to generate functional derived hematopoietic cells. The CAR B2M - / - CIITA - / - The iPSC and its derivative effector cells lack both HLA-I and HLA-II. In further embodiments, the HLA-I and HLA-II deficient CAR iPSC and its derivative effector cells are also CD38 null and can be used with a CD38 antibody that induces ADCC without causing effector cell elimination, thereby increasing the persistence and / or survival of the iPSC and its effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or survival.

[0193] From the above perspectives, provided herein are iPSCs comprising a CAR and optionally one, two, three, or more of CD38 knockout, hnCD16, a second CAR, an exogenous cytokine / receptor, and B2M / CIITA knockout, wherein when B2M is knocked out, a polynucleotide encoding HLA-G or at least one of CD58 and CD54 knockouts is optionally introduced, and the iPSCs are capable of differentiating to generate functional derived hematopoietic cells. Also included in the present application are functional iPSC-derived effector cells comprising a CAR and optionally one, two, three, or more of CD38 knockout, hnCD16, B2M / CIITA knockout, a second CAR, and an exogenous cytokine / receptor, wherein when B2M is knocked out, a polynucleotide encoding HLA-G or at least one of CD58 and CD54 knockouts is optionally introduced, and the derived effector cells include mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic system multipotent progenitor cells (MPP), 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, but are not limited thereto.

[0194] Another aspect provided herein includes iPSCs or iPSC-derived cells comprising a truncated fusion protein of IL15 and IL15Rα, wherein the fusion protein does not include an intracellular domain. As shown in FIG. 1 as "IL15Rα(ΔICD) fusion" and "IL5 / mb-Sushi", these embodiments are further collectively abbreviated as IL15Δ throughout this application and are one of the "IL" embodiments shown in Table 3. In some embodiments of "IL", the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 48, 51, or 50. In some embodiments of "IL", the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments of "IL", the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments of "IL", the truncated IL15 / IL15Rα fusion protein lacking an intracellular domain comprises the amino acid sequence of SEQ ID NO: 50.In some embodiments of iPSCs or iPSC-derived cells comprising a truncated IL15 / IL15Rα fusion protein lacking the intracellular domain (IL15Δ), the cells further comprise a CAR and optionally one or more of CD38 knockout, hnCD16, a second CAR, an exogenous cytokine / receptor, and B2M / CIITA knockout. When B2M is knocked out, one of a polynucleotide encoding HLA-G or CD58 and CD54 knockout is optionally introduced. The iPSCs can differentiate to generate functional derivative effector cells, and the derivative effector cells include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell 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 derivative immune effector cells having one or more functional properties not present in primary NK, T, and / or NKT cells.

[0195] Accordingly, the present application provides iPSCs and their functional derivative hematopoietic cells comprising any one of the following genotypes in Table 2. "CAR" provided in Table 2 of the present application (第2)" represents a CAR having a target specificity different from that of the first CAR. Non-limiting examples include CARs targeting at least one of CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1. As provided in Table 2, "IL" represents one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which specific cytokine / receptor expression is selected. Further, "IL" also encompasses embodiments of IL15Δ, which is detailed above as a truncated fusion protein of IL15 and IL15Rα and does not contain an intracellular domain. Additionally, in one embodiment of such cells, when iPSCs and their functional derivative hematopoietic cells have a genotype containing both a CAR (the first CAR or the second CAR) and IL, the CAR and IL are contained in a bicistronic expression cassette containing a 2A sequence. By comparison, in some other embodiments, the CAR and IL are in separate expression cassettes contained in iPSCs and their functional derivative hematopoietic cells. In a specific embodiment, the IL contained in iPSCs and their functional derivative effector cells that express both the CAR and IL is IL15 in construct 3 or 4 of Figure 1, and the IL15 construct is contained in an expression cassette either together with or separately from the CAR.

[0196]

Table 3-1

[0197]

Table 3-2

[0198]

Table 3-3

[0199] 7. Additional Modifications In some embodiments, iPSCs and their derived effector cells comprising any one of the genotypes in Table 2 may further comprise a deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, Fc receptor, engager, and introduced or increased expression in at least one of surface trigger receptors for binding to bispecific, multispecific or universal engagers.

[0200] Bispecific or multispecific engagers are fusion proteins consisting of two or more single-chain variable fragments (scFvs) of different antibodies, where at least one scFv binds to an effector cell surface molecule and at least one other binds to tumor cells via a tumor-specific surface molecule. Exemplary effector cell surface molecules or surface trigger receptors that can be used for bispecific or multispecific engager recognition or binding include, but are not limited to, CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, and the chimeric Fc receptors disclosed herein. In some embodiments, CD16 expressed on the surface of effector cells for engager recognition is CD16 (including F176V and optionally S197P) or the CD64 extracellular domain as described in Section I.2, and hnCD16 including a natural or non-natural transmembrane domain, stimulatory domain, and / or signaling domain. In some embodiments, CD16 expressed on the surface of effector cells for engager recognition is an hnCD16-based chimeric Fc receptor (CFcR). In some embodiments, the hnCD16-based CFcR includes the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, and the extracellular domain of hnCD16 is derived from the extracellular domain of the full-length or partial sequence of CD64 or CD16, and the extracellular domain of CD16 includes F176V and optionally S197P. Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, ROR1. In one embodiment, the bispecific antibody is 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. In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and the tumor cell antigen binding domain, for example, a modified IL15 (in some publications called TriKE, or trispecific killer engager) as a linker for effector NK cells that promotes effector cell proliferation. In one embodiment, the TriKE is CD16-IL15-EPCAM or CD64-IL15-EPCAM. In another embodiment, the TriKE is CD16-IL15-CD33 or CD64-IL15-CD33. In yet another embodiment, the TriKE is NKG2C-IL15-CD33 ("2C1533").

[0201] In some embodiments, the surface trigger receptor for the bispecific or multispecific engager can be endogenous to the effector cell, sometimes depending on the cell type. In some other embodiments, the methods and compositions provided herein are used, i.e., iPSCs comprising the genotypes listed in Table 2 are further engineered to direct the differentiation of iPSCs into T cells, NK cells, or any other effector cells comprising the same genotype and surface trigger receptor as the source iPSC, and one or more exogenous surface trigger receptors can be introduced into the effector cells.

[0202] 8. Antibodies for Immunotherapy In some embodiments, in addition to the genomically engineered effector cells provided herein, additional therapeutic agents including antibodies or antibody fragments that target antigens associated with a condition, disease, or indication can be used in combination therapy with these effector cells. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, as additional therapeutic agents for the administered iPSC-derived effector cells, antibodies suitable for combination therapy include CD20 antibodies (rituximab, belimumab, ofatumumab, ublituximab, ocrelizumab, obinutuzumab), HER2 antibodies (trastuzumab, pertuzumab), CD52 antibodies (alemtuzumab), EGFR antibodies (cetuximab), GD2 antibodies (dinutuximab), PDL1 antibodies (avelumab), CD38 antibodies (daratumumab, isatuximab, MOR202), CD123 antibodies (7G3, CSL362), SLAMF7 antibodies (elotuzumab), MICA / B antibodies (7C6, 6F11, 1C2), and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars, but are not limited thereto. In some embodiments, the iPSC-derived effector cells include hematopoietic lineage cells comprising the genotypes listed in Table 2. In some embodiments, the iPSC-derived effector cells include NK cells comprising the genotypes listed in Table 2. In some embodiments, the iPSC-derived effector cells include T cells comprising the genotypes listed in Table 2.

[0203] In some embodiments of combinations useful for the treatment of liquid or solid tumors, the combination comprises a preselected monoclonal antibody and iPSC-derived NK cells or T cells comprising at least a CAR having the provided endodomain. In some embodiments of combinations useful for the treatment of liquid or solid tumors, the combination comprises a preselected monoclonal antibody and iPSC-derived NK cells or T cells comprising at least hnCD16 and a CAR having the provided endodomain. In some embodiments of combinations useful for the treatment of liquid or solid tumors, the combination comprises a preselected monoclonal antibody and iPSC-derived NK cells or T cells comprising at least hnCD16 and a CAR comprising the provided endodomain. Without being limited by theory, hnCD16 provides enhanced ADCC of the monoclonal antibody, while the CAR not only targets a specific tumor antigen but also uses a dual-targeting strategy in combination with a monoclonal antibody targeting a different tumor antigen to prevent tumor antigen escape. In some embodiments of combinations useful for the treatment of liquid or solid tumors, the combination comprises at least a CD38-CAR comprising the endodomain provided herein, iPSC-derived NK cells or T cells comprising CD38 null, and a CD38 antibody. In one embodiment, the combination comprises at least a CD38-CAR comprising the endodomain provided herein, iPSC-derived NK cells comprising CD38 null and hnCD16, and one of daratumumab, isatuximab, and MOR202, which is a CD38 antibody. In one embodiment, the combination comprises a CD38-CAR comprising the endodomain provided herein, iPSC-derived NK cells comprising CD38 null and hnCD16, and daratumumab.In some further embodiments, the iPSC-derived NK cells included in the combination with daratumumab comprise a CAR targeting at least one of CD38-CAR, CD38 null, hnCD16, IL15, and MICA / B or CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1, and IL15 is co-expressed or expressed separately with the CAR, and IL15 is in any one of the forms presented in constructs 1-7 of FIG. 1. In some specific embodiments, IL15 is in the form of construct 3, 4, or 7 when expressed simultaneously or separately with the CAR.

[0204] 9. Checkpoint inhibitor Checkpoints are cell molecules, often cell surface molecules, that can suppress or downregulate the immune response when not inhibited. It is now clear that certain immune checkpoint pathways in tumors are exploited as a major mechanism of immune tolerance, particularly against T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can reduce checkpoint gene expression or gene products, or decrease 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 landscape of oncology, and these agents have produced long-term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a major concern. One aspect of the present application provides a therapeutic approach for overcoming CI resistance by including functionally derived cells that are genomically engineered to be provided in combination therapy with a CI. In one embodiment of the combination therapy, the derived cells are NK cells. In another embodiment of the combination therapy, the derived cells are T cells. In addition to exhibiting direct anti-tumor capabilities, the derived NK cells provided herein have been shown to be resistant to PDL1-PD1 mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at the tumor site. Thus, tumor infiltration of T cells promoted by functionally potent genomically engineered derived NK cells has been shown to synergize with T cell-targeted immunotherapy involving checkpoint inhibitors to alleviate local immunosuppression and reduce tumor burden.

[0205] In one embodiment, the derived NK cells for combination therapy with checkpoint inhibitors comprise a CAR comprising the endodomain provided herein, and optionally CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and one, two, three, or more of exogenous cell surface cytokine and / or receptor expression, and when B2M is knocked out, a polynucleotide encoding HLA-G, or at least one of CD58 or CD54 knockout is optionally included. In some embodiments, the derived NK cells comprise any one of the genotypes listed in Table 2. In some embodiments, the derived NK cells as described above further comprise a deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosomal 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, Fc receptor, engager, and further comprise an introduced or increased expression in at least one of a surface trigger receptor for binding to a bispecific, multispecific or universal engager.

[0206] In another embodiment, the derived T cells for combination therapy with checkpoint inhibitors comprise a CAR comprising the endodomains provided herein, and optionally CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and one, two, three, or more of exogenous cell surface cytokine and / or receptor expression, and when B2M is knocked out, a polynucleotide encoding HLA-G, or one of CD58 or CD54 knockout is optionally included. In some embodiments, the derived T cells comprise any one of the genotypes listed in Table 2. In some embodiments, the derived T cells described above further comprise a deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, Fc receptor, engager, and further comprise an introduced or increased expression in at least one of a surface trigger receptor for binding to a bispecific, multispecific or universal engager.

[0207] The above-mentioned derived NK cells or derived T cells are obtained by differentiating an iPSC clone strain comprising a CAR containing the endodomain provided herein, and optionally one, two, three, or all four of CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and exogenous cell surface cytokine expression. When B2M is knocked out, a polynucleotide encoding HLA-G, or at least one of CD58 and CD54 knockout is optionally introduced. In some embodiments, the above-mentioned iPSC clone strain further comprises a deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or an introduced or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A R, antigen-specific TCR, Fc receptor, engager, and an introduced or increased expression in at least one of a surface trigger receptor for binding to a bispecific, multispecific or universal engager.

[0208] Checkpoint inhibitors suitable for combination therapy with the derived NK cells or T cells provided in this specification include, but are not limited to, antagonists of PD1 (Pdcdl, CD279), PDL-1 (CD274), TIM3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG3 (Lag3, CD223), CTLA4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0209] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab)’2, F(ab)’3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one, two, or more than two checkpoint inhibitors include at least one of atezolizumab (PDL1 mAb), avelumab (PDL1 mAb), durvalumab (PDL1 mAb), tremelimumab (CTLA4 mAb), ipilimumab (CTLA4 mAb), IPH4102 (KIR antibody), IPH43 (MICA antibody), IPH33 (TLR3 antibody), lirilumab (KIR antibody), monalizumab (NKG2A antibody), nivolumab (PD1 mAb), pembrolizumab (PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0210] In some embodiments, since many miRNAs are found as regulators that control the expression of immune checkpoints, antagonists that inhibit any of the above checkpoint molecules are microRNA-based (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint antagonist miRNAs include, but are not limited to, miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197, miR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c.

[0211] Some embodiments of the provided combination therapies with derived NK cells or derived T cells include at least one checkpoint inhibitor that targets at least one checkpoint molecule, and the derived cells have the genotypes listed in Table 2. Some other embodiments of the provided combination therapies with derived NK cells or T cells include two, three or more checkpoint inhibitors such that two, three or more checkpoint molecules are targeted. In some embodiments of the combination therapy comprising at least one checkpoint inhibitor and derived cells having the genotypes listed in Table 2, the checkpoint inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the checkpoint inhibitor is produced by the derived cells by expressing an exogenous polynucleotide sequence encoding the antibody, or a fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding an antibody or a fragment or variant thereof that inhibits a checkpoint is co-expressed with a CAR in either a separate construct, or a bicistronic construct comprising both the CAR and the sequence encoding the antibody or a fragment thereof. In some further embodiments, the sequence encoding the antibody or a fragment thereof can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence illustrated, for example, as CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences of the checkpoint inhibitor and the CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered, expressed, and secreted as a payload by derived effector cells capable of infiltrating the tumor microenvironment (TME), binding to the TME neutralizes inhibitory checkpoint molecules and enables activation of effector cells by activating modalities such as CARs or activating receptors.In some embodiments, the checkpoint inhibitor co-expressed with the CAR inhibits at least one of the checkpoint molecules: PD1, PDL-1, TIM3, TIGIT, LAG3, CTLA4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR. In some embodiments, the checkpoint inhibitor co-expressed with the CAR in derivative cells having the genotypes listed in Table 2 is selected from the group consisting of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is atezolizumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is nivolumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar.

[0212] In some other embodiments of the combination therapy provided herein that includes at least one antibody that inhibits derived cells and checkpoint molecules, the antibody is not produced by or within the derived cells and is further administered before, simultaneously with, or after the administration of derived cells having the genotypes listed in Table 2. In some embodiments, the administration of one, two, three or more checkpoint inhibitors in combination therapy with the provided derived NK cells or T cells is simultaneous or sequential. In one embodiment of combination therapy comprising derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the treatment is atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and one or more of their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of combination therapy comprising derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the treatment is atezolizumab, or its humanized or Fc-modified variant, fragment, and its functional equivalent or biosimilar. In some embodiments of combination therapy comprising derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the treatment is nivolumab, or its humanized or Fc-modified variant, fragment, or its functional equivalent or biosimilar. In some embodiments of combination therapy comprising derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the treatment is pembrolizumab, or its humanized or Fc-modified variant, fragment, or its functional equivalent or biosimilar.

[0213] II. Methods of Targeted Genome Editing at Selected Loci of iPSCs Genome editing, also referred to as genomic editing or gene editing, which can be used interchangeably herein, is a type of genetic manipulation in which DNA is inserted, deleted, and / or replaced in the genome of a targeted cell. Targeted genome editing (which can be interchangeably referred to as "targeted genomic editing" or "targeted gene editing") enables insertion, deletion, and / or replacement at a preselected site within the genome. When an endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down due to the sequence deletion. Therefore, targeted editing can also be used to precisely disrupt the expression of an endogenous gene. The term "targeted integration" is used similarly herein and refers to a process involving the insertion of one or more exogenous sequences, regardless of the presence or absence of deletion of the endogenous sequence at the insertion site. In contrast, randomly integrated genes are subject to position effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomeric regions are particularly prone to transgene silencing. Conversely, newly integrated genes can affect the surrounding endogenous genes and chromatin, potentially altering cell behavior or supporting cell transformation. Therefore, inserting exogenous DNA into a preselected locus, such as a safe harbor locus or genomic safe harbor (GSH), is important for safe, efficient, copy number control, and reliable gene response control. Alternatively, the exogenous DNA can be inserted into a preselected locus where disruption of gene expression, including knockdown and knockout at the locus, is intended.

[0214] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach. In a nuclease-independent targeted editing approach, homologous recombination is induced by homologous sequences flanking the exogenous polynucleotide to be inserted, via the enzymatic machinery of the host cell.

[0215] Alternatively, targeted editing can be achieved at a higher frequency through the specific introduction of double-strand breaks (DSBs) by certain rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms that occur in response to DSBs, including non-homologous end joining (NHEJ). In the absence of a donor vector containing exogenous gene material, NHEJ often results in random insertions or deletions (indels) of a few endogenous nucleotides. In comparison, in the presence of a donor vector containing exogenous gene material flanked by a pair of homology arms, the exogenous gene material is introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration". In some cases, since the targeted integration site is intended to be within the coding region of the selected gene, targeted integration can disrupt gene expression and simultaneously result in knock-in and knock-out (KI / KO) in a single editing step.

[0216] One or more transgenes can be inserted at selected positions of a gene of interest (GOI) to simultaneously achieve gene knockout. Loci suitable for simultaneous knock-in and knockout (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant regions, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. Each site-specific targeting homology arm for position-selective insertion enables the transgene to be expressed either under the endogenous promoter of that site or under an exogenous promoter contained in the construct. When two or more transgenes are inserted at selected positions of the CD38 locus, a linker sequence, such as a 2A linker or IRES, is placed between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-I, and TaV (also referred to as "F2A", "E2A", "P2A", and "T2A" respectively), enabling the production of separate proteins from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter can be CAG, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters including, but not limited to, CMV, EF1α, PGK, and UBC.

[0217] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats) systems. In addition, the DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxb1 integrases is also a promising tool for targeted integration.

[0218] ZFN is a targeted nuclease that contains a nuclease fused to a zinc finger DNA binding domain. The "zinc finger DNA binding domain" or "ZFBD" means a polypeptide domain that binds to DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain, and its structure is stabilized by 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 whose design / composition results primarily from the application of rational criteria, such as substitution rules and computerized algorithms for processing information from existing ZFP designs and databases storing binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242, and 6,534,261, and also International Publications WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496. A "selected" zinc finger domain is a domain not found in nature and whose generation results primarily from empirical processes such as phage display, interaction trap, or hybrid selection. ZFN is described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most well-recognized example of ZFN in the art is the fusion of the FokI nuclease with a zinc finger DNA binding domain.

[0219] TALEN is a targeted nuclease that includes 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 a TAL effector protein that is involved in the binding of the TAL effector protein to DNA. TAL effector proteins are secreted from plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA binding domain depends on the effector variable number of imperfect 34-amino acid repeats that contain polymorphisms at select repeat positions called repeat variable diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.

[0220] Another example of a targeted nuclease found to be useful in the methods of the subject invention is a targeted Spo11 nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA binding domain having specificity for a DNA sequence of interest, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, and the like.

[0221] Additional examples of targeted nucleases suitable for use in the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used individually or in combination.

[0222] Other non-limiting examples of targeted nucleases include naturally occurring nucleases and recombinant nucleases; CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like.

[0223] Using Cas9 as an example, CRISPR / Cas9 requires two main components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to a target DNA sequence containing a PAM and a seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) that can direct Cas9 to target a selected sequence. These two components can be delivered into mammalian cells via transfection or transduction.

[0224] In the case of integration via DICE, a pair of recombinases, such as phiC31 and Bxb1, are used to provide unidirectional integration of exogenous DNA that is tightly restricted to small attB and attP recognition sites for each enzyme itself. Since these target att sites do not naturally occur in the mammalian genome, they need to be first introduced into the genome at the desired integration site. See, for example, U.S. Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0225] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct into a cell to enable site-specific homologous recombination by the cell host enzymatic machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to enable insertion via ZFN. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to enable insertion via TALEN. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into the cell, introducing a Cas9 expression cassette, and introducing a gRNA comprising a guide sequence specific to a desired integration site into the cell to enable insertion via Cas9. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases into a desired integration site of the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette of the DICE recombinase to enable targeted integration via DICE.

[0226] Promising sites for targeted integration are intragenic or extragenic regions of the human genome and include, but are not limited to, safe harbor loci or genomic safe harbors (GSHs) that can accommodate predictable expression of newly integrated DNA without theoretically adversely affecting the host cell or organism. A useful safe harbor needs to allow expression of the transgene sufficient to obtain the desired level of the protein or non-coding RNA encoded by the vector. A safe harbor also must not predispose cells to malignant transformation and must not alter cellular function. To be a potential safe harbor locus for integration, ideally, it needs to meet criteria including, but not limited to: no disruption of regulatory elements or genes as determined by sequence annotation; an intergenic region within a gene-dense region or a convergent position between two genes transcribed in opposite directions; maintaining distance to minimize the potential for long-range interactions between the gene adjacent to the transcriptional activator encoded by the vector, particularly the promoter of cancer-related genes and microRNA genes; and having clearly ubiquitous transcriptional activity as reflected by a broad spatial and temporal expression sequence tag (EST) expression pattern indicating ubiquitous transcriptional activity. This latter feature is particularly important in stem cells where chromatin remodeling during differentiation typically results in silencing of some loci and potential activation of other loci. Within a region suitable for exogenous insertion, the exact locus selected for insertion should lack repetitive elements and conserved sequences and should allow easy design of primers for homologous arm amplification.

[0227] Sites suitable for human genome editing, or specifically targeted integration, include, but are not limited to, the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the human ortholog of the mouse ROSA26 locus. In addition, the human ortholog of the mouse H11 locus may also be a suitable site for insertion using the compositions and targeted integration methods disclosed herein. Furthermore, the collagen and HTRP loci can also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be necessary, especially in stem cells for a particular integration event, and optimization of the insertion strategy, including promoter selection, exogenous gene sequence and placement, and construct design, is often required.

[0228] In the case of targeted indels, the editing site is often included in an endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted indel is related to the control and regulation of the immune response. In some other embodiments, the endogenous gene containing the targeted indel is related to a targeted modality, receptor, signaling molecule, transcription factor, drug target candidate, immune response control and regulation, or a protein that suppresses the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells, and their derived cells.

[0229] Accordingly, one aspect of the present invention provides a method of targeted integration at a selected locus, including a genomic safe harbor, or at another locus that is known or proven to be safe and well-controlled for constitutive or transient gene expression, such as AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, or that meets the criteria of a genomic safe harbor. In some embodiments, the targeted integration is at one of the loci where gene knockdown or knockout as a result of the integration is desired, such loci including, but not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0230] In one embodiment, the method of targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing a construct comprising a pair of homologous arms specific to a desired integration site and one or more exogenous sequences to enable site-specific homologous recombination by the cell host enzymatic machinery, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0231] In another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing into the cell a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site to enable ZFN-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, the TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing into the cell a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site to enable TALEN-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, the TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.In another embodiment, a method for targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell a gRNA comprising a CRISPR nuclease expression cassette and a guide sequence specific for a desired integration site to enable insertion via the CRISPR nuclease, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method for targeted integration in a cell comprises introducing into the desired integration site of the cell a construct comprising one or more att sites of a pair of DICE recombinases, introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing an expression cassette of the DICE recombinase to enable targeted integration via DICE, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0232] Furthermore, as provided herein, the above-described method for targeted integration in a safe harbor is used to insert any polynucleotide of interest, such as a safety switch protein, a targeting modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, and a polynucleotide encoding a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some other embodiments, a construct comprising one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the invention is a suicide gene encoding a safety switch protein. Suicide gene systems suitable for induced cell death include, but are not limited to, caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV); cytosine deaminase (CD) and 5-fluorocytosine (5-FC). In addition, some suicide gene systems are cell type-specific. For example, genetic modification of T lymphocytes with the B cell molecule CD20 allows them to be eliminated by administration of the mAb rituximab. Furthermore, the modified EGFR-containing epitope recognized by cetuximab can be used to deplete genetically engineered cells when the cells are exposed to cetuximab. Accordingly, one aspect of the invention provides a method for targeted integration of one or more suicide genes encoding a safety switch protein selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0233] In some embodiments, one or more exogenous polynucleotides incorporated by the methods herein are driven by an exogenous promoter operably linked in a construct for targeted integration. The promoter may be inducible or constitutive, and may be temporally specific, tissue specific, or cell type specific. Suitable constitutive promoters for the methods of the invention include, but are not limited to, the cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0234] The exogenous polynucleotides incorporated by the methods of this specification can be driven by endogenous promoters of the host genome at the integration site. In one embodiment, the methods of the invention are used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus of the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the methods of the invention are used for targeted integration at the ROSA26 locus of the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the methods of the invention are used for targeted integration at the H11 locus of the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the methods of the invention are used for targeted integration at the collagen locus of the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the methods of the invention are used for targeted integration at the HTRP locus of the genome of a cell. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous HTRP promoter. In theory, only correct insertion at the desired location will allow gene expression of an exogenous gene driven by an endogenous promoter.

[0235] In some embodiments, one or more exogenous polynucleotides included in a construct for a targeted integration method are driven by one promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to spread the physical separation between the parts and maximize access to the enzymatic machinery. The linker peptide of the linker sequence can consist of amino acids selected to make the physical separation between the parts (exogenous polynucleotide and / or the protein or peptide encoded thereby) more flexible or more rigid depending on the associated function. The linker sequence can be cleavable by a protease or chemically cleavable to yield separate parts. Examples of enzymatic cleavage sites in linkers include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is naturally produced by the host or is exogenously introduced. Alternatively, the cleavage site in the linker can be a site cleavable by a selected chemical, such as cyanogen bromide, hydroxylamine, or exposure to low pH. The optional linker sequence can serve purposes other than providing a cleavage site. The linker sequence should enable the effective placement of the parts relative to another adjacent part for the parts to function properly. The linker can also be a simple amino acid sequence of sufficient length to prevent steric hindrance between the parts. In addition, the linker sequence can provide post-translational modifications including, but not limited to, phosphorylation sites, biotinylation sites, sulfation sites, γ-carboxylation sites, etc. In some embodiments, the linker sequence is flexible so as not to hold a biologically active peptide in a single undesirable conformation. The linker can consist primarily of amino acids with small side chains such as glycine, alanine, and serine to provide flexibility. In some embodiments, about 80 or 90 percent or more of the linker sequence comprises glycine, alanine, or serine residues, particularly glycine and serine residues.In some 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 enables two separate proteins to be produced from a single translation. Suitable linker sequences can be readily identified empirically. Additionally, the suitable size and sequence of the linker sequence can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some embodiments, any two consecutive linker sequences are different.

[0236] Methods for introducing into cells a construct containing an exogenous polynucleotide for targeted integration can be achieved using methods of gene transfer into cells that are known per se. In one embodiment, the construct comprises the backbone of a viral vector such as an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a lentivirus vector, a Sendai virus vector. In some embodiments, plasmid vectors are used to deliver and / or express exogenous polynucleotides into target cells (such as pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo). In some other embodiments, episomal vectors are used to deliver exogenous polynucleotides into target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used for genetic manipulation to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV does not integrate into the host genome. In addition, episomal rAAV vectors mediate homologous-directed gene targeting at a much higher rate compared to transfection of conventional targeting plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions or substitutions into the target site of the genome of iPSCs. In some embodiments, the genome-modified iPSCs and their derivative cells obtained using the methods and compositions herein comprise at least one genotype listed in Table 2.

[0237] III. Methods for obtaining and maintaining genomically engineered iPSCs The present invention provides a method of obtaining and maintaining genomically engineered iPSCs that include one or more targeted edits at one or more desired sites, where the targeted edits continue to be intact and functional at each selected edit site in the expanded genomically engineered iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions, i.e., targeted integration and / or in / dels at the selected sites, into the genomes of the iPSCs and derivative cells therefrom. Among the many advantages of obtaining genomically engineered derivative cells by editing and differentiating iPSCs as provided herein, as compared to directly manipulating primary effector cells derived from a patient's peripheral blood, include, but are not limited to: an unlimited source of engineered effector cells; no need to repeatedly engineer effector cells, especially when multiple engineered modalities are included; the obtained effector cells have elongated telomeres and are rejuvenated due to low exhaustion; the effector cell population is homogeneous in terms of no editing site, copy number, and allelic modifications, random mutations, and expression diversity, mainly due to the ability to perform clonal selection on the engineered iPSCs provided herein.

[0238] In certain embodiments, genomically engineered iPSCs that include one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells in a cell culture medium shown in Table 3 as a Fate Maintenance Medium (FMM), and the iPSCs retain the targeted edits and functional modifications at the selected sites. The composition of the medium can be present in the medium in amounts within the optimal ranges shown in Table 3. iPSCs cultured in FMM have been shown to remain undifferentiated, maintain a basal or naive profile, and maintain genomic stability without the need to wash or select the culture, and readily give rise to in vitro differentiation through all three somatic lineages, embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation through teratoma formation. See, for example, International Publication No. WO2015 / 134652, the disclosure of which is incorporated herein by reference.

[0239]

Table 4

[0240] In some embodiments, genome-engineered iPSCs comprising one or more targeted integrations and / or indels are maintained, passaged, and expanded in a medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor and not or essentially free of a TGFβ receptor / ALK5 inhibitor, and the iPSCs retain the intact and functional targeted editing at the selected site.

[0241] Another aspect of the invention provides a method of generating genome-engineered iPSCs by generating non-pluripotent cells genome-engineered by targeted editing of iPSCs or first by targeted editing, and then reprogramming the selected / isolated genome-engineered non-pluripotent cells to obtain iPSCs comprising the same targeted editing as the non-pluripotent cells. A further aspect of the invention provides genome-engineered non-pluripotent cells that are undergoing reprogramming simultaneously by introducing targeted integrations and / or targeted indels into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and the reprogramming conditions include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genome engineering and reprogramming, targeted integrations and / or targeted indels can be introduced into the non-pluripotent cells before or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally small molecules.

[0242] In some embodiments, for the simultaneous genome engineering and reprogramming of non-pluripotent cells, targeted integration and / or indels may also be introduced into the non-pluripotent cells after the multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes including, but not limited to, SSEA4, Tra181, and CD30. A vector carrying the construct is introduced.

[0243] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor, and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 3). In some embodiments, genomically engineered iPSCs by any of the methods described above are further maintained and expanded using a mixture containing a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 3).

[0244] In some embodiments of methods for generating genomically engineered iPSCs, the method comprises genomically engineering the iPSCs by introducing one or more targeted integrations and / or indels into the iPSCs to obtain genomically engineered iPSCs having at least one genotype listed in Table 2. Alternatively, a method for generating genomically engineered iPSCs comprises: (a) introducing one or more targeted edits into non-pluripotent cells to obtain genomically engineered non-pluripotent cells comprising targeted integration and / or indels at selected sites; and (b) contacting the genomically engineered non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and optionally a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to obtain genomically engineered iPSCs comprising targeted integration and / or indels at selected sites. Alternatively, a method for generating genomically engineered iPSCs comprises: (a) contacting non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and optionally a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells; (b) introducing one or more targeted integrations and / or indels into the reprogramming non-pluripotent cells for genomic engineering; and (c) obtaining clonal genomically engineered iPSCs comprising targeted integration and / or indels at selected sites.

[0245] The reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in PCT / US2015 / 018801 and PCT / US16 / 57136 (the disclosures of which are incorporated herein by reference). One or more reprogramming factors can be in the form of polypeptides. Since the reprogramming factors can also be in the form of polynucleotides, they are introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai viral vector. In some embodiments, one or more polynucleotides are introduced by a combination of plasmids taking into account the stoichiometry of the various reprogramming factors. See, for example, International Publication No. WO2019 / 075057, the disclosure of which is incorporated herein by reference.

[0246] In some embodiments, non-pluripotent cells are introduced with multiple constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides can include suicide genes, or genes encoding targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNAs including, but not limited to, siRNA, shRNA, miRNA, and antisense nucleic acids. These exogenous polynucleotides can be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, temporally specific promoters, and tissue- or cell-type specific promoters. Thus, the polynucleotide is expressible under conditions that activate the promoter, for example, in the presence of an inducer, or in a particular differentiated cell type. In some embodiments, the polynucleotide is expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, one or more suicide genes are driven by a constitutive promoter, such as caspase-9 driven by CAG. These constructs containing different exogenous polynucleotides and / or different promoters can be introduced into non-pluripotent cells either simultaneously or sequentially. The non-pluripotent cells subjected to targeted integration of multiple constructs can be simultaneously contacted with one or more reprogramming factors to initiate reprogramming simultaneously with genome engineering, thereby obtaining genome-engineered iPSCs containing multiple targeted integrations in the same pool of cells. Thus, this robust method enables simultaneous reprogramming and engineering strategies to lead to clonal genome-engineered hiPSCs with multiple modalities integrated at one or more selected target sites.In some embodiments, the genome-edited iPSCs and their derivative cells obtained using the methods and compositions herein include at least one genotype listed in Table 2.

[0247] IV. Method for obtaining genetically engineered effector cells by differentiating genome-engineered iPSCs and by CAR end domain screening using an iPSC differentiation platform A further aspect of the invention provides a method for in vivo differentiation of genomically engineered iPSCs by teratoma formation, wherein the in vivo differentiated cells derived from the genomically engineered iPSCs carry intact and functional targeted editing including targeted integration and / or indels at desired sites. In some embodiments, the differentiated cells derived in vivo from genomically engineered iPSCs via teratomas contain one or more inducible suicide genes integrated at one or more desired sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci meeting genomic safe harbor criteria. In some other embodiments, the in vivo differentiated cells derived from genomically engineered iPSCs via teratomas contain a polynucleotide encoding a targeting modality, or a polynucleotide encoding a protein that promotes the transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some embodiments, the in vivo differentiated cells derived from genomically engineered iPSCs via teratomas containing one or more inducible suicide genes further contain one or more indels of endogenous genes associated with the control and mediation of the immune response. In some embodiments, the indels are contained in one or more endogenous checkpoint genes. In some embodiments, the indels are contained in one or more endogenous T cell receptor genes. In some embodiments, the indels are contained in one or more endogenous MHC class I suppressor genes. In some embodiments, the indels are contained in one or more endogenous genes associated with the major histocompatibility gene complex. In some embodiments, the indels are contained in one or more endogenous genes including, but not limited to, B2M, PD1, TAP1, TAP2, tapasin, TCR genes. In one embodiment, the genomically engineered iPSCs containing one or more exogenous polynucleotides at selected sites further contain targeted editing in the gene encoding B2M (beta-2-microglobulin).

[0248] In certain embodiments, genomic engineered iPSCs comprising one or more of the genetic modifications provided herein are used to direct hematopoietic cell lineages or any other specific cell type in vitro, and the derived non-pluripotent cells retain functional genetic modifications including targeted editing at selected sites. In one embodiment, cells derived from genomic engineered iPSCs include mesodermal cells with definitive hematopoietic endothelium (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, and macrophages, but are not limited thereto, and these cells derived from genomic engineered iPSCs retain functional genetic modifications including targeted editing at desired sites.

[0249] Differentiation methods and compositions applied to obtain hematopoietic cell lineages from iPSCs include, for example, those shown in International Publication No. WO2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, methods and compositions for generating hematopoietic cell lineages are in a serum-free, feeder-free, and / or stroma-free condition and in a culture platform that does not require scalable and monolayer EB formation, via secondary hematopoietic endothelium (HE) derived from pluripotent stem cells, including hiPSCs. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated cells and transdifferentiated cells, as well as various lineages of cells that have directly transitioned to a hematopoietic fate without passing through pluripotent intermediates. Similarly, cells produced by differentiating stem cells range from multipotent stem cells or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0250] Methods for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture involve contacting the pluripotent stem cells with a BMP pathway activator and optionally bFGF. As provided, mesodermal cells derived from pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with the potential to form secondary hematopoietic endothelium (HE) without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF and optionally a ROCK inhibitor and / or a WNT pathway activator causes the mesodermal cells with the potential to form secondary HE to differentiate into secondary HE cells, which also expand during differentiation.

[0251] The methods provided herein for obtaining hematopoietic lineage cells are superior to pluripotent stem cell differentiation via embryoid bodies (EBs) because EB formation results in moderate to minimal cell expansion and is important for many applications that require homogeneous expansion and monolayer culture, and does not allow for homogeneous differentiation of cells within the population, is difficult, and is inefficient.

[0252] The provided monolayer differentiation platform facilitates differentiation into secondary hematopoietic endothelium, which gives rise to hematopoietic stem cells and differentiated progeny such as T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion to enable the delivery of a therapeutically relevant number of pluripotent stem cell-derived effector cells for various therapeutic applications. Furthermore, monolayer culture using the methods provided herein yields functional hematopoietic lineage cells that enable the full spectrum of in vitro differentiation, ex vivo conditioning, and in vivo long-term hematopoietic self-renewal, reconstitution, and engraftment. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, secondary hematopoietic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0253] A method for directing the differentiation of pluripotent stem cells into cells of the secondary hematopoietic lineage, the method comprising: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate the differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor (the composition optionally not comprising a TGFβ receptor / ALK inhibitor) to initiate the differentiation and expansion of mesodermal cells with the potential for secondary HE from the mesodermal cells; and (iii) contacting the mesodermal cells with the potential for secondary HE with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator (the composition optionally not comprising a TGFβ receptor / ALK inhibitor) to initiate the differentiation and expansion of secondary hematopoietic endothelium from the pluripotent stem cell-derived mesodermal cells with the potential for secondary hematopoietic endothelium.

[0254] 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 (the composition not comprising a TGFβ receptor / ALK inhibitor) to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs comprising one or more gene imprints, and the one or more gene imprints comprised in the iPSCs are retained in the effector cells differentiated therefrom. In some embodiments of the method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of the hematopoietic lineage lacks the formation of embryoid bodies and is in a monolayer culture format.

[0255] In some embodiments of the above method, the obtained pluripotent stem cell-derived secondary hematopoietic endothelial cells are CD34+. In some embodiments, the obtained secondary hematopoietic endothelial cells are CD34+CD43-. In some embodiments, the secondary hematopoietic endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the secondary hematopoietic endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the secondary hematopoietic endothelial cells are CD34+CD43-CD93-. In some embodiments, the secondary hematopoietic endothelial cells are CD34+CD93-.

[0256] In some embodiments of the above method, the method further comprises: (i) contacting pluripotent stem cell-derived secondary hematopoietic endothelium with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, to initiate differentiation of the secondary hematopoietic endothelium into pre-T cell progenitor cells; and optionally, (ii) contacting the pre-T cell progenitor cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells. In some embodiments of this method, the pluripotent stem cell-derived T cell progenitor cells are CD34+CD45+CD7+. In some embodiments of this method, the pluripotent stem cell-derived T cell progenitor cells are CD45+CD7+.

[0257] In some further embodiments of the above method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the method comprises: (i) contacting secondary hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15 to initiate the differentiation of the secondary hematopoietic endothelium into pre-NK cell progenitor cells; and optionally, (ii) contacting pre-NK cell progenitor cells derived from pluripotent stem cells with a composition (the medium does not contain one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor) comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 to initiate the differentiation of the pre-NK cell progenitor cells into NK cell progenitor cells or NK cells. In some embodiments, the NK cell progenitor cells derived from pluripotent stem cells are CD3−CD45+CD56+CD7+. In some embodiments, the NK cells derived from pluripotent stem cells are CD3−CD45+CD56+ and are optionally further defined by NKp46+, CD57+, and CD16+.

[0258] Thus, using the above differentiation method, one or more populations of the following iPSC-derived hematopoietic cells can be obtained: (i) CD34+HE cells (iCD34) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2, (ii) secondary hematopoietic endothelium (iHE) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2, (iii) secondary HSCs using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2, (iv) multipotent progenitor cells (iMPP) using iMPP-A, (v) T-lineage cell progenitor cells (ipro-T) using one or more culture media selected from iTC-A2 and iTC-B2, (vi) T-lineage cells (iTC) using iTC-B2, (vii) NK-lineage cell progenitor cells (ipro-NK) using one or more culture media selected from iNK-A2 and iNK-B2, and / or (viii) NK-lineage cells (iNK), and iNK-B2. In some embodiments, the medium is as follows: a. iCD34-C contains one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator, and does not contain a TGFβ receptor / ALK inhibitor, b. iMPP-A contains a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11; c. iTC-A2 contains a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7, and optionally a BMP activator, d. iTC-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; e.iNK-A2 contains a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15; and f.iNK-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0259] In some embodiments, the genome-edited iPSC-derived cells obtained from the above methods contain one or more inducible suicide genes integrated at one or more desired integration sites including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In some other embodiments, the genome-edited iPSC-derived cells contain 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 trafficking, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some embodiments, the genome-edited iPSC-derived cells containing one or more suicide genes further contain one or more in / dels in one or more endogenous genes related to the control and mediation of the immune response, including but not limited to checkpoint genes, endogenous T cell receptor genes, and MHC class I inhibitory genes. In one embodiment, the genome-edited iPSC-derived cells containing one or more suicide genes further contain an in / del in the B2M gene and B2M is knocked out.

[0260] In addition, the applied dedifferentiation methods and compositions for obtaining genomically engineered hematopoietic cells of a second fate from genomically engineered hematopoietic cells of a first fate include, for example, those shown in International Publication No. WO2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided therein partially reprogram starting non-pluripotent cells into non-pluripotent intermediate cells by restricting the expression of the endogenous Nanog gene during reprogramming, and enable the non-pluripotent intermediate cells to be subjected to conditions for differentiating the intermediate cells into the desired cell type. In some embodiments, the genomically modified iPSCs and their derivative cells obtained using the methods and compositions herein include at least one genotype listed in Table 2.

[0261] V. Therapeutic uses of derivative immune cells having exogenous functional modalities differentiated from genetically engineered iPSCs In some embodiments, the present invention provides a composition comprising an isolated population or subpopulation of functionally enhanced derivative immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSCs comprise one or more targeted gene edits that can be retained in the iPSC-derived immune cells, and the genetically engineered iPSCs and their derivative cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived CD34 cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived proT or T lineage cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived proNK or NK lineage cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived immune regulatory cells or myeloid-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived genetically engineered immune cells are further modulated ex vivo for improved therapeutic potential. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells derived from iPSCs comprises an increase in the number or ratio of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells derived from iPSCs comprises an increase in the number or ratio of type I NKT cells. In another embodiment, the isolated population or subpopulation of genetically engineered immune cells derived from iPSCs comprises an increase in the number or ratio of adaptive NK cells. In some embodiments, the isolated population or subpopulation of iPSC-derived genetically engineered CD34 cells, HSC cells, T lineage cells, NK lineage cells, or myeloid-derived suppressor cells is allogeneic. In some other embodiments, the isolated population or subpopulation of iPSC-derived genetically engineered CD34 cells, HSC cells, T cells, NK cells, NKT cells, or MDSCs is autologous.

[0262] In some embodiments, the iPSCs for differentiation contain genetic imprints selected to confer desirable therapeutic attributes in effector cells, provided that the developmental biology of the cells during differentiation is not disrupted and the genetic imprints are retained and functional in the differentiated effector cells derived from the iPSCs.

[0263] In some embodiments, the genetic imprints of the pluripotent stem cells include (i) one or more genetically modified modalities obtained by genomic insertions, deletions, or substitutions in the genome of the pluripotent cells during or after reprogramming non-pluripotent cells to iPSCs, or (ii) one or more retainable therapeutic attributes of donor-specific, disease-specific, or treatment-response-specific source-specific immune cells, where the pluripotent cells are reprogrammed from source-specific immune cells and the iPSCs retain the therapeutic attributes of the source that are also included in the iPSC-derived hematopoietic lineage cells.

[0264] In some embodiments, the genetically modified modalities include one or more of a safety switch protein, a targeted modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a drug target candidate, or a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, regulation and modulation of the immune response, and / or survival of iPSCs or their derived cells. In some embodiments, the genetically modified iPSCs and their derived cells include the genotypes listed in Table 2. In some other embodiments, the genetically modified iPSCs and their derived cells that include the genotypes listed in Table 2 further include additional genetically modified modalities that include (1) deletion or reduced expression of one or more of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any gene in the chromosomal 6p21 region, and (2) introduced or increased expression of HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, an antigen-specific TCR, an Fc receptor, or a surface trigger receptor for binding to a bispecific or multispecific or universal engager.

[0265] In some other embodiments, hematopoietic lineage cells include therapeutic attributes of source-specific immune cells related to at least two combinations of (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) resistance to the tumor microenvironment, (iv) recruitment and immune modulation of bystander immune cells, (iv) improved target specificity by reduction of off-tumor effects, and (v) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0266] In some embodiments, iPSC-derived effector cells comprising the genotypes listed in Table 2, and the cells express at least one cytokine and / or its receptor, or any modified protein thereof, including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, and express at least a CAR. In some embodiments, the engineered expression of the cytokine and CAR is NK cell-specific. In some other embodiments, the engineered expression of the cytokine and CAR is T cell-specific. In one embodiment, the CAR comprises a MICA / B binding domain. In some embodiments, the iPSC-derived hematopoietic effector cells are antigen-specific. In some embodiments, the antigen-specific derived effector cells target liquid tumors. In some embodiments, the antigen-specific derived effector cells target solid tumors. In some embodiments, the antigen-specific iPSC-derived hematopoietic effector cells can rescue tumor antigen escape.

[0267] By introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy, various diseases can be recovered. In some embodiments, the provided iPSC-derived effector cells are for allogeneic adoptive cell therapy. In addition, in some embodiments, the present invention provides a therapeutic use of the above therapeutic composition by introducing the composition into a subject suitable for adoptive cell therapy, and the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin disease, multiple myeloma, and myelodysplastic syndrome). Examples of solid cancers include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpesvirus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella-zoster virus), adenovirus-, lentivirus-, BK polyomavirus-related disorders).

[0268] Treatment using the hematopoietic lineage cells of the embodiments disclosed herein can be carried out according to the symptoms or for prevention of recurrence. Terms such as "treating", "treatment", etc. are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in that it completely or partially prevents the disease, and / or can be therapeutic with respect to the partial or complete cure of the disease and / or the adverse effects resulting from the disease. As used herein, "treatment" encompasses any intervention in a disease in a subject and includes: preventing the occurrence of a disease in a subject who is susceptible to the disease but has not yet been diagnosed as having it, inhibiting the disease, i.e., preventing its onset, or alleviating the disease, i.e., causing the disease to regress. The therapeutic agent or composition can be administered before, during, or after the onset of the disease or injury. Treatment of an ongoing disease where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient is also of particular interest. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury that can be suppressed, restored, and / or improved by cell therapy with respect to at least one associated symptom. Certain specific embodiments contemplate that the subject in need of cell therapy includes, but is not limited to, a candidate for bone marrow or stem cell transplantation, a subject who has received chemotherapy or radiotherapy, a subject who has or is at risk of having a proliferative disorder or cancer, such as a hematopoietic proliferative disorder or cancer, a subject who has or is at risk of developing a tumor, such as a solid tumor, and a subject who has or is at risk of having a viral infection or a disease associated with a viral infection.

[0269] When evaluating the responsiveness to a treatment comprising the hematopoietic lineage cells of the embodiments disclosed herein, the response can be measured by criteria including at least one of clinical benefit rate, survival until death, pathological complete response, semi-quantitative measurement of pathological response, clinical complete remission, clinical partial remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Response Evaluation Criteria In Solid Tumors) criteria.

[0270] The disclosed therapeutic composition comprising hematopoietic lineage cells can be administered to a subject before, during, and / or after other treatments. Thus, a method of combination therapy can involve the administration or preparation of iPSC-derived immune cells before, during, and / or after the use of an additional therapeutic agent. As provided above, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or their substitutes, vectors containing one or more polynucleotides of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). Administration of iPSC-derived immune cells can be separated from administration of the additional therapeutic agent by time, days, or even weeks. Additionally, or alternatively, the administration can be combined with other biologically active agents or modalities such as, but not limited to, non-drug therapies such as anti-tumor agents, surgery, and the like.

[0271] In some embodiments of the combinatorial cell therapy, the therapeutic combination comprises the iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent that is an antibody or antibody fragment. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody can be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability. In some embodiments, as an additional therapeutic agent for the administered iPSC-derived hematopoietic lineage cells, suitable antibodies for combinatorial therapy include CD20 antibodies (e.g., rituximab, belimumab, ofatumumab, ublituximab, ocrelizumab, obinutuzumab), HER2 antibodies (e.g., trastuzumab, pertuzumab), CD52 antibodies (e.g., alemtuzumab), EGFR antibodies (e.g., cetuximab), GD2 antibodies (e.g., dinutuximab), PDL1 antibodies (e.g., avelumab), CD38 antibodies (e.g., daratumumab, isatuximab, MOR202), CD123 antibodies (e.g., 7G3, CSL362), SLAMF7 antibodies (elotuzumab), MICA / B antibodies (7C6, 6F11, 1C2), and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars, but are not limited thereto.

[0272] In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. Checkpoints are cell molecules, often cell surface molecules, that can suppress or downregulate the immune response when not inhibited. Checkpoint inhibitors are antagonists that can reduce checkpoint gene expression or gene products, or decrease the activity of checkpoint molecules. Checkpoint inhibitors suitable for combination therapy with derived effector cells including NK cells or T cells provided herein include, but are not limited to, antagonists of PD1 (Pdcdl, CD279), PDL-1 (CD274), TIM3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG3 (Lag3, CD223), CTLA4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0273] Some embodiments of the combination therapy comprising the provided derived effector cells further comprise at least one inhibitor that targets a checkpoint molecule. Some other embodiments of the combination therapy with the provided derived effector cells comprise two, three or more inhibitors such that two, three or more checkpoint molecules are targeted. In some embodiments, the effector cells for the combination therapy described herein are the provided derived NK cells. In some embodiments, the effector cells for the combination therapy described herein are derived T cells. In some embodiments, the derived NK cells or T cells for the combination therapy are functionally enhanced as provided herein. In some embodiments, two, three or more checkpoint inhibitors can be administered in the combination therapy before, or after administration, along with the administration of the derived effector cells. In some embodiments, two or more checkpoint inhibitors are administered simultaneously, or one at a time (sequentially).

[0274] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab’, F(ab)’2, F(ab)’3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one, or two, or three or more checkpoint inhibitors include at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0275] Combination therapy comprising derived effector cells and one or more check inhibitors is being investigated for the treatment of cutaneous T-cell lymphoma, non-Hodgkin's lymphoma (NHL), mycosis fungoides, Paget's reticulosis, Sézary syndrome, granulomatous flaccid skin, lymphomatoid papulosis, chronic pityriasis lichenoides, acute pityriasis lichenoides, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennart's lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, It is applied in the treatment of liquid and solid cancers, including but not limited to, Hodgkin's lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyosarcoma, Lewis lung carcinoma (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal carcinoma (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, liver cancer, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid cancer, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal carcinoma.

[0276] In some embodiments, in addition to the derived effector cells provided herein, the combination for therapeutic use includes one or more additional therapeutic agents, including chemotherapeutic agents or radioactive moieties. Chemotherapeutic agents refer to cytotoxic antitumor agents, i.e., chemical agents that are found to preferentially kill tumor cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the growth of neoplastic cells. Chemotherapeutic agents may also be referred to as antitumor or cytotoxic drugs or agents, and are well known in the art.

[0277] In some embodiments, the chemotherapeutic agent includes anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents, interferons, and interleukins. Exemplary chemotherapeutic agents include alkylating agents (cyclophosphamide, mechlorethamine, melphalan, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisantrene, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine, but are not limited thereto.Additional agents include aminoglutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzumab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, celecoxib, cetuximab, cladribine, clofarabine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, drostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nolfetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegademase, pegasparaginase, pentostatin, pipobroman, plicamycin, polyphepan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are drugs approved for use in humans, including those approved as chemotherapeutic or radiotherapeutic agents and known in the art.Such agents can be referenced through any of several standard physician and oncologist references (e.g., Goodman & Gilman’s The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, N.Y., 1995) or the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated as needed.

[0278] Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide stimulate both NK cells and T cells. As provided herein, IMiDs can be used with iPSC-derived therapeutic immune cells for cancer treatment.

[0279] In addition to the isolated population of iPSC-derived hematopoietic lineage cells included in the therapeutic composition, compositions suitable for administration to a patient can further comprise one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., a pharmaceutically acceptable medium, such as a cell culture medium), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the particular composition being administered and by the particular method used to administer the therapeutic composition. Thus, there are a variety of suitable formulations for the therapeutic compositions of the present invention (e.g., see Remington’s Pharmaceutical Sciences, 17 th th ed. 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0280] In one embodiment, the therapeutic composition comprises T cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises NK cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises CD34+HE cells derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises HSCs derived from pluripotent cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises MDSCs derived from pluripotent cells produced by the methods and compositions disclosed herein. A therapeutic composition comprising a population of iPSC-derived hematopoietic lineage cells disclosed herein can be administered separately by intravenous, intraperitoneal, enteral, or tracheal administration methods or in combination with other suitable compounds to affect the desired therapeutic goal.

[0281] These pharmaceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffer can be about 5% by weight of the total composition. Without limitation, electrolytes such as sodium chloride and potassium chloride can also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition ranges from about 4 to about 10. Alternatively, the pH of the therapeutic composition ranges from about 5 to about 9, from about 6 to about 9, or from about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH within one of these pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH ranging from about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0282] The present invention also provides, in part, the use of a pharmaceutically acceptable cell culture medium in certain compositions and / or cultures of the present invention. Such compositions are suitable for administration to a human subject. Generally speaking, any medium that supports the maintenance, growth, and / or health of iPSC-derived immune cells according to embodiments of the present invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, the serum-free medium does not contain animal substances and may optionally not contain proteins. Optionally, the medium may contain recombinant proteins acceptable for biological agents. A medium that does not contain animal substances refers to a medium whose components are derived from sources other than animals. Recombinant proteins replace natural animal-derived proteins in a medium that does not contain animal substances, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a protein-free medium is defined as substantially free of proteins. Those skilled in the art will understand that the above examples of media are illustrative and in no way limit the formulation of media suitable for use in the present invention, and that there are many suitable media available to those skilled in the art.

[0283] Isolated pluripotent stem cell-derived hematopoietic lineage cells can have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+HE cells, HSCs, B cells, myeloid-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, isolated pluripotent stem cell-derived hematopoietic lineage cells have from about 95% to about 100% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or myeloid-derived suppressor cells (MDSCs). In some embodiments, the present invention provides a therapeutic composition having purified T cells or NK cells, such as a composition having an isolated population of about 95% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or myeloid-derived suppressor cells (MDSCs) for treating a subject in need of cell therapy.

[0284] In one embodiment, the combination cell therapy comprises a population of NK cells derived from genomically engineered iPSCs comprising a therapeutic protein or peptide and the genotypes listed in Table 2, and the derived NK cells comprise a CAR having the provided end domain. In another embodiment, the combination cell therapy comprises a population of T cells derived from genomically engineered iPSCs comprising an antigen-specific therapeutic protein or peptide and the genotypes listed in Table 2, and the derived T cells comprise CD38 null and a CAR having the provided end domain. In some embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202, and a population of NK or T cells derived from genomically engineered iPSCs comprising the genotypes listed in Table 2, and the derived NK or T cells comprise a CAR having the provided end domain, CD38 null, and hnCD16. In some further other embodiments, the combination cell therapy comprises daratumumab, and a population of NK or T cells derived from genomically engineered iPSCs comprising the genotypes listed in Table 2, and the derived NK or T cells comprise a first CAR having the provided end domain, CD38 null, hnCD16, and a second CAR, and the first and / or second CAR targets at least one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1, and the first and second CARs target different antigens. In some further additional embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202, and a population of NK or T cells derived from genomically engineered iPSCs comprising the genotypes listed in Table 2, and the derived NK or T cells comprise a first CAR having the provided end domain, CD38 null, hnCD16, a second CAR, and one or more exogenous cytokines.In yet another embodiment, the combinatorial cell therapy comprises a population of NK cells derived from genomically engineered iPSCs comprising a therapeutic protein or peptide and the genotypes listed in Table 2, wherein the derived NK cells have a first CAR with a provided endodomain, CD38 null, hnCD16, a second CAR, and one or more exogenous cytokines, and B2M comprising at least one of HLA-G overexpression or CD58 knockout and CD54 knockout. - / - CIITA - / - comprises.

[0285] As will be appreciated by those skilled in the art, based on the methods and compositions herein, both autologous and allogeneic hematopoietic cells derived from iPSCs can be used in the cell therapies described above. In the case of autologous transplantation, the isolated population of derived hematopoietic cells is fully or partially HLA-matched to the patient. In another embodiment, the derived hematopoietic cells are not HLA-matched to the subject, and the derived hematopoietic cells are NK cells or T cells having HLA I and HLA II null.

[0286] In some embodiments, the number of derived hematopoietic cells in the therapeutic composition is at least 0.1×10 5 cells, at least 1×10 5 cells, at least 5×10 5 cells, at least 1×10 6 cells, at least 5×10 6 cells, at least 1×10 7 cells, at least 5×10 7 cells, at least 1×10 8 cells, at least 5×10 8 cells, at least 1×10 9 cells, or at least 5×10 9 cells. In some embodiments, the number of derived hematopoietic cells in the therapeutic composition is from about 0.1×10 5 cells to about 1×10 6 cells, from about 0.5×10 6 cells to about 1×10 7 cells, from about 0.5×10 7Cells ~ about 1×10 8 Cells, about 0.5×10 8 Cells ~ about 1×10 9 Cells, about 1×10 9 Cells ~ about 5×10 9 Cells, about 0.5×10 9 Cells ~ about 8×10 9 Cells, about 3×10 9 Cells ~ about 3×10 10 Cells, or any range therebetween. Generally, for a 60 kg patient, 1×10 8 Cells / dose is converted to 1.67×10 6 Cells / kg.

[0287] In one embodiment, the number of hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a partial or single umbilical cord of blood, or at least 0.1×10 5 Cells / kg body weight, at least 0.5×10 5 Cells / kg body weight, at least 1×10 5 Cells / kg body weight, at least 5×10 5 Cells / kg body weight, at least 10×10 5 Cells / kg body weight, at least 0.75×10 6 Cells / kg body weight, at least 1.25×10 6 Cells / kg body weight, at least 1.5×10 6 Cells / kg body weight, at least 1.75×10 6 Cells / kg body weight, at least 2×10 6 Cells / kg body weight, at least 2.5×10 6 Cells / kg body weight, at least 3×10 6 Cells / kg body weight, at least 4×10 6 Cells / kg body weight, at least 5×10 6 Cells / kg body weight, at least 10×10 6 Cells / kg body weight, at least 15×10 6 Cells / kg body weight, at least 20×10 6 Cells / kg body weight, at least 25×10 6 Cells / kg body weight, at least 30×10 6 Cells / kg body weight, 1×108 cells / kg body weight, 5×10 8 cells / kg body weight, or 1×10 9 cells / kg body weight.

[0288] In one embodiment, a dose of hematopoietic lineage cells is delivered to a subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2×10 6 cells / kg, at least 3×10 6 cells / kg, at least 4×10 6 cells / kg, at least 5×10 6 cells / kg, at least 6×10 6 cells / kg, at least 7×10 6 cells / kg, at least 8×10 6 cells / kg, at least 9×10 6 cells / kg, or at least 10×10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0289] In another exemplary embodiment, the effective amount of cells provided to the subject is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, or about 10×10 6 cells / kg, or more cells / kg (including all intervening cell doses).

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

[0291] In some embodiments, the therapeutic use of hematopoietic lineage-derived cells is a single-dose treatment. In some embodiments, the therapeutic use of hematopoietic lineage-derived cells is a multi-dose treatment. In some embodiments, the multi-dose treatment is one dose per day, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days, every 45 days, or every 50 days, or one dose at any number of days in between.

[0292] Compositions containing a population of progenitor hematopoietic cells of the invention can be sterile, suitable for administration to a human patient, and ready for administration (i.e., can be administered without further processing). A cell-based composition that is ready for administration means that the composition does not require any further processing or manipulation prior to transplantation or administration to a subject. In other embodiments, the invention provides an isolated population of progenitor hematopoietic cells that are expanded and / or conditioned prior to administration of one or more agents. In the case of progenitor hematopoietic cells genetically engineered to express a recombinant TCR or CAR, for example, the cells can be activated and expanded using the methods described in U.S. Patent No. 6,352,694.

[0293] In certain embodiments, the primary and co-stimulatory signals for progenitor hematopoietic cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in "cis" formation), or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent can be bound to a surface and the other agent can be present in solution. In one embodiment, the agent providing the co-stimulatory signal can be bound to the cell surface and the agent providing the primary activation signal can be in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents are in soluble form and then cross-linked to a surface, such as an antibody or other binding agent that binds to agents disclosed in, for example, U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 for cells expressing Fc receptors or artificial antigen presenting cells (aAPCs) contemplated for use in activating and expanding T lymphocytes in embodiments of the invention.

[0294] Some variation in dosage, frequency, and protocol will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage, frequency, and protocol for an individual subject.

Example

[0295] The following examples are provided for illustration and not for limitation.

[0296] Example 1 - Materials and Methods To effectively select and test suicide systems in combination with different safe harbor locus integration strategies under the control of various promoters, the applicant's own hiPSC platform is used, which enables single-cell passage and high-throughput 96-well plate-based fluorescence-activated cell sorting (FACS) to allow the induction of clonal hiPSCs by single or multiple gene regulations.

[0297] Maintenance of hiPSCs in small molecule culture: When the confluence of the culture reached 75% - 90%, hiPSCs were routinely passaged as single cells. For single-cell dissociation, hiPSCs were washed once with PBS (Mediatech), treated with Accutase (Millipore) at 37°C for 3 - 5 minutes, and then pipetted to ensure single-cell dissociation. Subsequently, the single-cell suspension was mixed with an equal volume of the conventional medium, centrifuged at 225×g for 4 minutes, resuspended in FMM, and plated on a surface coated with Matrigel. Passage was typically at a ratio of 1:6 - 1:8, transferred to a tissue culture plate pre-coated with Matrigel at 37°C for 2 - 4 hours, and supplied with FMM every 2 - 3 days. Cell culture was maintained in a humidified incubator set at 37°C and 5% CO2.

[0298] Human iPSC manipulation by ZFN, CRISPR for targeted editing of the modality of interest: For genome editing via ZFN, for AAVS1-targeted insertion, a mixture of 2.5 μg of ZFN-L (FTV893), 2.5 μg of ZFN-R (FTV894), and 5 μg of donor construct was transfected into 2 million iPSCs. For genome editing via CRISPR, for ROSA26-targeted insertion, a mixture of 5 μg of ROSA26-gRNA / Cas9 (FTV922) and 5 μg of donor construct was transfected into 2 million iPSCs. Transfection was performed using the Neon transfection system (Life Technologies) using parameters 1500V, 10 ms, 3 pulses. On the second or third day after transfection, if the plasmid contained an artificial promoter driver GFP and / or RFP expression cassette, flow cytometry was used to measure the transfection efficiency. On the fourth day after transfection, puromycin was added to the medium at a concentration of 0.1 μg / ml for the first 7 days and 0.2 μg / ml after 7 days to select for targeted cells. During puromycin selection, cells were passaged into new wells coated with Matrigel on day 10. After day 16 of puromycin selection, viable cells were analyzed by flow cytometry for the proportion of GFP+ iPS cells.

[0299] Bulk and clonal selection of genome-edited iPSCs: iPSCs with genome-targeted editing using ZFN or CRISPR-Cas9 were bulk and clonally selected for GFP+SSEA4+TRA181+ iPSCs 20 days after puromycin selection. The targeted iPSC pool dissociated into single cells was resuspended in a chilled staining buffer newly prepared for optimal performance, containing Hank's balanced salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1x penicillin / streptomycin (Mediatech), and 10 mM Hepes (Mediatech). Conjugated primary antibodies, including SSEA4-PE and TRA181-Alexa Fluor-647 (BD Biosciences), were added to the cell solution and incubated on ice for 15 minutes. All antibodies were used at 7 μL in 100 μL of staining buffer per 1 million cells. The solution was washed once with staining buffer, spun down at 225 g for 4 minutes, resuspended in staining buffer containing 10 μM thiazobibenzene, and maintained on ice for flow cytometry sorting. Flow cytometry sorting was performed on a FACS Aria II (BD Biosciences). For bulk selection, GFP+SSEA4+TRA181+ cells were gated and sorted into a 15 mL standard tube filled with 7 mL of FMM. For clonal selection, the sorted cells were directly dispensed into a 96-well plate using a 100 μM nozzle at a concentration of 3 events per well. Each well was pre-filled with 200 μL of FMM supplemented with 5 μg / mL fibronectin and 1x penicillin / streptomycin (Mediatech) and pre-coated overnight with 5x Matrigel. 5x Matrigel pre-coating included adding 1 aliquot of Matrigel to 5 mL of DMEM / F12, then incubating overnight at 4 °C to allow proper resuspension, and finally adding 50 μL per well to the 96-well plate and incubating overnight at 37 °C. 5x Matrigel was aspirated immediately before adding medium to each well. Once sorting was complete, the 96-well plate was centrifuged at 225 g for 1 - 2 minutes before incubation. The plate was left standing for 7 days.On the 7th day, 150 μL of the medium was removed from each well and replaced with 100 μL of FMM. On the 10th day after sorting, an additional 100 μL of FMM was replenished to the wells. Colony formation was detected as early as the 2nd day, and most colonies expanded between 7 and 10 days after sorting. In the first passage, the wells were washed with PBS and dissociated with 30 μL of Accutase at 37 °C for approximately 10 minutes. The need for extended Accutase treatment reflects the compactness of colonies that were in an idling state during long-term culture. After the cells were observed to be dissociated, 200 μL of FMM was added to each well and pipetted several times to disrupt the colonies. The dissociated colonies were transferred to another well of a 96-well plate pre-coated with 5x Matrigel and then centrifuged at 225 g for 2 minutes before incubation. This 1:1 passage was performed to expand the initial colonies before they grew larger. Subsequent passages were routinely performed with a 3 - 5 minute Accutase treatment and a 1:4 - 1:8 expansion at 75 - 90% confluence into larger wells pre-coated with 1x Matrigel in FMM. Each clonal cell line was analyzed for GFP fluorescence levels and TRA1-81 expression levels. Clonal lines with GFP+ and TRA1-81+ approaching 100% were selected for further PCR screening and analysis. Flow cytometry analysis was performed on a Guava EasyCyte 8 HT (Millipore) and analyzed using FlowJo (FlowJo, LLC).

[0300] Example 2 - Functional Profiling of CAR Candidates and Derived NK or T Cells Expressing CARs Containing Novel Endodomains To screen for functional chimeric antigen receptors (CARs), candidate CARs (CARs) with the same antigen specificity but different endodomains and / or transmembrane domains ネオ) A group of are expressed in primary NK cells and T cells, respectively, to examine their cell-specific surface expression profiles. As shown in FIGS. 2A-C, these 29 constructs have the same scFv and CD8 hinge region, and only differ in the signaling components including the endodomain. By comparing the expression profiles of these 29 different CAR constructs targeting the same specific antigen, this assay was performed to determine which construct, more specifically, which endodomain component gives efficient and detectable CAR expression on the cell surface. In one example, all candidate CARs are constructed to be specific for MICA / B. Similarly, functional screening can also utilize, for example, CD19 scFV for CAR specificity. Derived NK lineage cells were transduced with lentivirus carrying each CAR construct. Each CAR construct in FIGS. 2A-C contains a Thy1.1 marker at the C-terminus, which is separated from the construct by a P2A peptide (not shown). Approximately 10 days after transduction, the transduced cells were assayed for CAR and Thy1.1 expression by FACS. Successfully transduced cells were sorted based on Thy1.1 expression, while CAR staining was performed using an antibody specific for the scFv region of the CAR. As shown in FIGS. 3A-I, the results show distinct but changing CAR expression patterns at the time of the assay, and certain transmembrane regions (i.e., CD28, CD8) appear to give enhanced CAR expression. However, constructs 3 and 23 were not detectable on the cell surface at that time, which may be due to the cell stage and / or biology of the constructs.

[0301] To show antigen-specific killing mediated by the CAR candidate, MICA / B-CAR ネオNK or T cells are co-cultured with tumor cells that express MICA / B or in which MICA / B is null or low. T cells expressing MICA / B-CD28-CD3ζ1XXCAR and NK cells expressing MICA / B-NKG2D-2B4-CD3ζ CAR, as well as CAR-free T cells and NK cells, are used as positive and negative controls. Then, each MICA / B-CAR ネオ showing specific killing ability is transduced into iPSCs. All CAR ネオ -iPSC lines are examined for CAR expression, karyotypic abnormalities, and genomic stability. Regardless of the presence or absence of expression in iPSCs, each CAR ネオ -iPSC line is continued for the differentiation of both T cells and NK cells according to the methods described herein. Cells at day 10, intermediate cells at day 20, and cells at other time points during differentiation are characterized for marker expression profiles and cell proliferation. Cell proliferation at important time points and at the end of the differentiation process is also evaluated.

[0302] MICA / B-CAR ネオ To determine the functional profile of derived NK or T cells expressing MICA / B-CAR candidates, stabilization of cell surface MICA / B by MICA / B-CAR ネオ is examined.

[0303] MICA / B-CAR ネオ An iPSC-derived NK cell expressing MICA / B-CAR (MICA / B-CAR ネオ iNK) and a tumor cell line cell expressing MICA / B (target cell) are used in a co-culture system. The resulting activation and enhanced function of MICA / B-CAR ネオ iNK are also tested using this co-culture system. Co-culture of MICA / B-positive tumors with MICA / B-CAR ネオ iNK is examined for the level of soluble MICA / B released into the culture supernatant using ELISA. Compared with co-culture with unmodified NK cells, target cells are treated with MICA / B-CAR ネオCo - culturing with iNK cells, the reduction of soluble MICA / B released into the culture supernatant supports the discovery of MICA / B stabilization on the tumor cell surface. The positive control for this test uses co - culturing of target cells with mAb7C6.

[0304] Under the same co - culture conditions, MICA / B - CAR ネオ Activation of iNK cells is examined by production of cytokines IFNγ and TNFα, degranulation by evaluation of surface CD107a, and direct killing of target cell lines using caspase - based flow assays. Compared with the observed lack of difference in activity when co - cultured with MICA / B - negative, the increased levels of cytokines and degranulation, and MICA / B - CAR compared to unmodified NK cells in respon...

Claims

**Claim 1** A chimeric antigen receptor comprising an ectodomain comprising at least one antigen recognition domain, a transmembrane domain, and an endodomain comprising at least one signaling domain, wherein the at least one signaling domain is derived from the cytoplasmic domain of a signaling protein specific for the activation or function of T and / or NK cells, and when the chimeric antigen receptor is contained in induced pluripotent stem cells (iPSCs), promoting the differentiation of the iPSCs towards desired effector cells, and the iPSC-derived effector cells differentiated from the iPSCs have, compared to primary immune cells obtained from peripheral blood, cord blood, or any other donor tissue, (i) improved persistence and / or survival, (ii) improved cell proliferation, (iii) increased cytotoxicity, (iv) increased resistance to allogeneic rejection, (v) improved tumor infiltration, (vi) an enhanced ability to migrate, activate and / or mobilize bystander immune cells to the tumor site, and (vii) an enhanced ability to reduce tumor immunosuppression, a chimeric antigen receptor having at least one of the characteristics. **Claim 2** (a) the signal transduction protein includes any one of 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell-specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain-containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain), DAP10 (hematopoietic cell signal transducer), DAP12 (TYRO protein tyrosine kinase-binding protein), DNAM1 (CD226 antigen), FcERIγ (high-affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta), IL-2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D type II integral membrane protein), NKp30 (natural cytotoxicity triggering receptor 3), NKp44 (natural cytotoxicity triggering receptor 2), NKp46 (natural cytotoxicity triggering receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain), and / or (b) each of the at least one signal transduction domain includes an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a part thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8, which is represented by SEQ ID NOs: 21-41, 54, and 56, and / or (c) The chimeric antigen receptor according to claim 1, wherein the at least one signal transduction domain comprises an amino acid sequence having about 85%, about 95%, about 97%, about 98% or about 99% identity to the cytoplasmic domain of 2B4, CD28H, CD3ζ, DAP10, FcERIγ, KIR2DS2, NKG2D, CD3ζ, CD3ζ1XX, DNAM1, CS1, or a combination thereof, or a part thereof.

3. The at least one signal transduction domain each comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8, represented by SEQ ID NOs: 21-41, 54, and 56, respectively, and a part of the cytoplasmic domain comprises an ITAM (immunoreceptor tyrosine-based activation motif), YxxM motif, TxYxxV / I motif, FcRγ, hemi-ITAM, and / or ITT-like motif. The chimeric antigen receptor according to claim 2.

4. The endodomain comprises a first signal transduction domain, a second signal transduction domain, and optionally a third signal transduction domain, and the first, second, and third signal transduction domains are different. The chimeric antigen receptor according to claim 1.

5. The chimeric antigen receptor according to claim 4, wherein the second or third signal transduction domain has an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a part thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8, which are represented by SEQ ID NOs: 21-41, 54, and 56, respectively.

6. The chimeric antigen receptor according to claim 1, wherein the endodomain comprises only one signal transduction domain, and the endodomain has an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a part thereof of DNAM1, CD28H, KIR2DS2, DAP12, or DAP10.

7. The chimeric antigen receptor according to claim 4, wherein the endodomain comprises two different signal transduction domains, and the endodomain domain comprises a fused cytoplasmic domain or a part thereof in any one of the forms of 2B4-CD3ζ / 1XX, 2B4-DNAM1, 2B4-FcERIγ, 2B4-DAP10, CD16-DNAM1, CD16-DAP10, CD16-DAP12, CD2-CD3ζ / 1XX, CD2-DNAM1, CD2-FcERIγ, CD2-DAP10, CD28-DNAM1, CD28-FcERIγ, CD28-DAP10, CD28-DAP12, CD28H-CD3ζ / 1XX, DAP10-CD3ζ / 1XX, DAP10-DAP12, DAP12-CD3ζ / 1XX, DAP12-DAP10, DNAM1-CD3ζ / 1XX, KIR2DS2-CD3ζ / 1XX, KIR2DS2-DAP10, KIR2DS2-2B4, and NKp46-2B4.

8. The end domain includes three different signaling domains, and the end domain includes a fusion cytoplasmic domain or a part thereof in any one of the forms including 2B4-DAP10-CD3ζ / 1XX, 2B4-IL21R-DAP10, 2B4-IL2RB-DAP10, 2B4-IL2RB-CD3ζ / 1XX, 2B4-41BB-DAP10, CD16-2B4-DAP10, and KIR2DS2-2B4-CD3ζ / 1XX. The chimeric antigen receptor according to claim 4.

9. The transmembrane domain has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the transmembrane region or a part thereof of CD2, CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide. The chimeric antigen receptor according to claim 1.

10. The transmembrane domain, respectively, (a) is represented by SEQ ID NOs: 1-20, 53, and 55, 2B4, CD2, CD16, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, or (b) has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the transmembrane region or a part thereof of DAP10, KIR2DS2, 2B4, NKG2D, CD28H, or DNAM1. The chimeric antigen receptor according to claim 1.

11. The transmembrane domain and its directly linked signaling domain are derived from the same protein or different proteins. The chimeric antigen receptor according to claim 1.

12. The chimeric antigen receptor includes a transmembrane domain and an endodomain (TM-(endodomain)), and the chimeric antigen receptor is (i) in the form of NKG2D-(2B4-IL2RB-CD3ζ), CD8-(41BB-CD3ζ1XX), CD28-(CD28-2B4-CD3ζ), CD28H-(CD28H-CD3ζ), CD28H-(CD28H-2B4), CD28H-(CD28H-2B4-CD3ζ), DNAM1-(DNAM1-CD3ζ), DNAM1-(DNAM1-CS1), DAP10-(DAP10-CD3ζ), KIR2DS2-(KIR2DS2-CD3ζ), KIR2DS2-(KIR2DS2-DAP10), KIR2DS2-(KIR2DS2-DAP10-CD3ζ), KIR2DS2-(KIR2DS2-2B4), CD16-(CD16-2B4-DAP10), CD16-(CD16-DNAM1), NKp46-(NKp46-2B4), NKp46-(NKp46-2B4-CD3ζ), NKp46-(NKp46-CD2-DAP10), CD2-(CD2-CD3ζ), 2B4-(2B4-CD3ζ), 2B4-(2B4-FcERIγ), CS1-(CS1-CD3ζ), NKG2D-(CS1), NKG2D-(2B4-CS1), and NKG2D-(2B4-CS1-CD3ζ), or (ii) in the form of DAP10-(DAP10-CD3ζ), KIR2DS2-(KIR2DS2-CD3ζ), KIR2DS2-(KIR2DS2-DAP10), KIR2DS2-(KIR2DS2-2B4), 2B4-(2B4-CD3ζ), 2B4-(2B4-FcERIγ), NKG2D-(2B4-CS1), CD28H-(CD28H-2B4), CD28H-(CD28H-2B4-CD3ζ), and DNAM1-(DNAM1-CS1), or (iii) an amino acid sequence having about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the sequence represented by each of SEQ ID NOs: 57 to 74, the chimeric antigen receptor according to claim 1.

13. The chimeric antigen receptor according to claim 1, wherein the antigen recognition domain specifically binds to an antigen associated with a disease, pathogen, liquid tumor, or solid tumor.

14. The antigen recognition domain is (i) any one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGFR-2, PSMA, and PDL1, or (ii) ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFR, EGFR-VIII, ERBB folic acid binding protein (FBP), fetal acetylcholine receptor (AChR), folic acid receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCS1, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGFR-2), Wilms tumor protein (WT-1), and is specific for any one of pathogen antigens, the chimeric antigen receptor according to claim 1. **Claim 15** The ectodomain is (i) two antigen recognition domains, (ii) a signal peptide, and / or The chimeric antigen receptor according to claim 1, comprising one or more of (iii) spacer / hinge.

16. The chimeric antigen receptor is included in a bicistronic construct that co-expresses a cell surface-expressed exogenous cytokine or a partial or full-length peptide of its receptor, and the exogenous cytokine or its receptor is (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or (b) (i) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein having an intracellular domain of cleaved or excluded 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 common receptor γC, wherein the common receptor γC is natural or modified, and (vii) the chimeric antigen receptor according to claim 1, comprising at least one of homodimers of IL15Rβ.

17. The derived effector cells from iPSC differentiation include one or more of 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 cells, derived NKT cells, derived NK cells, derived B cells, or derived immune effector cells, and the chimeric antigen receptor according to claim 1.

18. The iPSC-derived immune effector cells express the chimeric antigen receptor and include at least one functional property that is not present in primary T, NK, and / or NKT cells, and the chimeric antigen receptor according to claim 17.

19. A cell or a population thereof, (i) the cell is an immune cell, an induced pluripotent cell (iPSC), a cloned iPSC, or an iPS cell line cell, or the cell is a derived effector cell obtained from differentiating the iPSC, (ii) the cell includes at least one chimeric antigen receptor (CAR) according to any one of claims 1 to 18, and the cell or its population.

20. The derived effector cells are hematopoietic cells and contain longer telomeres compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, or the CAR has the following characteristics: (i) being specific for T cells or NK cells, (ii) being bispecific in antigen binding, (iii) being a switchable CAR, (iv) being a dimerized CAR, (v) being a split CAR, (vi) being a multi-chain CAR, (vii) being an inducible CAR, and (viii) being inserted into one of the gene loci of B2M, TAP1, TAP2, tapasin, NLRRC5, CII TA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL - B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, wherein the insertion knocks out or reduces the expression of the gene at the locus, the cell or population according to claim 19 having at least one of the insertions.

21. The cell is (i) CD38 knockout, (ii) B2M null or low, optionally CII TA null or low, compared to its corresponding primary cell, (iii) introduction of the expression of HLA - G or non - cleavable HLA - G, or knockout of one or both of CD58 and CD54, (iv) CD16 or its variant, (v) a second CAR having different targeting specificities, (vi) a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, (vii) at least one of the genotypes listed in Table 2, (viii) deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRRC5, CII TA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL - B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT compared to its corresponding primary cell, or (ix) Compared to its corresponding primary cell, HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ R, antigen-specific TCR, Fc receptor, an engager, and the cell or population thereof according to claim 19, further comprising one or more of the introduced or increased expression in at least one of the surface trigger receptors for binding an agonist.

22. The cell is a derived effector cell and has, compared to its corresponding primary cell obtained from peripheral blood, umbilical cord blood, or any other donor tissue, (i) improved persistence and / or survival, (ii) increased resistance to alloreactive recipient immune cells, (iii) increased cytotoxicity, (iv) improved tumor infiltration, (v) enhanced or acquired ADCC, (vi) an enhanced ability to migrate, activate, and / or mobilize bystander immune cells to the tumor site, (vii) an enhanced ability to reduce tumor immunosuppression, (viii) an improved ability to rescue tumor antigen escape, (ix) the ability to stabilize tumor antigens, and (x) the ability to avoid fratricide, The cell or population thereof according to claim 19, having at least one of the characteristics including.

23. The cell or population thereof according to claim 21, wherein the cell further comprises high-affinity non-cleavable CD16 (hnCD16) or a variant thereof.

24. The CD16 or a variant thereof is (a) F176V and S197P of the extracellular domain of CD16, (b) the complete or partial extracellular domain derived from CD64, (c) a non-natural (or non-CD16) transmembrane domain, (d) a non-natural (or non-CD16) intracellular domain, (e) a non-natural (or non-CD16) signaling domain, (f) a non-natural stimulatory domain, and (g) The cell or population thereof according to claim 21, comprising at least one of a transmembrane, signaling, and stimulatory domain that is not derived from CD16 and is derived from the same or different polypeptides.

25. (a) The non-natural transmembrane domain is derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor (TCR) polypeptide, or (b) the non-natural stimulatory domain is derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide, (c) the non-natural signaling domain is derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide, or (d) the non-natural transmembrane domain is derived from NKG2D, the non-natural stimulatory domain is derived from 2B4, and the non-natural signaling domain is derived from CD3ζ, the cell or population of cells according to claim 24.

26. the cell further comprises a second CAR, the second CAR being (i) T cell-specific or NK cell-specific, (ii) a bispecific antigen-binding CAR, (iii) a switchable CAR, (iv) a dimerized CAR, (v) a split CAR, (vi) a multi-chain CAR, (vii) an inducible CAR, (viii) co-expressed, optionally in a separate construct or a bicistronic construct, with a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor, (xi) co-expressed, optionally in a separate construct or a bicistronic construct, with a checkpoint inhibitor, (xii) specific for at least one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGFR-2, PSMA, and PDL1, and / or (xiii)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, CD8, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2, 3, 4, EGFR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-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), NKCS1, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and a cell or population thereof according to claim 21, which is specific for any one of pathogen antigens.

27. The cell comprises a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor, and the exogenous cytokine or its receptor is a) including at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors, or b) Co-expression of IL15 and IL15Rα by using a self-cleaving peptide, a fusion protein of IL15 and IL15Rα, an IL15 / IL15Rα fusion protein having an intracellular domain of cleaved or excluded IL15Rα, a fusion protein of IL15 and a membrane-bound Sushi domain of IL15Rα, a fusion protein of IL15 and IL15Rβ, a fusion protein of IL15 and common receptor γC, wherein the common receptor γC is natural or modified, and at least one of homodimers of IL15Rβ, any one of (i) to (vii) can be co-expressed with a CAR in a separate construct or a bicistronic construct, optionally, the cell or population thereof according to claim 21, which is transiently expressed.

28. The cell or population thereof according to claim 19, wherein the derived effector cells can mobilize and / or migrate T cells to a tumor site, and the derived effector cells can reduce immunosuppression of a tumor in the presence of one or more checkpoint inhibitors.

29. The cell or population thereof according to claim 26 or 28, wherein the checkpoint inhibitor is an antagonist to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR.

30. The checkpoint inhibitor is one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or The cell or population thereof according to claim 29, comprising at least one of atezolizumab, nivolumab, and pembrolizumab.

31. The cell or population thereof according to claim 20, wherein the derived effector cell comprises a derived CD34 cell, a derived hematopoietic stem cell and progenitor cell, a derived hematopoietic multipotent progenitor cell, a derived T cell progenitor cell, a derived NK cell progenitor cell, a derived T cell, a derived NKT cell, a derived NK cell, a derived B cell, or a derived immune effector cell.

32. The cell is (i) one exogenous polynucleotide integrated into one safe harbor locus or a selected locus, or (ii) more than two exogenous polynucleotides integrated into different safe harbor loci or two or more selected loci, The cell or population thereof according to claim 19.

33. The safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, and the selected locus is B2M, TAP1, TAP2, tapasin, NLRRC5, 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 the integration of the exogenous polynucleotide knocks out the expression of the gene at the locus. The cell or population thereof according to claim 32.

34. The cell or population thereof according to claim 33, wherein the TCR locus is the constant region of TCR alpha or TCR beta.

35. A composition comprising the cell or population thereof according to any one of claims 19 to 34.

36. A composition for therapeutic use, comprising the derived effector cell according to any one of claims 19 to 34 and one or more therapeutic agents.

37. The composition according to claim 36, wherein the one or more therapeutic agents include a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector containing one or more polynucleic acids of interest, an antibody or a functional variant or fragment thereof, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

38. (1) The checkpoint inhibitor is (a) one or more antagonists against a checkpoint molecule including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or an inhibitory KIR, (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or (2) The composition according to claim 37, wherein the one or more therapeutic agents include one or more of venetoclax, azacitidine, and pomalidomide.

39. The antibody is (a) an anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibody, (b) Rituximab, belimumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, abemaciclib, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and one or more of their humanized or Fc-modified variants or fragments, and functional equivalents and biosimilars thereof, or (c) The composition according to claim 37, comprising daratumumab, wherein the derived effector cells comprise CD38 knockout, optionally including the expression of CD16 or its variant. (Claim 40) (d) The therapeutic use of the composition according to any one of claims 35 to 39, by introducing the composition into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection. (Claim 41) (e) A method for producing derived effector cells comprising the CAR according to claim 1, the method comprising differentiating genetically engineered iPSCs, the iPSCs comprising a polynucleotide encoding the CAR, and optionally, by one or more edits, (i) CD38 knockout, (ii) B2M null or low, optionally CII TA null or low, compared to its corresponding primary cells, (iii) Introduction of the expression of HLA-G or non-cleavable HLA-G, or knockout of one or both of CD58 and CD54, (iv) CD16 or its variant, (v) A chimeric antigen receptor (CAR) having different targeting specificities, (vi) A partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface, (vii) At least one of the genotypes listed in Table 2 (viii) Deletion or reduced expression in at least one of TAP1, TAP2, tapasin, NLRRC5, CIITA, RFXANK, RFX5, RFXAP, TCR α or β constant region, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL - B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT as compared to its corresponding primary cell, and / or (ix) An introduced or increased expression in at least one of HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ R, antigen-specific TCR, Fc receptor, an engager, and a surface trigger receptor for binding to a bispecific or multispecific or universal engager, resulting in a method.

42. Genome - engineering the cloned iPSC to knock - in the polynucleotide encoding the CAR, optionally, (i) Knocking out CD38, (ii) Knocking out B2M and CIITA, (iii) Knocking out one or both of CD58 and CD54, and / or (iv) Introducing the expression of HLA - G or non - cleavable HLA - G, high - affinity non - cleavable CD16 or its variant, a second CAR, and / or a partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface, the method according to claim 41, further comprising.

43. The method according to claim 42, wherein the genome - engineering comprises targeted editing.

44. The targeted editing comprises deletions, insertions, or indels, and the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variant of these methods, the method according to claim 43.

45. The editing is CRISPR - mediated editing of the cloned iPSC, wherein the editing comprises knocking - in the polynucleotide encoding the CAR of claim 1.

46. (a) The editing of the cloned iPSC further comprises knocking out CD38, or (b) the CRISPR-mediated editing according to claim 45, wherein the CAR is inserted into one of gene loci including B2M, TAP1, TAP2, tapasin, NLRRC5, CII TA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and the insertion knocks out the expression of the gene at the locus.

47. A method of treating a disease or condition, comprising administering to a subject in need of treatment a cell comprising the CAR according to any one of claims 1 to 18.

48. The cell comprises CD38 knockout, a derivative effector cell comprising CD16 or a variant thereof, and optionally, (i) B2M and CII TA knockout, (ii) introduction of the expression of HLA-G or non-cleavable HLA-G, or knockout of one or both of CD58 and CD54, (iii) introduction of a second CAR, and / or introduction of the expression of a partial or complete peptide of an exogenous cytokine or its receptor on the cell surface, and / or (iii) the method according to claim 47, comprising at least one of the genotypes listed in Table 2.

49. The administration of the cell results in one or more of the following compared to a treatment using effector cells without the CAR according to claim 1: (i) reducing shedding of the MICA / B antigen on the surface of tumor cells, (ii) increasing the density of MICA / B on the surface of tumor cells, (iii) preventing tumor antigen escape, (iv) overcoming tumor microenvironment suppression, (v) enhancing the activation and killing functions of effector cells, and (vi) controlling in vivo tumor progression, reducing tumor cell load, tumor clearance, and / or improving survival rate. The method according to claim 47.

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