Enhanced Chimeric Antigen Receptor Effector Cell Manipulation and its Use for Immunity

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 on 17 October 2019, and to International Application PCT / US20 / 54601, filed on 7 October 2020, the disclosures thereof being incorporated herein by reference in their entirety.

[0002] References to electronically submitted sequence listings This application incorporates, by reference, the computer-readable format (CRF) of the sequence listing in ASCII text format filed with this application, titled 056932-530001WO_SEQUENCE_LISTING_ST25.txt, created on 19 October 2020, and measuring 92,883 bytes.

[0003] This disclosure broadly relates to the field of ready-made immune cell products. More specifically, this disclosure relates to strategies for developing multifunctional effector cells that can deliver therapeutically relevant properties in vivo. Cell products developed under this disclosure address significant limitations of patient-derived cell therapies. [Background technology]

[0004] The field of adoptive cell therapy currently focuses on the use of patient-derived and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapy and deliver treatment to all patients who could potentially benefit. To promote favorable patient outcomes, there is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes. Lymphocytes such as T cells and natural killer (NK) cells are potent anti-tumor effectors that play a crucial role in innate and adaptive immunity. However, the use of these immune cells for adoptive cell therapy remains challenging, and there is an unmet need for improvement. Therefore, there remains a significant opportunity to fully utilize the potential of T cells, NK cells, or other immune effector cells in adoptive immunotherapy. [Overview of the Initiative]

[0005] Functionally improved effector cells are needed to address issues related to the effectiveness against solid tumors, namely, problems extending to the tumor microenvironment and associated immunosuppression, recruitment, transport, and invasion, including response rates, cell depletion, loss of transfused cells (survival and / or persistence), tumor escape due to target loss or lineage change, accuracy of tumor targeting, extra-target toxicity, and extratumor effects.

[0006] The object of the present invention is to provide a method and composition for generating derived non-pluripotent cells differentiated from a single-cell derived iPSC (induced pluripotent stem cell) clone line, the iPSC containing one or more genetic modifications in its genome. These one or more genetic modifications include DNA insertions, deletions, and substitutions, and these modifications are retained and continue to function in subsequent derived cells after differentiation, expansion, passage, and / or transplantation.

[0007] The iPSC-derived non-pluripotent cells of this application include, but are not limited to, CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, 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 this application have one or more genetic modifications in their genomes through differentiation from iPSCs containing the same genetic modifications. In engineered clonal iPSC differentiation strategies for obtaining genetically modified derived cells, it is also necessary that the potential for iPSC development in differentiation is not adversely affected by the engineered modality of the iPSC, and that the engineered modality functions as intended in the derived cells. Furthermore, this strategy overcomes the current barriers to manipulating primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely, the difficulty in manipulating such cells due to their often lack of reproducibility and homogeneity, resulting in cells with poor cellular persistence accompanied by high cell death and low cell proliferation. Moreover, this strategy avoids the generation of heterogeneous effector cell populations obtained by other methods, using initially heterogeneous primary cell sources.

[0008] Several aspects of the present invention provide genome-engineered iPSCs obtained using methods comprising (I), (II), or (III), respectively, which reflect the strategies of genome manipulation after, concurrently with, and prior to the reprogramming process. (I): Genetically engineer iPSCs using one or both of (i) and (ii) in any order: (i) introduce one or more constructs into the iPSCs to enable targeted incorporation at selected sites; (ii)(a) introduce one or more double-strand breaks into the iPSCs at selected sites using one or more endonucleases capable of recognizing selected sites; (b) culture the iPSCs from step (I)(ii)(a) to enable endogenous DNA repair to generate targeted in / dels at selected sites, thereby obtaining genomically engineered iPSCs capable of differentiating into partially or fully differentiated cells. (II): Reprogramming non-pluripotent cells to obtain genetically engineered iPSCs: This includes (i) contacting non-pluripotent cells with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells, and (ii) introducing either or both of the following into the reprogrammed non-pluripotent cells of step (II)(i): (a) one or more constructs enabling targeted incorporation at a selected site, and (b) one or more double-strand breaks at a selected site using at least one endonuclease capable of recognizing the selected site, and then culturing the cells of step (II)(ii)(b) so that they can generate targeted in / del at the selected site by endogenous DNA repair, and so the resulting genetically engineered iPSCs contain at least one functional targeted genome edit, and the genetically engineered iPSCs can differentiate into partially or fully differentiated cells. (III): Genetically engineer non-pluripotent cells to reprogram them to obtain genetically engineered iPSCs: This involves (i) introducing either or both of the following into non-pluripotent cells in any order: (a) one or more constructs that enable targeted incorporation at selected sites, and (b) one or more double-strand breaks at selected sites using at least one endonuclease capable of recognizing selected sites (culturing the cells from step (III)(i)(b) so that they can generate in / del targeted at selected sites by endogenous DNA repair). (i) and (ii) the cells of step (III)(i) are brought into contact with one or more reprogramming factors and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to obtain a genome-engineered iPSC having targeted editing at a selected site, thereby obtaining a genome-engineered iPSC having at least one functionally targeted genome edit, the genome-engineered iPSC being able to differentiate into partially differentiated or fully differentiated cells.

[0009] In one embodiment of the method described above, at least one targeted genome edit at one or more selected sites includes the insertion of one or more exogenous polynucleotides encoding a safety switch protein, a targeted modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a candidate drug target, or a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of a genome-engineered iPSC or its derived cell. In some embodiments, the exogenous polynucleotide for insertion is operably ligated to one or more exogenous promoters, including (1) CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, transient, tissue-specific, or cell-type-specific promoters; or (2) one or more endogenous promoters contained in a selected site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genome-safe harbor. In some embodiments, the genome-engineered iPSCs produced using the methods described above comprise one or more distinct exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, where, if the genome-engineered iPSC comprises two or more suicide genes, the suicide genes are incorporated into different safe harbor loci, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotides encode partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors. In some embodiments, the partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors, encoded by exogenous polynucleotides, are in the form of a fusion protein.

[0010] In some other embodiments, the genome-engineered iPSCs produced using the methods provided herein include in / dels in one or more endogenous genes related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response modulation and regulation, or proteins that suppress engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of the iPSC or its derived 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 genes in the chromosome 6p21 region.

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

[0012] In some other embodiments, approaches (I), (II), and / or (III) involve contacting the genetically engineered iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain the pluripotency of the genetically engineered iPSCs. In one embodiment, the resulting genetically engineered iPSCs, comprising at least one targeted genome edit, are functional, differentiateable, 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 may be derived from the cytoplasmic domain of a signaling protein specific to the activation or function of T and / or NK cells, and, if comprising induced pluripotent stem cells (iPSCs), the chimeric antigen receptor promotes the differentiation of iPSCs directed toward desired derived effector cells, and the iPSC-derived effector cells differentiated from the iPSCs have at least one of the following features compared to primary immune cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue: (i) improved persistence and / or survival, (ii) improved cell proliferation, (iii) increased cytotoxicity, (iv) increased resistance to allo-rejection, (v) improved tumor infiltration, (vi) enhanced ability to migrate and / or activate or mobilize bystander immune cells to tumor sites, and (vii) enhanced ability to reduce tumor immunosuppression.In various embodiments, (a) the signaling proteins include 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 signaling 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), and IL-7R (interleukin (a) comprising any one of the following: (a) receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D type II integral membrane protein), NKp30 (native cytotoxicity-inducing receptor 3), NKp44 (native cytotoxicity-inducing receptor 2), NKp46 (native cytotoxicity-inducing receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain), and / or (b) at least one signaling domain represented by (a) SEQ ID NOs: 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, respectively. The amino acid sequence contains at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the cytoplasmic domain or a portion thereof of IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, and / or (b)2B4, CD28H, CD3ζ, DAP10, FcERIγ, KIR2DS2, NKG2D, CD3ζ, CD3ζ1XX, DNAM1, or CS1.In certain embodiments, at least one signaling domain is represented by SEQ ID NOs: 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, and FcERIγ, respectively, as indicated by SEQ ID NOs: 21-41, 54, and 56. The amino acid sequence comprises an amino acid sequence having at least approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% identity with the cytoplasmic domain or portion thereof of 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 contains an ITAM (immune receptor tyrosine-based activation motif), a YxxM motif, a TxYxxV / I motif, FcRγ, a hemi-ITAM, and / or an ITT-like motif.

[0014] In various embodiments, the end domain includes a first signaling domain, a second signaling domain, and optionally a third signaling domain, wherein the first, second, and third signaling domains are distinct. In some of these embodiments, the endodomain comprises a second signaling domain and optionally a 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 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, respectively. In various embodiments, the end domain comprises two different signaling domains, and these end domain domains include 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, and CD28-DA Includes, but not limited to, P10, 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, comprising a fused cytoplasmic domain or a portion thereof in any one of these morphologies.In various embodiments, the endodomain comprises three distinct signaling domains, the endodomain domain further comprises a fused cytoplasmic domain or a portion 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 endodomain comprises only one signaling domain, and the endodomain contains an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the cytoplasmic domain or portion thereof of DNAM1, CD28H, KIR2DS2, DAP12, or DAP10.

[0016] In various embodiments of the chimeric antigen receptor, the transmembrane domain is 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, The amino acid sequence contains at least approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% identity with the transmembrane domain or portion thereof of FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide. In various embodiments, the transmembrane domains each contain (a) amino acid sequences represented by SEQ ID NOs: 1-20, 53, and 55, such as 2B4, CD2, CD16, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8; or (b) amino acid sequences having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the transmembrane region or portion thereof of DAP10, KIR2DS2, 2B4, NKG2D, CD28H, and DNAM1. In various embodiments, the transmembrane domain and its directly linked signaling domain originate 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 is (i)NKG2D-(2B4-IL2RB-CD3ζ), CD8-(41BB-CD3 ζ1XX), CD28-(CD28-2B4-CD3ζ), CD28H-(CD28H-CD3ζ), CD28H-(CD28H-2B4), CD28H-(CD28H-2B4-CD3ζ), DNAM1-(DNAM1-CD3ζ), DNAM1-(DNAM 1-CS1), DAP10-(DAP10-CD3ζ), KIR2DS2-(KIR2DS2-CD3ζ), KIR2DS2-(KIR2DS2-DAP10), KIR2DS2-(KIR2DS2-DAP10-CD3ζ), KIR2DS2-(KIR2DS2 One of the following forms: -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 following forms: 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) Containing an amino acid sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence represented by each of sequence numbers 57-74.

[0018] In various embodiments of the chimeric antigen receptor, the antigen recognition domain specifically binds to antigens associated with disease, pathogens, liquid tumors, or solid tumors. 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, CD4 4V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigens 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 insertion 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 (M It may be specific to any one of the following: uc-1), mucin 16 (Muc-16), mesoserin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testicular antigen NY-ESO-1, tumor embryonic 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.

[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 may be contained in a bisistronic construct co-expressing a cell surface-expressed exogenous cytokine or a partial or full-length peptide of its receptor, the exogenous cytokine or its receptor being (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 using a self-cleaving peptide, or (ii) IL15 and IL (iii) an IL15 / IL15Rα fusion protein having a cleaved or excluded intracellular domain of IL15Rα, (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either native or modified, and (vii) at least one of the homodimers of IL15Rβ.

[0020] In some of these embodiments, the chimeric antigen receptor is incorporated into induced pluripotent stem cells (iPSCs) and promotes the differentiation of iPSCs directed toward desired derived effector cells. In some of these embodiments, the derived effector cells from iPSC differentiation include one or more derived CD34 cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic pluripotent 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 characteristic not present in primary T, NK, and / or NKT cells.

[0021] In another embodiment, the present invention provides cells or populations thereof, where (i) the cells may be immune cells, induced pluripotent cells (iPSCs), cloned iPSCs, or iPS cell lines, or the cells may be derived effector cells obtained from differentiating iPSCs, and (ii) the cells may comprise at least one chimeric antigen receptor (CAR) provided herein. In various embodiments, cells may (i) have CD38 knockout, (ii) be B2M null or low, optionally CIITA null or low, compared to their corresponding primary cells, (iii) have introduced expression of HLA-G or an uncleavable HLA-G, or knockout of one or both CD58 and CD54, (iv) have CD16 or a variant thereof, (v) have a second CAR with different targeting specificity, (vi) have cell surface-expressed exogenous cytokines and / or partial or complete peptides of their receptors, (vii) have at least one of the genotypes listed in Table 2, and (viii) have TAP1, TAP2, Tapasin, NLRC5, or CIITA compared to their corresponding primary cells. (ix) further comprising deletion or reduced expression of at least one of RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (ix) introduced or increased expression of 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 agonists, compared to its corresponding cell.

[0022] In various embodiments, the cells further comprise high-affinity, uncleavable CD16 (hnCD16) or a variant thereof. Some embodiments of high-affinity, uncleavable CD16 or a variant thereof comprise at least one of the following: (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 stimulating domain, and (g) transmembrane, signaling, and stimulating domains not derived from CD16 but derived from the same or a different polypeptide. In certain embodiments, (a) the non-native transmembrane domain may 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, and (b) the non-native stimulating domain may be derived from CD27, CD28, 4-1BB, O (c) The non-natural signaling domain may be derived from the polypeptides X40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D; (d) The non-natural transmembrane domain may be derived from NKG2D, the non-natural stimulating domain may be derived from 2B4, and the non-natural signaling domain may be derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D; or (d) The non-natural transmembrane domain may be derived from NKG2D, the non-natural stimulating domain may be derived from 2B4, and the non-natural signaling domain may be derived from CD3ζ.

[0023] In various embodiments, the cells further comprise a second CAR, the second CAR being optionally co-expressed in separate constructs or bicistronic constructs with (i) T cell-specific or NK cell-specific, (ii) bispecific antigen-binding CARs, (iii) switchable CARs, (iv) dimerized CARs, (v) split CARs, (vi) multi-chain CARs, (vii) inducible CARs, (viii) cell surface-expressed exogenous cytokines and / or their receptors, in partial or complete peptides. xi) Co-expressed with a checkpoint inhibitor in an optionally separate construct or bicistronic construct, (xii) specific to 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, antigens 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-α, 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 insertion domain receptor (KDR), Lewis A (CA19).9) It is specific to one of the following: 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), mesoserin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncoemetic 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 oncoprotein (WT-1), and pathogen antigens.

[0024] In various embodiments, the cell comprises a partial or complete peptide of exogenous cytokines and / or their receptors expressed on the cell surface, the exogenous cytokine or its receptor comprising (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, or (iii) the 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 either native or modified, and (vii) a homodimer of IL15Rβ, wherein any one of (i) to (vii) may be co-expressed with CAR in a separate construct or in a bicistronic construct, and may be optionally (c) transiently expressed.

[0025] In some embodiments where the cells or population are derived effector cells, the derived effector cells may be hematopoietic cells containing longer telomeres compared to the corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, or the CAR may have the following characteristics: (i) specific to T or NK cells, (ii) bispecific in antigen binding, (iii) switchable CAR, (iv) dimerized CAR, (v) splintered CAR, (vi) multichain CAR, (vii) inducible CAR (viii) being present, and 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. In various embodiments, derived effector cells may be able to recruit and / or migrate T cells to the tumor site, and derived effector cells may be able to reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. In various embodiments, derived effector cells include derived CD34 cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic pluripotent 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 cells further contain a second CAR co-expressed with the checkpoint inhibitor, or the derived effector cells can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors, the checkpoint inhibitors may be antagonists 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 KIRs. In various embodiments, the checkpoint inhibitor comprises (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0027] In some of these embodiments of cells or populations, the derived effector cells have, compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, at least one of the following characteristics: (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 and / or activate or mobilize bystander immune cells to tumor sites; (vii) enhanced ability to reduce tumor immunosuppression; (viii) improved ability in rescuing tumor antigen evasion; (ix) ability to stabilize tumor antigens; and (x) ability to evade flutorides.

[0028] In some embodiments of the cells or populations thereof, the cells comprise (i) one or more exogenous polynucleotides incorporated into a safe harbor locus or a selected locus, or (ii) two or more exogenous polynucleotides incorporated into different safe harbor loci or two or more selected loci. In certain embodiments, the safe harbor locus may include at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, and the selected locus may 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 incorporation of an exogenous polynucleotide knocks out the expression of the gene at the locus. In embodiments where the locus is TCR, the TCR locus may be a constant region of TCR alpha or TCR beta.

[0029] In another embodiment, the present invention provides compositions comprising cells or populations thereof as described herein. In a related embodiment, the present invention provides compositions for therapeutic use comprising derived effector cells provided herein and one or more therapeutic agents. In various embodiments, one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies or functional variants or fragments thereof, chemotherapeutic agents or radioactive moieties, or immunomodulatory agents (IMiDs). In some of these embodiments, the composition comprises a checkpoint inhibitor, where the checkpoint inhibitor is (a) 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), T (b) one or more antagonists to checkpoint molecules including LR3, VISTA, NKG2A / HLA-E, or inhibitory KIRs, (c) one or more of the following: atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents, or (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or one or more therapeutic agents including one or more of the following: venetoclax, azacitidine, and pomalidomide.In some of these embodiments in which the composition contains antibodies, the antibodies are (a) anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies, (b) rituximab, bertuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cerltuximab (c) one or more of the following: (c) daratumumab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, as well as their functional equivalents and biosimilars, or (c) derived effector cells containing daratumumab and CD38 knockout, and optionally the expression of CD16 or its variants.

[0030] In another aspect, the present invention provides therapeutic uses of the compositions provided herein by introducing the compositions into subjects suitable for adoptive cell therapy, the subjects having autoimmune disorders, hematological malignancies, solid tumors, cancer, or viral infections.

[0031] In yet another aspect, the present invention provides a method for producing derived effector cells containing the CAR described herein, the method comprising differentiating a genetically engineered iPSC, the iPSC comprising a polynucleotide encoding the CAR, and optionally, by editing one or more of the following: (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 an uncleavable HLA-G, or knockout of one or both CD58 and CD54, (iv) CD16 or a variant thereof, (v) a chimeric antigen receptor (CAR) having different targeting specificities, (vi) a cell surface-expressed exogenous cytokine or a portion or complete peptide of its receptor, (vii) at least one of the genotypes listed in Table 2, (viii) This results in deletion or reduced expression of at least one of the following compared to the corresponding primary cells: 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, and / or (ix) introduced or increased expression of at least one of the following compared to the corresponding primary cells: 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 bispecific or multispecific or universal engagers.In various embodiments, the method further comprises genomically manipulating a cloned iPSC 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 a partial or complete peptide of HLA-G or an uncleavable HLA-G, a high-affinity uncleavable CD16 or a variant thereof, a second CAR, and / or a cell surface-expressed exogenous cytokine or its receptor. In some embodiments, the genomic manipulation includes targeted editing. In certain embodiments, targeted editing includes deletion, insertion, or in / del, and targeted editing may be carried out by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional modification of these methods.

[0032] In yet another embodiment, the present invention provides CRISPR-mediated editing of cloned iPSCs, the editing comprising knock-in of a polynucleotide encoding a CAR as described herein. In various embodiments, the editing of a cloned iPSC further comprises knocking out CD38, the CAR may 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, the insertion knocks out the expression of the gene at the locus.

[0033] In yet another embodiment, the present invention provides a method for treating a disease or condition, comprising administering cells containing the CARs described herein to a subject in need of treatment. In various embodiments, the cells include derived effector cells containing CD38 knockout, CD16 or a variant thereof, and may optionally include (i) B2M and CIITA knockout, (ii) transfected expression of HLA-G or an uncleavable HLA-G, or knockout of one or both CD58 and CD54, (iii) transfected expression of a second CAR, and / or cell surface-expressed exogenous cytokines or partial or complete peptides of their receptors, and / or (iii) at least one of the genotypes listed in Table 2. In various embodiments, cell administration results in, compared to treatment using effector cells without CAR as described herein, (i) reducing tumor cell surface shedding of MICA / B antigens, (ii) increasing tumor cell surface MICA / B density, (iii) preventing tumor antigen escape, (iv) overcoming tumor microenvironment suppression, (v) enhancing the activation and killing functions of effector cells, and (vi) in vivo control of tumor progression, reduction of tumor cell load, tumor clearance, and / or improved survival rates.

[0034] Various purposes and advantages of the compositions and methods provided herein will become apparent from the following description, together with the accompanying drawings illustrating certain embodiments of the invention, as examples and illustrations. [Brief explanation of the drawing]

[0035] [Figure 1] This diagram illustrates several construct designs for cytokines expressed on the cell surface in iPSC-derived cells. IL15 is used as an illustrative example and can be replaced with other desirable cytokines. [Figure 2A] This example illustrates CAR constructs that have identical scFv and CD8 hinge regions, differing only in their signal transduction components, including the endodomain. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3A] This study demonstrates that iPSC-derived cells stably express target-specific CARs after lentiviral transduction using FACS sorting and CAR antibody staining of Thy1.1-expressing cells. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 4] Figure 4A shows a multiple myeloma antigen-specific cytotoxicity assay of various CAR constructs constructed in iPSC-induced effector cells against MM.1S target cells. Figure 4B shows the 1 / EC50 ratio, which indicates the high killing rate of the CARs shown. [Figure 5] This graph shows telomere lengths determined by flow cytometry, indicating that mature derived NK cells from iPSCs maintain longer telomeres compared to adult peripheral blood NK cells. [Modes for carrying out the invention]

[0036] Genome modification of iPSCs (induced pluripotent stem cells) includes polynucleotide insertions, deletions, and substitutions. Exogenous gene expression in genetically engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genetically engineered iPSC, after cell differentiation, and in dedifferentiated cell types from cells derived from genetically engineered iPSCs. On the other hand, directly manipulating primary immune cells such as T cells or NK cells is difficult, hindering the preparation and delivery of engineered immune cells for adoptive cell therapy. This invention provides an efficient and reliable targeted approach for the stable incorporation of one or more exogenous genes, including suicide genes and other functional modalities, which impart improved therapeutic properties with respect to engraftment, transport, 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] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular forms shall include plural forms and plural forms shall include singular forms.

[0038] The present invention is not limited to, and therefore may differ from, the specific methodologies, protocols, and reagents described herein. The terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

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

[0040] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination thereof of the alternatives.

[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, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to the quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of reference. In one embodiment, the terms “about” or “approximately” refer to a range of a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of reference.

[0043] As used herein, the terms “substantially” or “essentially” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “essentially the same” or “substantially the same” refer to a range of quantities, levels, values, numbers, frequencies, percentages, dimensions, size, volume, weight, or length that is approximately the same as the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0044] As used herein, the terms “substantially absent” and “essentially absent” are interchangeable and, when used to describe a composition such as a cell population or culture medium, refer to a composition that does not contain a particular substance or its source, for example, a composition that is 95%, 96%, 97%, 98%, 99% absent of a particular substance or its source, or undetectable when measured by conventional means. The terms “absent” or “essentially absent” of a particular component or substance in a composition also mean that such component or substance is (1) not present in the composition at any concentration, or (2) present in the composition at a low concentration, but functionally inactive. A similar meaning can be applied to the term “absent,” which refers to the absence of a particular substance or its source in a composition.

[0045] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” are understood to mean including the steps or elements or groups of steps or elements described, but not to mean excluding any other steps or elements or groups of steps or elements. In certain embodiments, the terms “include,” “have,” “contain,” and “comprise” are used as synonyms.

[0046] The phrase "consists of" means that what follows the phrase is included and limited to it. Therefore, the phrase "consists of" indicates that the listed elements are necessary or essential, and that other elements cannot exist.

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

[0048] Throughout this specification, references to “one embodiment,” “embodiment,” “a particular embodiment,” “related embodiment,” “a certain embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, mean that any particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, any particular feature, structure, or characteristic can be combined in any preferred manner in one or more embodiments.

[0049] The term “ex vivo” generally refers to activities performed outside of a living organism, such as experiments or measurements conducted in or on living tissue in an artificial environment outside of a living organism, preferably with minimal alteration of natural conditions. In certain embodiments, “ex vivo” procedures involve living cells or tissues taken from a living organism and cultured in an experimental apparatus, usually under sterile conditions, for typically several hours or up to about 24 hours (but depending on the circumstances, up to 48 or 72 hours or more). In certain embodiments, such tissues or cells may be collected and frozen and later thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissues that last longer than several days are typically considered “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,” and “increased developmental potential” refer to methods of increasing a cell’s differentiation potential 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 that has not been reprogrammed. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell that has not been reprogrammed.

[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 blood cells or muscle cells. Differentiated or induced-differentiation cells are cells that have taken on a more specialized (“committed”) position within a cell lineage. When applied to the process of differentiation, the term “committed” refers to a cell that, under normal circumstances, has progressed along the differentiation pathway to the point where it continues to differentiate into a particular 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: the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, ranging from incomplete or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce complete organisms to more primitive and pluripotent cells that can produce complete organisms (e.g., embryonic stem cells).

[0053] As used herein, the terms “induced pluripotent stem cells” or iPSCs mean stem cells produced from induced or modified, differentiated adult, neonatal, or fetal cells, i.e., cells that have been reprogrammed to differentiate into all tissues of all three germ layers or dermis: mesoderm, endoderm, and ectoderm. Produced iPSCs do not refer to cells found in nature.

[0054] As used herein, the term “embryonic stem cells” refers to the naturally occurring pluripotent stem cells in the inner cell mass of a 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 membrane or placenta; that is, 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 called “partially differentiated cells.” Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells such as hematopoietic stem cells and neural stem cells. “Multipotency” indicates that a cell may form many cell types in a given lineage, but not cells in other lineages. For example, a multipotent hematopoietic cell may form many different blood cell types (red, white, platelet, etc.), but not neurons. Thus, the term “multipotency” refers to a state of cell development that has a lower degree of developmental potential than totipotency and pluripotency.

[0056] Pluripotency can be determined in part by evaluating the pluripotent features of cells. These features 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 cell lineages (ectoderm, mesoderm, and endoderm), (v) the formation of teratomas consisting of the three somatic cell lineages, and (vi) the formation of embryoid bodies consisting of cells from the three somatic cell lineages.

[0057] Two types of pluripotency have been previously described: a pluripotent "priming" or "metastable" state similar to epiblast stem cells (EpiSCs) of late blastocysts, and a pluripotent "naive" or "grounded" state similar to cell masses of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or grounded 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 in DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks to developmental regulatory gene promoters, and (v) reduced expression of differentiation markers compared to pluripotent cells in the priming state. Standard methodologies of cell reprogramming in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or removed from the resulting pluripotent cells are generally considered to have the characteristics of the priming state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in a priming state and exhibit ground state characteristics unless the expression of exogenous transgenes that exhibit ground state characteristics is maintained.

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

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

[0060] A “pluripotency factor” or “reprogramming factor” refers to a drug that can increase the developmental potential of a cell, either alone or in combination with other drugs. 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"), "supplementary components," and "medium supplementary components" refer to the nutritional composition used to culture cells.

[0062] "Culture" or "maintain" refers to maintaining, proliferating (growing), and / or differentiating cells outside of tissue or in vitro, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culture" or "maintain" may utilize the culture medium as a source of nutrients, hormones, and / or other factors that help proliferate and / or maintain the cells.

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

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

[0065] The terms “hematopoietic stem cells and progenitor cells,” “hematopoietic stem cells,” “hematopoietic progenitor cells,” or “hematopoietic progenitor cells” refer to cells that are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation, including pluripotent hematopoietic stem cells (hemoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including bone marrow (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). As used herein, the term “secondary hematopoietic stem cells” refers to CD34+ hematopoietic cells that can give rise to both mature bone marrow and lymphoid cell types, including T lineage cells, NK lineage cells, and B lineage cells. Hematopoietic cells include various subsets of primordial hematopoietic cells that give rise to primitive red blood cells, megakaryocytes, and macrophages.

[0066] As used herein, the terms “T lymphocyte” and “T cell” are interchangeable and refer to the major type of leukocyte that completes maturation in the thymus and has various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells in a manner restricted by MHC class I. 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., Jarcutt, SupT1, etc., or T cells obtained from mammals. 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), and others, and can be at any developmental stage. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also differentiate from stem cells or progenitor cells. T cell-like derived effector cells may have T cell lineage in some respects, but at the same time possess one or more functional characteristics that are not present in primary T cells.

[0067] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a post-stimulation secretion profile, 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 the MHC (major histocompatibility complex) class II restriction immune response. In T lymphocytes, they define a helper / inducer subset.

[0068] "CD8+ T cells" refer 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 in thymocytes, as well as cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a relevant recognition element in major histocompatibility complex class I restriction 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” refer to interchangeable cells that are phenotypically CD3- and CD56+, express at least one of NKG2C and CD57, and optionally express CD16, but not PLZF, SYK,

[0070]

number

[0071] As used herein, the terms “NKT cells” or “natural killer T cells” refer to CD1d-restricted T cells that express a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire, i.e., standard α chains (Vα24-Jα18 in humans) associated with a limited spectral β chain (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, exhibits a more heterogeneous use of TCRαβ. Type I NKT cells are considered preferable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one 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 cells or populations of cells separated from their original environment; that is, the environment of isolated cells substantially does not contain at least one component found in the environment in which “unisolated” reference cells exist. This term includes cells isolated from some or all components found in a natural environment, for example, cells isolated from tissue or biopsy specimens. This term also includes cells isolated from at least one, some or all components, such as cells isolated from a cell culture or cell suspension, where cells are found in an environment in which they do not naturally exist, for example. Thus, isolated cells are partially or completely separated from at least one component, including other substances, cells, or populations of cells, where they would be found in nature or grow, preserve, or survive in an environment in which they do not naturally exist. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in media that do not naturally exist. Isolated cells can be obtained by separating desired cells or populations from other substances or cells in an environment, or by removing one or more other cell populations or subpopulations from an environment.

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

[0074] As used herein, the term “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that serve 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 derived therefrom. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0075] "Construction" refers to a macromolecule or molecular complex containing polynucleotides that is delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct that can be directed to the delivery or transfer of foreign genetic material to a target cell and can be replicated and / or expressed in the target cell. As used herein, the term "vector" includes the construct being delivered. A vector can be a linear or cyclic molecule. A vector can be embedded or unembedded. Major 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, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, and Sendai virus vectors.

[0076] "Integration" means that one or more nucleotides of a construct are stably inserted into the cellular genome, i.e., covalently bonded to a nucleic acid sequence within the cell's chromosomal DNA. "Targeted integration" means that the nucleotides of a construct are inserted into the cell's chromosome or mitochondrial DNA at a pre-selected site or "integration site." As used herein, the term "integration" further refers to the process of inserting one or more exogenous sequences or nucleotides of a construct, with or without deletion of endogenous sequences or nucleotides 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. The molecule may be introduced, for example, by introducing the encoding nucleic acid into the host's genetic material, for example, by incorporating it into the host's chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in relation to the expression of encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid into the cell in an expressible form. The term “endogenous” refers to the referenced molecule or activity that is present in the host cell. Similarly, when used in relation to the expression of encoding nucleic acid, the term refers to the expression of encoding nucleic acid that is present in the cell and not introduced exogenously.

[0078] As used herein, “Gene of Interest” or “Polynucleotide Sequence of Interest” is a DNA sequence that, under the control of an appropriate regulatory sequence, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo. Genes of Interest or polynucleotides of Interest may include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from DNA of eukaryotes (e.g., mammals), and synthetic DNA sequences. For example, genes of Interest may encode miRNAs, shRNAs, natural polypeptides (i.e., polypeptides found in nature) or fragments thereof; variant polypeptides (i.e., variants of natural polypeptides having less than 100% sequence identity with the natural polypeptide) or fragments thereof; engineered polypeptides or peptide fragments, therapeutic peptides or polypeptides, imaging markers, selectable markers, etc.

[0079] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, which is either a deoxyribonucleotide or a ribonucleotide or an analog thereof. The sequence of a polynucleotide consists of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) for thymine. Polynucleotides may 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 interchangeable and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, also commonly referred to in the art as polypeptides or proteins. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0081] "Operatively linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is influenced by the other. For example, a promoter is operationally linked to a coding sequence or functional RNA if it can influence 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 operationally linked to a regulatory sequence in sense or antisense orientation.

[0082] As used herein, the term “genetic imprint” refers to genetic or epigenetic information that contributes to preferred therapeutic attributes in source cells or iPSCs and that can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic cells. As used herein, “source cell” is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can be further differentiated into specific cell types, including any hematopoietic cell lineage. Source cell-derived iPSCs, and cells differentiated therefrom, are sometimes collectively referred to as “derived” or “derived” cells, depending on the context. For example, derived effector cells, or derived NK lineage cells or derived T lineage cells as used throughout this specification, are cells differentiated from iPSCs compared to their corresponding primary cells obtained from natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissue. As used herein, gene imprints conferring preferred therapeutic attributes are incorporated into iPSCs either by reprogramming donor-specific, disease-specific, or treatment-response-specific selected source cells, or by introducing a genetically modified modality into iPSCs using genome editing. In the case of source cells obtained from a specifically selected donor, disease, or treatment situation, the gene imprints contributing to preferred therapeutic attributes may include situation-specific genetic or epigenetic modifications that represent a retainable phenotype, i.e., preferred therapeutic attribute, which are passed on to derived cells of the selected source cells, regardless of whether the underlying molecular event has been identified. Donor-specific, disease-specific, or treatment-response-specific source cells may include genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, which may include, but are not limited to, pre-configured single-specific TCRs from virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, such as homozygosity for point mutations encoding high-affinity CD16 receptors in selected donors; and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting an increased haplotype in the population.When used herein, preferred therapeutic attributes include improved engraftment, transport, homing, viability, self-renewal, persistence, control and regulation of immune responses, survival, and cytotoxicity of derived cells. Preferred therapeutic attributes may also relate to antigen-targeting receptor expression, HLA presentation or absence, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity through reduced extratumor effects, and resistance to therapies such as chemotherapy.

[0083] As used herein, the term “enhanced therapeutic properties” refers to the therapeutic properties of a cell that are enhanced compared to a typical immune cell of the same common cell type. For example, NK cells with “enhanced therapeutic properties” have enhanced, improved, and / or increased therapeutic properties compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, 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 absence, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity by reducing extratumor effects, and resistance to treatments such as chemotherapy.

[0084] As used herein, the term “engager” refers to molecules, such as fusion polypeptides, that can form links between immune cells, such as T cells, NK cells, NKT cells, B cells, macrophages, neutrophils, and tumor cells, and activate immune cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), triplicate killer cell engagers, or multispecific killer cell engagers, or universal engagers 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. Surface trigger receptors can be manipulated and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, surface trigger receptors facilitate the binding of bispecific or multispecific antibodies between effector cells and specific target cells, such as tumor cells, regardless of the effector cell’s native receptor and cell type. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and then differentiated into populations of various effector cell types expressing the universal surface trigger receptor. “Universal” means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind to or ligate to engagers having the same epitope recognizable by the surface trigger receptor, regardless of the engager’s tumor-binding specificity. In some embodiments, engagers having the same tumor-targeting specificity are used to bind to the universal surface trigger receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to universal surface trigger receptors. Therefore, one or more effector cell types may be used to kill one specific type of tumor cell, or two or more types of tumor cells. Surface trigger receptors generally include a co-stimulatory domain for effector cell activation and an epitope-binding region specific to the engager's epitope. A bispecific engager is specific to the epitope-binding region of the surface trigger receptor at one end and specific to the 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 adverse effects of cell therapy. In some cases, the expression of a safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells, in which the gene encoding the safety switch protein is permanently incorporated into its genome. This conditional control may be variable and may include control by post-translational activation via small molecules as well as by tissue-specific and / or transient transcriptional regulation. Safety switches may mediate the induction of apoptosis, inhibition of protein synthesis, arrest of DNA replication and growth, transcription and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some cases, safety switch proteins are activated by exogenous molecules, such as prodrugs, which, upon activation, induce apoptosis and / or cell death in 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, a prodrug administered in the event of an adverse event is activated by a suicide gene product, killing the transduced cells.

[0087] As used herein, the term “pharmaceutically active protein or peptide” refers to a protein or peptide capable of achieving biological and / or pharmaceutical effects on an organism. Pharmacologically active proteins may have curative, radical, or palliative properties to a disease and may be administered to restore, alleviate, reduce, reverse, or mitigate the severity of the disease. Pharmacologically active proteins may also have prophylactic properties and may be used to prevent the onset of a disease or, when it appears, to mitigate the severity of such a disease or pathological condition. Pharmacologically active proteins include whole proteins or peptides, or their pharmaceutically active fragments. It also includes pharmaceutically active analogues of proteins or peptides, or analogues of protein or peptide fragments. The term pharmaceutically active protein may also refer to multiple proteins or peptides that act synergistically or cooperatively to provide therapeutic benefits. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitor proteins, antibodies or their fragments, growth factors, and / or cytokines.

[0088] As used herein, the term “signaling molecule” refers to any molecule that modulates, participates in, inhibits, activates, reduces, or increases cellular signaling. Signaling refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along pathways that ultimately lead to biochemical events within cells. Signaling pathways are well known in the art and include, but are not limited to, G protein-bound receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Tollgate signaling, ligand-dependent ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0089] As used herein, the term “targeting modality” refers to molecules, e.g., polypeptides, that are genetically incorporated into cells and promote antigen and / or epitope specificity, including but not limited to: i) antigen specificity when related to a unique chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when related to a monoclonal antibody or bispecific engager; iii) targeting of transformed cells; iv) targeting of cancer stem cells; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0090] As used herein, the terms “specific” or “singularity” may be used to refer to the ability of a molecule, such as a receptor or engager, to selectively bind to a target molecule, as opposed to nonspecific or nonselective binding.

[0091] As used herein, the term “adoptive cell therapy” means, as used herein, a cell-based immunotherapy associated with the infusion of autologous or allogeneic lymphocytes, which are identified as T cells or B cells that have been expanded ex vivo prior to infusion, whether genetically modified or not.

[0092] As used herein, “therapeutably sufficient amount” means, to the extent of its meaning, a sufficient and / or effective amount of the particular therapeutic and / or pharmaceutical composition it refers to, that is nontoxic but provides the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient’s general health, the patient’s age, and the stage and severity of the condition. In particular 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 being treated.

[0093] 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 utilizes the formation of embryoid bodies (EBs) as a general and important intermediate to initiate lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryogenesis, giving rise to numerous lineages within a three-dimensional region. Through a differentiation process that typically lasts from hours to days, simple EBs (e.g., aggregated pluripotent stem cells that induce differentiation) continue to mature and develop into cystic EBs, at which point (typically days to weeks) they are further processed and continue to differentiate. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other within a three-dimensional multilayer cell cluster, typically achieved by one of several methods, including settling the pluripotent cells into droplets, settling the cells into a "U" bottom well plate, or by mechanical agitation. Aggregates maintained in pluripotent culture maintenance medium do not form proper EBs, so pluripotent stem cell aggregates require further differentiation cues to promote EB development. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cues for induction toward the selected lineage. EB-based culture of pluripotent stem cells typically results in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm) with moderate proliferation within EB cell clusters. While proven to promote cell differentiation, EBs produce heterogeneous cells in different differentiation states because the three-dimensional cells are not consistently exposed to differentiation cues from the environment. In addition, EBs are difficult to produce and maintain. Furthermore, EB-mediated cell differentiation involves moderate cell expansion, which also contributes to reduced differentiation efficiency.

[0094] In contrast, "aggregate formation," distinct from "EB formation," can be used to expand populations of pluripotent stem cell-derived cells. For example, during aggregate-based expansion of pluripotent stem cells, culture media are selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of aggregates, which form larger aggregates, and these aggregates can routinely dissociate into smaller aggregates mechanically or enzymatically to maintain cell proliferation in culture and increase cell number. Unlike EB culture, cells cultured in aggregates under maintenance culture retain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0095] As used herein, “monolayer differentiation” is a term that refers to a differentiation method distinct from differentiation by three-dimensional multilayer clustering of cells, i.e., “EB formation.” Among other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Because monolayer culture does not mimic embryonic development such as EB formation, differentiation into a particular lineage is considered minimal compared to the differentiation of all three germ layers in EB.

[0096] As used herein, “dissociated” cells refer to cells that have been substantially separated or purified from other cells or from a surface (e.g., the surface of a culture plate). For example, cells may be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro may be dissociated from one another 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 may 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 “feeder” is a term describing one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, expand, or differentiate, by providing stimuli, growth factors, and nutrients to support the second cell type. Feeder cells are, arbitrarily, 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 irradiation or treatment with mitotic antagonists such as mitomycin to prevent them from growing more than the cells they support when co-cultured with other cells. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type may be a human feeder, such as human dermal fibroblasts. Another feeder cell type may be a mouse embryonic fibroblast (MEF). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation to a particular lineage, enhance proliferative capacity, and promote maturation to specialized cell types such as effector cells.

[0098] As used herein, “feeder-free” (FF) environment refers to an environment such as culture conditions, cell cultures, or culture media that are essentially free from feeder cells or stromal cells and / or are not pre-conditioned by feeder cell culture. “Pre-conditioned” medium refers to a medium that has been harvested after feeder cells have been cultured in the medium for a period of time, such as at least one day. Pre-conditioned mediums contain many mediator substances, including growth factors and cytokines secreted by feeder cells cultured in the medium. In some embodiments, a feeder-free environment is free from both feeder cells and stromal cells and is not pre-conditioned by feeder cell culture.

[0099] When used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, “functional” means (1) good knock-in, knock-out, knock-down gene expression, transgenic or controlled gene expression at the gene level, such as inducible or transient expression at a desired cell developmental stage, achieved by direct genome editing or modification, or by “transmission” through differentiation or reprogramming from the initiating cell that is first genome-edited, or (2) (i) 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 initial genome-edited starting cell, (iii) downstream gene regulation in the cell as a result of modification of gene expression that appears only in the early developmental stages of the cell, or only in the starting cell that gives rise to the cell through differentiation or reprogramming, or (iv) removal, addition, or modification of favorable cellular functions / characteristics at the cellular level by enhanced or newly acquired cellular functions or attributes presented in the mature cell product, which originate from genome editing or modification performed initially on an iPSC, progenitor cell, or dedifferentiated cell origin.

[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, no longer maintained, or has a reduction in level such that the decrease or reduction is lower than the level naturally detectable by other cells or synthetic methods.

[0101] As used herein, “modified HLA-deficient iPSC” refers to an HLA-deficient iPSC that is further modified by introducing genes expressing proteins related to, but not limited to, improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, co-stimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3ζ, 41BBL, CD47, CD113, and PDL1. “Modified HLA-deficient” cells also include cells other than iPSCs.

[0102] Fc receptors, abbreviated as FcR, are classified based on the type of antibody they recognize. 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 signaling characteristics 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 their 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 are modified or replaced with non-native transmembrane and / or intracellular signaling domains. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane and / or signaling domains being non-native, one or more stimulating domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance cell activation, expansion, and function through receptor induction. Unlike chimeric antigen receptors (CARs) that contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to Fc fragments, or the Fc region of an antibody, or the Fc region contained in an engager or binding molecule, activating cells regardless of whether the targeted cell is in proximity. For example, the Fcγ receptor may be engineered to contain a selected transmembrane domain, a stimulating domain, and / or signaling domains within the intracellular region that respond to the binding of IgG at the extracellular domain, thereby generating a CFcR. In one example, CFcRs are generated by manipulating the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domains. To further improve the binding affinity of CD16-based CFcRs, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcRs with a high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is eliminated, or the extracellular domain of the receptor is replaced in a way that makes it uncleavable, i.e., unaffected by shedding, thereby yielding an hnCD16-based CFcR.

[0104] The FcγR receptor CD16 has been identified to have two isoforms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and facilitating antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, “high affinity CD16,” “uncleavable CD16,” or “high affinity uncleavable CD16 (hnCD16)” refers to native or non-native variants of CD16. Wild-type CD16 has low affinity and, upon activation of NK cells, is subjected to external domain shedding, a proteolytic cleavage process that modulates 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) is modified or eliminated are not subject to shedding. The cleavage site and membrane proximal region are described in detail in WO2015 / 148926, the full disclosure of which is incorporated herein by reference. The CD16 S197P variant is an engineered non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high affinity and non-cleavable. Another exemplary high affinity and non-cleavable CD16 (hnCD16) variant is engineered CD16 containing an external domain derived from one or more of the three exons of the CD64 external domain.

[0105] I. Cells and compositions useful for adoptive cell therapy with enhanced properties Provided herein are strategies for systematically manipulating the regulatory circuits of clonal iPSCs without affecting the differentiation potential of iPSCs or the cellular developmental biology of iPSCs and their derived cells, while enhancing the therapeutic properties of derived cells. The derived cells are functionally improved and are suitable for adoptive cell therapy after a combination of selective modalities has been introduced into the cells at the iPSC level via genomic engineering. Prior to the present invention, it was unclear whether modified iPSCs, including one or more provided gene edits, still possessed the ability to enter cell development and / or to mature and generate functionally differentiated cells while retaining regulated activity. Unexpected failures during targeted cell differentiation from iPSCs are due to embodiments including, but not limited to, developmental stage-specific gene expression or its absence, requirements for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure differentiation protocols to allow for changes in the cellular phenotype and / or function. This application demonstrates that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from the modified iPSCs possess enhanced and / or acquired therapeutic properties resulting from the individual or combined genomic modifications, which are retained in the effector cells after iPSC differentiation.

[0106] 1. CAR with a novel end domain In embodiments, a chimeric antigen receptor (CAR) is a fusion protein generally comprising an external domain including an antigen recognition domain and a transmembrane domain, and an internal domain including one or more signaling domains. In embodiments, the CARs described herein are designed to be expressed and function in induced pluripotent stem cells (iPSCs) and derived effector cells differentiated 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 directed toward a desired effector cell type. In embodiments, the CAR enhances effector cell proliferation, persistence, survival, cytotoxicity, resistance to allo-rejection, tumor infiltration, migration, ability to activate and / or mobilize bystander immune cells, and / or ability to overcome tumor suppression. In embodiments, the CARs provided herein may also be directly expressed in cell line cells and primary sources (primary cells), i.e., cells from natural / natural sources such as peripheral blood, umbilical 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 may include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or a combination thereof. 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, the CAR containing an NK cell-specific signaling component is NK cell-specific. In some embodiments, the CAR containing an NK cell-specific signaling component is also suitable for T cells or other cell types. In some embodiments, the CAR containing a T cell-specific signaling component is T cell-specific. In some embodiments, the CAR containing a T cell-specific signaling component is also suitable for NK cells or other cell types. In some embodiments, the CAR containing an NKT cell-specific signaling component is NKT cell-specific. In some embodiments, the CAR containing an NKT cell-specific signaling component is also suitable for NK or T cells, or other cell types.

[0108] In embodiments, the CAR described herein comprises at least an ectodomain, a transmembrane domain, and an endodomain. The endodomain of the CAR includes at least one signaling domain that influences the proliferation 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 called additional signaling domains) are further included to influence cell lifespan, memory differentiation, and metabolic properties. Here, a signaling protein specific to T cells and / or NK cells is used to supply the components of the CAR fusion protein, e.g., one or more signaling domains contained in 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. A description of exemplary signaling proteins, including their transmembrane and cytoplasmic sequences, is further provided below in Table 1A.

[0109] 2B4 (natural killer cell receptor 2B4) is a receptor for CD48, a signaling lymphocyte-activating molecule (SLAM). Upon ligand binding, 2B4 is involved in the regulation and interconnection of both innate and adaptive immune responses, as it modulates the activation and differentiation of a wide variety of immune cells. Acting as an activated NK cell receptor, 2B4 stimulates NK cell cytotoxicity, IFN-γ production, and granular exocytosis. Optimal NK cell proliferation and activation appear to depend on the involvement of CD48 and 2B4 expressed on adjacent NK cells. 2B4 is also involved in the regulation of CD8+ T cell proliferation. 2B4 expression on activated T cells and its binding to CD48 provide a co-stimulatory function to adjacent T cells. Furthermore, 2B4 is involved in leukocyte migration.

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

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

[0112] CD2 (T cell surface antigen CD2) interacts with lymphocyte function-associated antigens CD58 (LFA-3) and CD48 / BCM1 to mediate adhesion between T cells and other cell types. The cytoplasmic domain of CD2 is involved in signaling that triggers T cell activation. CD2 is also involved in leukocyte migration, NK cell activation, T cell differentiation, and the regulation of IFN-γ and IL-8 secretion.

[0113] CD28 (T cell-specific surface glycoprotein CD28) is involved in the T cell receptor signaling pathway and influences T cell activation and co-stimulation, induction of cell proliferation, cytokine production, and promotion of T cell survival. CD28 also modulates regulatory T cell differentiation and enhances IL4 and IL10 production 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 via 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 lymphocytes, playing a crucial role in adaptive immune responses. When antigen-presenting cells (APCs) activate the T cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by CD3 chains CD3D, CD3E, CD3G, and CD3Z. All CD3 chains contain an immune receptor tyrosine-based activation motif (ITAM) in their cytoplasmic domain. Upon TCR involvement, these motifs are phosphorylated, leading to the activation of downstream signaling pathways. CD3Z plays a vital role in the differentiation of intrathymic T cells.

[0116] DAP10 (hematopoietic cell signaling transducer) is a transmembrane adapter protein that, in association with KLRK1, forms the activated receptor KLRK1-HCST in lymphoid and myeloid cells. The KLRK1-HCST receptor plays a role in immunological surveillance against tumors and is typically required for the cellular breakdown of tumor cells expressing cell surface ligands, such as MHC class I chain-associated MICA and MICB, as well as UL16-binding protein (ULBP), which 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-inducing 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, mediating signaling and cell activation after ligand binding by the receptor. DAP12 is associated with natural killer (NK) cell receptors such as KIR2DS2 and KLRD1 / KLRC2 heterodimers, and mediates the activation of NK cells. DAP12 also enhances the transport and cell surface expression of NK cell receptors KIR2DS1, KIR2DS2, and KIR2DS4, ensuring their stability on the cell surface. Furthermore, DAP12 negatively regulates B cell proliferation.

[0118] DNAM1 (CD226 antigen) is involved in immune responses, cell-cell adhesion, lymphocyte signaling, and cytotoxicity and lymphokine secretion mediated by cytotoxic T lymphocytes (CTLs) and NK cells. DNAM1 also modulates T cell receptor signaling, stimulating T cell proliferation and cytokine production, including those 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 classes 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 activating natural killer cells.

[0121] IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta) is the beta subunit of the interleukin-2 receptor, associated with IL15RA, and is involved in receptor-mediated endocytosis, transducing IL2 signaling. IL-2Rβ may influence cell persistence through negative regulation of the apoptotic process.

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

[0123] IL-7R (interleukin-7 receptor subunit alpha) is the receptor for interleukin-7 and is involved in IL-7 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 the receptor for natural killer (NK) cells of the HLA-C allele. KIR2DS2 does not inhibit NK cell activity and is involved in the regulation of innate immune responses and immune responses.

[0125] NKG2D (NKG2-D type II endogenous membrane protein) functions as an activating and co-stimulating 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, providing both stimulative and co-stimulating innate immune responses to activated killer (NK) cells, resulting in cytotoxic activity. By amplifying T cell activation, NKG2D functions as a co-stimulating receptor for the T cell receptor (TCR) in CD8+ T cell-mediated adaptive immune responses. NKG2D stimulates perforin-mediated efflux from ligand-expressing tumor cells. NKG2D is also involved in signaling with calcium influx, leading to peak TNF-α expression and involvement in NK cell-mediated bone marrow graft rejection. NKG2D may also play a regulatory role in NK cell differentiation and survival.

[0126] NKp30 (Natural Cytotoxicity-Inducing Receptor 3) is a cell membrane receptor for natural killer cells and is activated by the binding of extracellular ligands, including BAG6 and NCR3LG1. NKp30 is involved in cell recognition, immune responses, and the regulation of immune responses. 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-Inducing Receptor 2) and NKp46 (Natural Cytotoxicity-Inducing Receptor 1) are receptors that activate cytotoxicity and may contribute to improved efficiency of activated natural killer (NK) cells that mediate tumor cell lysis. Both NKp44 and NKp46 are involved in cellular defense responses, innate immune responses, and their regulation.

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

[0129] CD8 (T cell surface glycoprotein CD8 alpha chain) is an endogenous membrane glycoprotein that plays an essential role in the immune response, performing multiple functions in responses to both external and internal attacks. In T cells, CD8 primarily 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 conjugation 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 provided CARs, the end domain of the CAR comprises 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 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, respectively. In some embodiments, the signaling domain of the CAR 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, the 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 ITAM (immune receptor tyrosine-based activation motif), YxxM motif, TxYxxV / I motif, FcRγ, hemi-ITAM, and / or ITT-like motifs.

[0134] In some embodiments of the provided CARs, the end domain of the CAR containing a first signaling domain further comprises a second signaling domain containing 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 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, and the second signaling domain is distinct from the first signaling domain.

[0135] In some embodiments of the provided CAR, the end domains of the CAR, including the first and second signaling domains, are represented by SEQ ID NOs: 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, and FcERIγ, respectively, as indicated by SEQ ID NOs: 21-41, 54, and 56. The third signaling domain further comprises an amino acid sequence that is at least approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% identical to the cytoplasmic domain or portion thereof of IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ / 1XX (i.e., CD3ζ or CD3ζ1XX), CS1, or CD8, wherein the third signaling domain is distinct 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 includes 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 portion thereof of a protein, including but not limited 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 is 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-FcERI This includes, but is not limited to, γ, 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, and comprises a morphological fusion cytoplasmic domain or a portion thereof.

[0138] In some exemplary embodiments of a CAR having an end domain consisting of three different signaling domains, the end domain includes, but is not limited to, 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, and comprises a morphologically fused cytoplasmic domain or a portion thereof.

[0139] In some embodiments, the transmembrane domain of CAR contains 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 partial transmembrane domain 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 contains 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 portion thereof of the transmembrane domain of DAP10, KIR2DS2, 2B4, NKG2D, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, CD28H, or DNAM1, respectively, as represented by SEQ ID NOs.1-20, 53, and 55.

[0140] Table 1B provides non-limiting examples of CAR constructs containing transmembrane domains and endodomains (indicated as TM-(endodomain)). Generally, each exemplary CAR construct includes a transmembrane domain and an endodomain containing one or more signaling domains derived from the cytoplasmic region of one or more signaling proteins. In embodiments, the one or more signaling domains contained in the CAR endodomain are derived from the same or different proteins from which the TM is derived. As shown in Table 1B, the portion of the CAR presenting the transmembrane domain (TM) is underlined, the domains contained in the endodomain are indicated in parentheses "()", and each of the TM and 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 may be approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% identical to the full length or portion of the corresponding transmembrane or cytoplasmic region of a given signaling protein. Exemplary CAR constructs containing transmembrane domains and endodomains provided herein include: [Sequence Listing 2] This includes, but is not limited to, TIFF0007861191000005.tif48165. In some embodiments, each of the above exemplary CAR constructs, including transmembrane and endodomains, contains an amino acid sequence of approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequences represented by each of SEQ ID NOs. 57–74 in Table 1B. The exemplary sequences for each construct provided in Table 1B have formatted text to match the formatting (i.e., underlined, normal, or bold text) of the corresponding region in the diagram to the left of the sequence. In most of the exemplary constructs in Table 1B, TM is the first sequence region, but the construct may include an extracellular domain preceding TM (see construct 6, e.g.,) and may originate from the same or a different protein as TM. In some embodiments, two or more signaling domains contained within a CAR endodomain may be separated by one or more additional sequences, such as spacers or linkers.

[0141] [Table 2-1]

[0142] [Table 2-2]

[0143] [Table 2-3]

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

[0145] In certain embodiments, the ectodomain of the provided CAR includes an antigen recognition domain containing a mouse antibody, human antibody, humanized antibody, camel Ig, shark heavy chain-only antibody (VNAR), Ig NAR, chimeric antibody, recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, antigen-binding single-chain variable 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.

[0146] Non-limiting examples of antigens that may be targeted by CARs in genetically engineered iPSCs and derived effector cells include ADGRE2, carbonic anhydrase IX (CAIX), CCRI, 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), and epithelial cells. 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 insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A Examples include MAGE-A1, mucin 1 (Muc-1), mucin 16 (Muc-16), mesoserin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryonic 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 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 ectodomain of the provided CAR further comprises a signal peptide. The signal peptide guides the CAR polypeptide to the endoplasmic reticulum (ER) for appropriate glycosylation and fixation to the prammasa membrane. In general, any eukaryotic signal sequence that targets secreted proteins to the ER pathway can be used. Suitable example signal peptides include, but are not limited to, IL-2 signal sequences, kappa reader sequences, CD8α reader sequences, albumin signal sequences, prolactin signal sequences, and IgG signal peptides, and GM-CSF signal peptides.

[0148] In some embodiments, the ectodomain of the provided CAR may optionally include a hinge (also called 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 hinge of the CAR includes 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 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 hinge region of the provided CAR contains 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 CH2 / CH3 domain of the immunoglobulin.

[0149] In some embodiments, effector cells containing one or more CARs provided may be used to treat autoimmune disorders, hematological malignancies, solid tumors, or infections 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 tumors include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), several forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, several forms of myocarditis, and multiple Examples of viral infections include, but are not limited to, HIV-(human immunodeficiency virus), HSV-(herpes simplex virus), KSHV-(herpes zoster virus), RSV-(respiratory syncytial virus), EBV-(Epstein-Barr virus), CMV-(cytomegalovirus), VZV-(varicella-zoster virus), adenovirus-(adenovirus), lentivirus-(adenovirus), and BK-polyomavirus-associated disorders).

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

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

[0152] Non-restrictive CAR strategies include conditionally activated heterodimer CARs via dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9,587,020); split CARs which are homologous recombinations of antigen-binding, hinge, and endo-domains to generate the CAR (see, e.g., U.S. Publication No. 2017 / 0183407); multi-chain CARs which enable non-covalent bonding between two transmembrane domains linked to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Publication No. 2014 / 0134142); CARs with bispecific antigen-binding domains (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). See Invest.2016;126(8):3036-3052); inductive CARs (see, e.g., U.S. Publication Nos. 2016 / 0046700, 2016 / 0058857, and 2017 / 0166877); switchable CARs (see, e.g., U.S. Publication No. 2014 / 0219975); and any other designs known in the art.

[0153] Suitable genomic loci for CAR insertion provided herein include loci that meet the criteria for a genome-safe harbor, and / or loci where knockdown or knockout of a gene at the selected locus as a result of insertion is desired. 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 region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

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

[0155] As will be described in more detail herein, further embodiments provided include derived effector cells obtained from differentiating genomically engineered iPSCs, the iPSCs and derived cells including the CARs described herein, and further including 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, HLA-G overexpression and knockout of one or both of CD58 and CD54, TCR null, CD3-presenting surface, antigen-specific TCR, NKG2C, DAP10 / 12, NKG2C-IL15-CD33 ("2C1533").

[0156] 2. CD38 Knockout The cell surface molecule CD38 is highly upregulated in several hematological malignancies originating from both lymphoid and myeloid lines, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapies that deplete cancer cells. Antibody-mediated cancer cell depletion typically results from a combination of direct induction of cell apoptosis and activation of immunoeffector mechanisms such as ADCC (antibody-dependent cell-mediated toxicity). In addition to ADCC, immunoeffector mechanisms that work in conjunction 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 on CD34 + CD38 is expressed in stem cells, as well as in progenitor cells committed to lymphoid, erythrocyte, and bone marrow lineages, during the final stage of maturation which continues to the plasma cell stage. As a type II transmembrane glycoprotein, CD38 performs cellular functions as both a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 is involved in NAD + It catalyzes the synthesis and hydrolysis of the reaction from ADP to ribose, thereby producing the secondary messengers CADPR and NAADP, which stimulate the release of calcium from the endoplasmic reticulum and lysosomes, crucial for the cell adhesion process (a calcium-dependent process). As a receptor, CD38 recognizes CD31 and regulates cytokine release and cytotoxicity in activated NK cells. CD38 associates with cell surface proteins of lipid rafts and releases cytoplasmic Ca 2+ It has also been reported that it regulates flux and mediates signaling in lymphoid and myeloid cells.

[0158] In malignant tumor treatment, systemic use of T cells transduced with the CD38 antigen-binding receptor has been shown to lyse the CD38+ fractions of CD34+ hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells, resulting in an incomplete therapeutic response and reduced or eliminated efficacy due to recipient immune effector cell dysfunction. In addition, in multiple myeloma patients treated with daratumumab, a CD38-specific antibody, a reduction in NK cells was observed in both bone marrow and peripheral blood, while other immune cell types such as T cells and B cells were unaffected despite CD38 expression (Casneuf et al., Blood Advances. 2017;1(23):2105-2114). Without being limited by theory, CD38 null effector cells containing MICA / B-CARs could overcome CD38-mediated fracturing and avoid effector cell depletion or reduction induced by specific antibodies and / or CD38 antigen-binding domains. Furthermore, since CD38 is upregulated by activated lymphocytes such as T cells or B cells, CD38-specific antibodies such as daratumumab can be used to eliminate activated lymphocytes or suppress the activation of these lymphocytes in recipients of adaptive allogeneic effector cells that are CD38 null, thereby reducing and / or preventing allo-rejection by host lymphocytes against these effector cells and increasing the viability and persistence of these effector cells despite the presence of CD38 antibodies used for lymphocyte depletion. Thus, this application also provides strategies for reducing or preventing allo-rejection by using CD38-specific antibodies, secreted CD38-specific engagers, or CD38 CARs (chimeric antigen receptors) against and / or eliminating activated recipient T cells and B cells, while enhancing the persistence and / or viability of effector cells.

[0159] In one embodiment provided herein, CD38 knockout in iPSCs is a biallelic knockout. As disclosed herein, the provided CD38 null iPSCs can differentiate to produce functional derived effector cells, including, but not limited to, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional characteristics not present in primary NK, T, and / or NKT cells. In some embodiments, when ADCC is induced using a CD38 antibody or when CD38-CAR is used for targeted cell killing, CD38 - / - iPSCs and / or their derived 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 in vivo persistence and / or survival in the presence and / or after exposure to such therapeutic agents. 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 derived cells include one or more additional genome editing methods described herein, including but not limited to hnCD16 expression, CAR expression, cytokine / cytokine receptor expression, HLA I and / or HLA II knockout, and additional modalities provided herein.

[0160] In another embodiment, inserting one or more transgenes provided herein at a selected location of CD38 while simultaneously knocking out CD38 can be achieved, for example, by CD38-targeted knock-in / knockout (CD38-KI / KO) constructs. In some embodiments of such constructs, 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 located 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 the exogenous promoter contained in the construct. When two or more transgenes are inserted at a selected location of the CD38 locus, a linker sequence, e.g., a 2A linker or IRES, is positioned 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 distinct proteins from a single translation. In some embodiments, the 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 may 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.

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

[0162] Accordingly, in some embodiments, the high-affinity, non-cleavable CD16 receptor (hnCD16) comprises both F176V and S197P, and in some embodiments, it comprises F176V with the cleavage region excluded. In some other embodiments, hnCD16 comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, when compared to any of the exemplary sequences SEQ ID NOs 42, 43, and 44, each comprising at least a portion of the CD64 ectodomain. SEQ ID NOs 42, 43, and 44 are coded by illustrating SEQ ID NOs 45-47, respectively. As used herein and throughout this application, the identity percentage 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). The comparison of sequences and the determination of the identity percentage between two sequences can be achieved using mathematical algorithms recognized in the art. [Sequence Listing 3] TIFF0007861191000009.tif46167[Sequence Listing 4] TIFF0007861191000010.tif46169[Sequence Listing 5] TIFF0007861191000011.tif51169[Sequence Listing 6] TIFF0007861191000012.tif92169 [Sequence Listing 7] TIFF0007861191000013.tif97169 [Sequence Listing 8] TIFF0007861191000014.tif92169

[0163] Accordingly, among other edits intended and described herein, genetically engineered cloned iPSCs containing 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 iPSC. In some embodiments, the derived effector cells containing hnCD16 are NK cells. In some embodiments, the derived effector cells containing hnCD16 are T cells. Exogenous hnCD16 expressed in iPSCs or derived cells exhibits high affinity not only for ADCC antibodies or fragments thereof, but also for binding to bispecific, triplicate, or multispecific engagers or binders that recognize the extracellular binding domain of CD16 or CD64 of said hnCD16. Bispecific, triplicate, or multispecific engagers or binders are described further below in this application (see below). Thus, this application provides derived effector cells or a population of cells pre-loaded with one or more pre-selected ADCC antibodies via high-affinity binding to the extracellular domain of hnCD16 expressed on derived effector cells, in amounts sufficient for therapeutic use in the treatment of conditions, diseases, or infections as further detailed in the following sections, wherein the hnCD16 comprises the extracellular binding domain of CD64, or of 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 so that the chimeric Fc receptor (CFcR) is produced to include a non-native transmembrane domain, a non-native stimulating domain, and / or a non-native signaling domain. As used herein, the term “non-native” means that the transmembrane domain, stimulating domain, or signaling domain is derived from a different receptor other than the receptor that provides the extracellular domain. In the figures herein, CFcRs based on CD16 or a variant thereof do not have a transmembrane domain, stimulating domain, or signaling domain derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR includes a non-innate 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, or T cell receptor polypeptides. In some embodiments, the exogenous hnCD16-based CFcR includes a non-innate stimulative / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD1, LAG3, 2B4, BTLA, DAP10, DAP12, CTLA4, or NKG2D polypeptides. In some embodiments, the exogenous hnCD16-based CFcR includes a non-natural signaling domain derived from the CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of hnCD16, the provided chimeric receptor includes a transmembrane domain and a signaling domain, both derived from one of the IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides.One particular embodiment of the hnCD16-based chimeric Fc receptor comprises a transmembrane domain of NKG2D, a stimulating domain of 2B4, and a signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the extracellular domain of CD64 or a full-length or partial sequence of CD16, and the extracellular domain of CD16 comprises F176V and S197P. Another embodiment of the hnCD16-based chimeric Fc receptor comprises a transmembrane domain and a signaling domain of CD3ζ, wherein the extracellular domain of hnCD16 is derived from the extracellular domain of CD64 or a full-length or partial sequence of CD16, and the extracellular domain of CD16 comprises F176V and S197P.

[0165] The various embodiments of the hnCD16-based chimeric Fc receptor described above can bind with high affinity to the Fc region of an antibody or its fragment, or to the Fc region of a bispecific, triplicate, or multispecific engager or binder. Upon binding, the stimulating and / or signaling domains of the chimeric receptor enable activation of effector cells and cytokine secretion, as well as the killing of tumor cells targeted by the antibody or tumor antigen-binding component and the bispecific, triplicate, or multispecific engager or binder having its Fc region. Without being limited by theory, CFcRs contribute to the effector cell killing ability and increase the proliferation and / or potential of effector cells through the non-native transmembrane, stimulating, and / or signaling domains of the hnCD16-based chimeric Fc receptor, or through the binding of an engager to its external domain. Antibodies and engagers can bring tumor cells expressing the antigen and effector cells expressing CFcR into close proximity, which also contributes to the enhancement of tumor cell killing. Exemplary tumor antigens that are bispecific, tripspecific, or multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, tripspecific, or multispecific engagers or binders suitable for binding effector cells expressing hnCD16-based CFcRs to attack 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, which is cleaved from the cell surface after NK cell activation, various non-cleavable versions of CD16 in derived NK cells avoid CD16 shedding and maintain constant expression. In derived NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, which exhibit improved cellular function. Non-cleavable CD16 also enhances antibody-dependent cell-mediated toxicity (ADCC) and the binding of bispecific, tripspecific, 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 introduced hnCD16 in derived NK cells also allow for in vitro loading of ADCC antibodies to hnCD16-mediated NK cells before administering the cells to subjects requiring cell therapy. As provided, hnCD16 may, in some embodiments, comprise F176V and S197P, or a complete or partial ectodomain derived from CD64 as exemplified by SEQ ID NOs. 42, 43, or 44, or further comprise at least one of a non-natural transmembrane domain, a stimulating domain, and a signaling domain. As disclosed, the application also provides derived NK or a population of its cells pre-loaded with one or more pre-selected ADCC antibodies in quantities sufficient for therapeutic use in the treatment of a condition, disease, or infection, as will be detailed in the following sections.

[0167] Unlike primary NK cells, mature T cells from primary sources (i.e., natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing expressed exogenous, uncleavable CD16 could differentiate into functional derived T cells that not only express exogenous CD16 but also perform functions via the acquired ADCC mechanism, without compromising the developmental biology of the T cells. This acquired ADCC in derived T cells can additionally be used as an approach for dual targeting and / or as an approach to rescue antigen escape, which often occurs in CAR-T cell therapy, where tumors relapse with reduced or lost expression of CAR-T targeted antigens, or with mutated antigens that evade recognition by the CAR (chimeric antigen receptor). If the derived 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 regurgitation of targeted tumors commonly seen in CAR-T therapy. Such strategies for reducing and / or preventing antigen escape while achieving dual targeting are equally applicable to NK cells expressing one or more CARs. A variety of CARs that can be used in this antigen escape reduction and prevention strategy include the CARs described in this application.

[0168] Accordingly, in embodiments, the present invention provides derived T cells comprising exogenous CD16 in addition to at least one CAR provided. In further embodiments provided, the derived T cells obtained herein comprise CD38 knockout in addition to hnCD16 and CAR expression. In some embodiments, the hnCD16 contained in the derived T cells comprises F176V and S197P. In some other embodiments, the hnCD16 contained in the derived T cells comprises a complete or partial ectodomain derived from CD64, as exemplified by SEQ ID NOs. 42, 43, or 44, or may further comprise at least one of a non-natural transmembrane domain, a stimulating domain, and a signaling domain. As described, such derived T cells have an acquisition mechanism that targets tumors with monoclonal antibodies meditated by ADCC to enhance the therapeutic effect of the antibodies. As disclosed, the application also provides derived T cells or cell populations pre-filled with one or more pre-selected ADCC antibodies in amounts 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, and as a result, the cells can avoid being eliminated 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 containing 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 associated with such practices is reduced, while cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional soluble cytokine administration, 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 cytokines themselves, 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, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or ephemeral.

[0170] Figure 1 shows several structural designs using IL15 as an example. The transmembrane (TM) domain in any of the designs in Figure 1 is either naturally occurring for the IL15 receptor or modified or substituted with a transmembrane domain of any other membrane-bound protein.

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

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

[0173] Design 3: IL15Rα with a cleaved 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 excluding 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 crucial for the receptor to be expressed in IL15-responsive cells, and for these cells to expand and function. Such a cleaved construct contains an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 48, which may be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 49. In one embodiment of cleaved IL15 / IL15Rα, the construct does not contain the last four amino acids "KSRQ" of SEQ ID NO: 48 and contains an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 50. [Sequence Listing 9] TIFF0007861191000015.tif50169[Sequence Listing 10] TIFF0007861191000016.tif109167[Sequence Listing 11] TIFF0007861191000017.tif52169

[0174] Those skilled in the art will understand that the above-described signal peptide and linker sequences are illustrative and in no way limit their modifications suitable for use as signal peptides or linkers. Many suitable signal peptide or linker sequences exist that are known to and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence may be substituted with another sequence without altering 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, it has been shown that the cytoplasmic domain of IL15Rα can be omitted in such designs without adversely affecting the autonomous function of effector cells with IL15. Design 4 is a construct that provides another functional alternative to Design 3, in which essentially the entire IL15Rα is removed except for the Sushi domain, and IL15 is fused at one end with the other transmembrane domain (mb-Sushi), and optionally 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, unwanted signaling via IL15Rα, including cis presentation, is eliminated, while only the desired trans presentation of IL15 is retained. In some embodiments, the component comprising IL15 fused with the Sushi domain contains an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 51, which may be encoded by an exemplary nucleic acid sequence represented by SEQ ID NO: 52. [Sequence Listing 12] TIFF0007861191000018.tif46169[Sequence Listing 13] TIFF0007861191000019.tif82169

[0176] Those skilled in the art will understand that the above-described signal peptide and linker sequences are illustrative and in no way limit their modifications suitable for use as signal peptides or linkers. Many suitable signal peptide or linker sequences exist that are known to and available to those skilled in the art. Those skilled in the art will understand that the signal peptide and / or linker sequence may be substituted with another sequence without altering the activity of the functional peptide provided by the signal peptide or linked by the linker.

[0177] Design 5: Natural or modified IL15Rβ is fused to IL15 at its C-terminus via a linker, enabling constitutive signaling and maintaining IL15 membrane binding and trans presentation.

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

[0179] Design 7: Manipulated IL15Rβ, which forms homodimers in the absence of IL15, is useful for generating constitutive signaling of cytokines.

[0180] In some embodiments, cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or one or more of their receptors, can be introduced into iPSCs and their derived cells during iPSC differentiation using one or more of the designs in Figure 1. In some embodiments, IL2 or IL15 cell surface expression and signaling are mediated through the constructs illustrated in any one of designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling are mediated through the constructs illustrated in designs 5, 6, or 7, by using either a common receptor or a cytokine-specific receptor. In some embodiments, IL7 surface expression and signaling are mediated through the constructs illustrated in designs 5, 6, or 7, by using either a common receptor or a cytokine-specific receptor (such as the IL4 receptor). The transmembrane (TM) domains in any of the designs in Figure 1 may be naturally occurring for the corresponding cytokine receptor or may be modified or substituted with the transmembrane domains of any other membrane-bound protein.

[0181] In iPSCs and derived cells containing both CAR and exogenous cytokine and / or cytokine receptor signaling, CAR and IL may be expressed in separate constructs or co-expressed in a bicistronic construct containing both CAR and IL. In some further embodiments, IL15 in the form represented by any of the construct designs in Figure 1 may be ligated to either the 5' or 3' end of a CAR expression construct via an autocleaved 2A coding sequence, exemplified as CAR-2A-IL15 or IL15-2A-CAR. Thus, IL15 and CAR reside in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct contains IL15 of design 3 in Figure 1. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct contains IL15 of design 3 in Figure 1. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct contains IL15 of design 7 in Figure 1. When CAR-2A-IL15 or IL15-2A-CAR is expressed, the expressed CAR and IL15 dissociate via the autocleavage 2A peptide, and the dissociated IL15 is presented on the cell surface. The CAR-2A-IL15 or IL15-2A-CAR bisistron design enables coordinated expression of CAR and IL15 in terms of both timing and quantity, and under the same regulatory mechanisms that can be selected to incorporate, for example, an inducible promoter for the expression of a single ORF. Self-cleaving peptides are found in foot-and-mouth disease viruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tesiovirus-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 cardioviruses such as tyrovirus (e.g., Tyler mouse encephalomyelitis virus) and encephalomyocarditis virus.The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively.

[0182] Embodiments of the bisistronic CAR-2A-IL15 or IL15-2A-CAR disclosed herein for IL15 also intend to express any other cytokines 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 histocompatibility in allogeneic recipients. iPSC cell lines and their derivative cells differentiated therefrom in which the expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced are provided herein. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or by deletion or reduction of expression levels of HLA class I-related genes, including but not limited to the beta-2 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 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, which is 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 to lack 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 avoiding recognition and killing by host (allogeneic) T cells.

[0184] However, in some cell types, the absence of class I expression leads to lysis by NK cells. To overcome this "self-loss" response, HLA-G can be selectively knocked in to evade 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 HLA-G knock-in. Alternatively, in one embodiment, the provided HLA-I-deficient iPSCs and their derived cells further comprise one or both of CD58 knockout and CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and facilitate cell migration, including immune cells. It was previously unclear whether, and how, disruption of CD58 and / or CD54 in iPSCs affects pluripotency and developmental biology in iPSC differentiation into functional immune effector cells, including T cells and NK cells. Furthermore, it was previously unclear whether CD58 and / or CD54 knockout could effectively and / or sufficiently reduce the sensitivity of effector cells derived from HLA-I-deficient iPSCs to allogeneic NK cell killing. Here, we showed that CD58 knockout was more efficient than CD54 knockout in reducing allogeneic NK cell activation, while double knockout of both CD58 and CD54 most effectively enhanced the reduction in NK cell activation. In some observations, CD58 and CD54 double knockout is even 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 derived cells have an exogenous polynucleotide encoding HLA-G. In some embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD58 null. In some other embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD54 null. In some yet other embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD58 null and CD54 null.

[0186] In some embodiments, engineering for HLA-I and / or HLA-II deficiencies can be bypassed or kept intact by expressing an inactivated CAR that targets upregulated surface proteins in activated recipient immune cells to avoid allo-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 cells express such an inactivated CAR, it is preferable that the cells do not express or knock out the same surface proteins targeted by the CAR.

[0187] 6. Genetically modified iPSCs and derived cells provided herein In light of the foregoing, this application provides iPSCs, iPS cell lines, or populations thereof, and derived effector cells obtained from differentiating such iPSCs, each cell comprising at least a CAR having an endodomain as described herein. In some embodiments, derived effector cells include, but are not limited to, mesodermal cells having definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional properties not present in primary NK, T, and / or NKT cells.

[0188] Furthermore, CARs further comprising CD38- / - (also referred herein as "CD38 null" or CD38 knockout) are provided herein, and the cells are derived functional effector cells comprising CARs and CD38 knockout obtained from iPSCs, iPS cell lines, or iPSC differentiation. In some embodiments, derived effector cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional characteristics not present in primary NK, T, and / or NKT cells.

[0189] iPSCs containing a polynucleotide encoding CAR and a polynucleotide encoding high-affinity, uncleavable CD16 (hnCD16) are further provided herein, and the iPSCs can differentiate to produce functionally derived hematopoietic cells. Cells containing both CAR and hnCD16 are suitable for dual targeting by CAR binding and CD16-mediated ADCC, increasing the precision of tumor targeting, promoting tumor killing, and minimizing the effects of tumor antigen evasion. Furthermore, in some embodiments, iPSCs and / or their derived effector cells containing CD38-CAR and hnCD16 having the endodomain provided herein are also CD38 null, and consequently, when hnCD16-mediated enhanced ADCC is induced using a CD38 antibody, iPSCs and / or their derived effector cells containing CD38 knockout, CD38-CAR, and hnCD16-CD38 can target CD38-expressing (tumor) cells and / or allo-reactivation recipient cells without causing the elimination of effector cells, 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, possess ADCC (in the case of T cells, acquire ADCC), and result in further multi-mechanisms of tumor killing while improving cell persistence.

[0190] iPSCs comprising a first CAR provided herein may comprise a polynucleotide encoding a second chimeric antigen receptor (CAR) having target specificity other than the first CAR, and the iPSCs can differentiate to produce functional derived effector cells having two CARs targeting two different tumor antigens. In one embodiment, the two different antigens targeted by the CARs contained in the iPSC and its 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 iPSC and / or its derived effector cells have a CAR targeting CD38, CD25, CD69, or CD44, and the cells are also null in the targeted protein.

[0191] Further provided are iPSCs comprising polynucleotides encoding CARs provided herein, as well as polynucleotides encoding at least one exogenous cytokine and / or receptor (IL) that enable cytokine signaling contributing to cell viability, persistence, and / or proliferation, and the iPSCs can be differentiated to produce functionally derived effector cells with improved viability, persistence, proliferation, and effector cell function. The exogenously introduced cytokine signaling includes the signaling of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or complete peptides of cytokines and / or their respective receptors for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or ephemeral. In some embodiments, transient / temporary expression of cell surface cytokines / cytokine receptors is mediated by retroviruses, Sendai viruses, adenoviruses, episomes, minicircles, or RNA, including mRNA. In some embodiments, exogenous cell surface cytokines and / or receptors containing iPSCs or their derivative cells within the CAR enable IL7 signaling. In some embodiments, exogenous cell surface cytokines and / or receptors containing iPSCs or their derivative cells within the CAR enable IL10 signaling. In some embodiments, exogenous cell surface cytokines and / or receptors containing iPSCs or their derivative cells within the CAR enable IL15 signaling. In some embodiments of the CAR IL iPSC, IL15 expression is by construct 3 in Figure 1. In some embodiments of the CAR IL iPSC, IL15 expression is by construct 4 in Figure 1.The CAR IL iPSCs and their derived cells in the embodiments described above 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 iPSCs and their derived effector cells, the cells are CD38 null and can be used with a CD38 antibody to induce ADCC without causing elimination of effector cells, thereby synergistically increasing the persistence and / or survival of the iPSCs and their effector cells.

[0192] The provided CAR, B2M knockout, and CIITA knockout, as well as iPSCs containing, optionally, one of HLA-G overexpression, CD58 knockout, and CD54 knockout, can be differentiated to produce functionally derived hematopoietic cells. - / - CIITA - / - iPSCs and their derived effector cells are deficient in both HLA-I and HLA-II. In further embodiments, HLA-I and HLA-II deficient CAR iPSCs and their derived effector cells are also CD38 null and can be used with a CD38 antibody to induce ADCC without causing elimination of effector cells, thereby increasing the persistence and / or survival of the iPSCs and their effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or survival.

[0193] From the above perspective, provided herein are iPSCs comprising a CAR, and optionally one, two, three, or more of the following: CD38 knockout, hnCD16, a second CAR, an exogenous cytokine / receptor, and B2M / CIITA knockout, wherein if B2M is knocked out, an HLA-G encoding polynucleotide or at least one of the CD58 and CD54 knockouts is optionally introduced, and the iPSC can be differentiated to produce functional derived hematopoietic cells. In this application, also provided are functional iPSC derived effector cells comprising a CAR, and optionally one, two, three, or more of the following: CD38 knockout, hnCD16, B2M / CIITA knockout, a second CAR, and exogenous cytokine / receptor, wherein if B2M is knocked out, an HLA-G encoding polynucleotide or at least one of the CD58 and CD54 knockouts is optionally introduced, and the derived effector cells can be differentiated to produce definitive hematopoietic endothelial cells (HE) This includes, but is not limited to, mesodermal cells with potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional characteristics not present in primary NK, T, and / or NKT cells.

[0194] Another embodiment provided herein comprises iPSCs or iPSC-derived cells containing cleaved fusion proteins of IL15 and IL15Rα, the fusion proteins lacking an intracellular domain. As shown in Figure 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 embodiments of “IL” shown in Table 3. In some embodiments of “IL”, the cleaved IL15 / IL15Rα fusion protein lacking an intracellular domain contains an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NOs. 48, 51, or 50. In some embodiments of “IL”, the cleaved IL15 / IL15Rα fusion protein lacking an intracellular domain contains the amino acid sequence of SEQ ID NOs. 48. In some embodiments of “IL”, the cleaved IL15 / IL15Rα fusion protein lacking an intracellular domain contains the amino acid sequence of SEQ ID NOs. 51. In some embodiments of "IL," the cleaved IL15 / IL15Rα fusion protein lacking an intracellular domain contains the amino acid sequence of SEQ ID NO: 50.In some embodiments of iPSCs or iPSC-derived cells containing a cleaved IL15 / IL15Rα fusion protein (IL15Δ) lacking an intracellular domain, the cells further contain a CAR, and optionally, one or more of the following: CD38 knockout, hnCD16, a second CAR, an exogenous cytokine / receptor, and B2M / CIITA knockout, wherein if B2M is knocked out, an HLA-G encoding polynucleotide or one of the CD58 and CD54 knockouts is optionally introduced, and the iPSCs differentiate to produce functional derived effector cells. Derived effector cells may include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional characteristics not present in primary NK, T, and / or NKT cells.

[0195] Therefore, this application provides iPSCs and their functionally derived hematopoietic cells, comprising any one of the following genotypes in Table 2. The "CAR" provided in Table 2 of this application (第2)"IL" represents a CAR with a different target specificity than the first CAR, and non-limiting examples include CARs that target 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 particular cytokine / receptor expression is selected. Furthermore, "IL" also encompasses the IL15Δ embodiment, which is detailed above as a cleaved fusion protein of IL15 and IL15Rα, but does not contain an intracellular domain. Furthermore, if the iPSC and its functionally derived hematopoietic cells have a genotype containing both CAR (first CAR or second CAR) and IL, in one embodiment of the cells, CAR and IL are contained in a bicistromic expression cassette containing a 2A sequence. For comparison, in some other embodiments, CAR and IL are in separate expression cassettes contained in the iPSC and its functionally derived hematopoietic cells. In a particular embodiment, the iPSC and its functionally derived effector cells expressing both CAR and IL contain IL15 in construct 3 or 4 of Figure 1, and the IL15 construct is contained in an expression cassette together with or separately from 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 containing any one of the genotypes in Table 2 may further include deletion or reduced expression in at least one of the following genes: 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 This may further include introduced or increased expression in at least one of the following: R, antigen-specific TCRs, Fc receptors, engagers, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers.

[0200] A bispecific or multispecific engager is a fusion protein comprising two or more single-chain variable fragments (scFv) of different antibodies, where at least one scFv binds to an effector cell surface molecule and at least one other binds to a tumor cell 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, the CD16 expressed on the surface of effector cells for engager recognition is hnCD16, which includes the CD16 (containing F176V and optionally S197P) or CD64 extracellular domain as described in Section I.2, as well as native or non-native transmembrane domains, stimulating domains, and / or signaling domains. 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 comprises a transmembrane domain of NKG2D, a stimulating domain of 2B4, and a signaling domain of CD3ζ, the extracellular domain of hnCD16 is derived from the extracellular domain of CD64 or a full-length or partial sequence of CD16, and the extracellular domain of CD16 comprises F176V and optionally S197P. Exemplary tumor cell surface molecules that recognize bispecific or multispecific engagers include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. In one embodiment, the bispecific antibody is CD3-CD19.In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In yet 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 includes a linker between the effector cell and tumor cell antigen-binding domain, for example, a modified IL15 (referred to in some publications as TriKE, or triplicate killer enhancer) as a linker for effector NK cells that promote effector cell proliferation. In one embodiment, TriKE is CD16-IL15-EPCAM or CD64-IL15-EPCAM. In another embodiment, TriKE is CD16-IL15-CD33 or CD64-IL15-CD33. In yet another embodiment, TriKE is NKG2C-IL15-CD33 ("2C1533").

[0201] In some embodiments, surface trigger receptors for bispecific or multispecific engagers may be endogenous to effector cells, sometimes depending on the cell type. In some other embodiments, using the methods and compositions provided herein, iPSCs containing the genotypes listed in Table 2 can be further manipulated to direct the differentiation of the iPSCs into T cells, NK cells, or any other effector cells containing the same genotype and surface trigger receptor as the source iPSCs, thereby introducing one or more exogenous surface trigger receptors into the effector cells.

[0202] 8. Antibodies for immunotherapy In some embodiments, in addition to the genome-engineered effector cells provided herein, additional therapeutic agents, including antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication, can be used in combination therapy with these effector cells. In some embodiments, 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, suitable antibodies for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include, but are not limited to, CD20 antibodies (rituximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), HER2 antibodies (trastuzumab, partuzumab), CD52 antibodies (alemtuzumab), EGFR antibodies (certuximab), 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. In some embodiments, iPSC-derived effector cells include hematopoietic lineage cells containing the genotypes listed in Table 2. In some embodiments, iPSC-derived effector cells include NK cells containing the genotypes listed in Table 2. In some embodiments, iPSC-derived effector cells include T cells containing the genotypes listed in Table 2.

[0203] In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises a pre-selected monoclonal antibody and iPSC-derived NK cells or T cells containing at least hnCD16 and a CAR having the provided endodomain. In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises a pre-selected monoclonal antibody and iPSC-derived NK cells or T cells containing at least hnCD16 and a CAR having the provided endodomain. In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises a pre-selected monoclonal antibody and iPSC-derived NK cells or T cells containing at least hnCD16 and a CAR having the provided endodomain. Without being limited by theory, hnCD16 provides an enhanced ADCC of the monoclonal antibody, but the CAR not only targets a specific tumor antigen but also prevents tumor antigen evasion using a dual-targeting strategy in combination with a monoclonal antibody targeting a different tumor antigen. In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises at least CD38-CAR containing an endodomain provided herein, iPSC-derived NK cells or T cells containing a CD38 null, and a CD38 antibody. In one embodiment, the combination comprises at least CD38-CAR containing an endodomain provided herein, iPSC-derived NK cells containing a CD38 null and hnCD16, and one of the CD38 antibodies, daratumumab, isatuximab, and MOR202. In one embodiment, the combination comprises iPSC-derived NK cells containing a CD38-CAR containing an endodomain provided herein, a CD38 null and hnCD16, and daratumumab.In some further embodiments, the iPSC-derived NK cells included in the combination with daratumumab include CD38-CAR, CD38 null, hnCD16, IL15, and a CAR targeting at least one of MICA / B or CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1, wherein IL15 is expressed co-expressed or separately with the CAR, and IL15 is in any of the forms presented in constructs 1-7 of Figure 1. In some specific embodiments, when IL15 is expressed concurrently or separately with the CAR, it is in the form of construct 3, 4, or 7.

[0204] 9. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that, if not inhibited, can suppress or downregulate the immune response. It is now clear that tumors utilize certain immune checkpoint pathways, particularly as a primary mechanism for immune resistance to tumor antigen-specific T cells. Checkpoint inhibitors (CIs) are antagonists that can reduce checkpoint gene expression or gene product, 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 changed the oncological landscape, with these drugs resulting in long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a significant concern. One aspect of this application provides a therapeutic approach to overcome CI resistance by including genomically engineered functional derived cells, as provided in combination therapy with CIs. 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 antitumor 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 tumor sites. Therefore, T cell tumor infiltration promoted by functionally potent genome-engineered derived NK cells indicates that these NK cells can synergistically interact with T cell-targeted immunotherapy, including checkpoint inhibitors, to alleviate local immunosuppression and reduce tumor burden.

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

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

[0208] Suitable checkpoint inhibitors for combination therapy with derived NK cells or T cells provided herein include 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, and CD73 (NT5E). This includes, but is not limited to, antagonists of 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 KIRs (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 three or more 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, many miRNAs are found to be regulators that control the expression of immune checkpoints, so 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 combination therapy with the provided derived NK cells or derived T cells include at least one checkpoint inhibitor targeting at least one checkpoint molecule, and the derived cells have the genotypes listed in Table 2. Some other embodiments of the combination therapy with the provided derived NK cells or T cells include two or more checkpoint inhibitors so that two or three or more checkpoint molecules are targeted. In some embodiments of the combination therapy including 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 the fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the antibody or fragment or variant that inhibits the checkpoint is co-expressed with the CAR in either a separate construct or a bisistronic construct containing both the CAR and the sequence encoding the antibody or fragment thereof. In some further embodiments, the sequence encoding the antibody or a fragment thereof may be ligated to either the 5' or 3' end of the CAR expression construct via an autocleaved 2A coding sequence, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences of the checkpoint inhibitor and the CAR reside 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), it binds to the TME, counteracting the inhibitory checkpoint molecule and enabling the activation of the effector cells by activating modalities such as the CAR or activating receptor.In some embodiments, checkpoint inhibitors co-expressed with CAR inhibit at least one of the following 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 KIRs. In some embodiments, the checkpoint inhibitors co-expressed with CAR in derived cells having the genotypes listed in Table 2 are selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is atezolizumab, or its humanized or Fc-modified variants, fragments, or their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is nivolumab, or its humanized or Fc-modified variants, fragments, or their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is pembrolizumab, or a humanized or Fc-modified variant, fragment thereof, or a functional equivalent or biosimilar thereof.

[0212] In some other embodiments of the combination therapy comprising the derived cells provided herein and at least one antibody that inhibits checkpoint molecules, the antibody is not produced by, or within, the derived cells and is further administered before, simultaneously with, or after administration of the 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 therapy is one or more 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 of combination therapy comprising derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the therapy 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 therapy 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 therapy 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, genomic editing, or gene editing as 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 (interchangeable with “targeted genomic editing” or “targeted gene editing”) allows for insertion, deletion, and / or replacement at pre-selected sites within the genome. If 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 by the deletion of the sequence. Thus, targeted editing can also be used to precisely disrupt the expression of endogenous genes. The term “targeted integration” is used similarly herein and refers to a process involving the insertion of one or more exogenous sequences, with or without the deletion of an endogenous sequence at the insertion site. In comparison, randomly integrated genes are subject to positional effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomere regions are particularly susceptible to transgene silencing. Mutually, newly incorporated genes can affect surrounding endogenous genes and chromatin, potentially altering cellular behavior or supporting cellular transformation. Therefore, inserting exogenous DNA into pre-selected loci, such as safe harbor loci or genome-safe harbors (GSHs), is crucial for safe, efficient, copy number control, and reliable gene response regulation. Alternatively, exogenous DNA may be inserted into pre-selected loci where disruption of gene expression, including knockdown and knockout, is intended.

[0214] Targeted editing can be achieved by either a nuclease-independent or nuclease-dependent approach. In nuclease-independent targeted editing approaches, homologous recombination is induced by a homologous sequence adjacent to the inserted exogenous polynucleotide via an enzymatic mechanism in the host cell.

[0215] Alternatively, targeted editing can be achieved more frequently through the specific introduction of double-strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. In the absence of a donor vector containing exogenous gene material, NHEJ often results in random insertions or deletions (in / del) of a small number of endogenous nucleotides. In comparison, when a donor vector containing exogenous gene material adjacent to a pair of homologous arms is present, the exogenous gene material can be introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration." In some cases, because the targeted integration site is intended to be within the coding region of a selected gene, targeted integration can disrupt gene expression, resulting in simultaneous knock-in and knock-out (KI / KO) in a single editing step.

[0216] Simultaneous gene knockout can be achieved by inserting one or more transgenes at a selected location of a target gene locus (GOI). Suitable loci 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 region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. Each site-specific targeted homology arm for site-selective insertion allows the transgene to be expressed either under the endogenous promoter of that site or under the exogenous promoter included in the construct. When two or more transgenes are inserted at a selected location on 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), allowing distinct proteins to be produced from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter may 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), and RNA-guided CRISPR (clustered regular-arranged palindromic sequence repeat) systems. In addition, DICE (dual integrase cassette exchange) systems utilizing phiC31 and Bxb1 integrases are also promising tools for targeted incorporation.

[0218] ZFNs are targeted nucleases containing a nuclease fused to a zinc finger DNA-binding domain. A "zinc finger DNA-binding domain" or "ZFBD" refers to a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within a zinc finger-binding domain, whose structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2, C3H, and C4 zinc fingers. A "designed" zinc finger domain is a domain that does not exist in nature, whose design / composition arises primarily from rational criteria, e.g., the application of substitution rules and computerized algorithms to process information from databases storing information on existing ZFP designs and binding data. See, for example, U.S. Patents 6,140,081, 6,453,242, and 6,534,261, as well as International Publications WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496. “Selected” zinc finger domains are domains not found in nature, and their production arises primarily from empirical processes such as phage display, interaction trapping, or hybrid selection. ZFNs are described in detail in U.S. Patents 7,888,121 and 7,972,854, the full disclosure of which is incorporated herein by reference. The most recognized example of a ZFN in the art is the fusion of a FokI nuclease with a zinc finger DNA-binding domain.

[0219] TALEN is a targeted nuclease containing a nuclease fused to the TAL effector DNA-binding domain. “Transcription activator-like effector DNA-binding domain,” “TAL effector DNA-binding domain,” or “TALE DNA-binding domain” refers to the polypeptide domain of the TAL effector protein 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 and bind to effector-specific DNA sequences via their DNA-binding domains, activating 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 incomplete 34-amino acid repeats containing polymorphism at a selective repeat position called a repeat variable two residue (RVD). TALEN is described in detail in U.S. Patent Application No. 2011 / 0145940, incorporated herein by reference. The most well-known example of a TALEN in this field is a fusion polypeptide of the FokI nuclease to the TAL effector DNA-binding domain.

[0220] Another example of a targeted nuclease found to be used in the subjective method is the targeted Spo11 nuclease, which is a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain specific to the DNA sequence of interest, such as a zinc finger DNA-binding domain or a TAL effector DNA-binding domain.

[0221] Additional examples of targeted nucleases suitable for 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 and recombinant nucleases; CRISPR-related nucleases from families including Cas, CPF, CSE, CSY, CSN, CSD, CST, CSH, CSA, CSM, and CMR; restriction endonucleases; meganucleases; homing endonucleases, etc.

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

[0224] DICE-mediated insertions utilize a pair of recombinases, e.g., phiC31 and Bxb1, to provide unidirectional integration of exogenous DNA strictly restricted to the small attB and attP recognition sites of each enzyme itself. Since these target att sites are not naturally present in the mammalian genome, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Publication 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 targeted integration method comprises introducing the construct into a cell to enable site-directed homologous recombination by a cell-host enzyme mechanism. In another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site into the cell to enable ZFN-mediated insertion. In yet another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site into the cell to enable TALEN-mediated insertion. In yet another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a 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 Cas9-mediated insertion. In another embodiment, a targeted integration method in cells includes introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in a cell, introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing a DICE recombinase expression cassette to enable DICE-mediated targeted integration.

[0226] Promising sites for targeted integration include, but are not limited to, intragenetic or extragenetic regions of the human genome that, theoretically, can accommodate the predictable expression of newly integrated DNA without adverse effects on the host cell or organism, safe harbor loci or genome-safe harbors (GSHs). A useful safe harbor must allow sufficient expression of the transgene to obtain the desired level of the protein or non-coding RNA encoded by the vector. The safe harbor must also not make cells more susceptible to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it must ideally meet criteria including, but not limited to,: no disruption of regulatory elements or genes as determined by sequence annotation; being an intergeneric region within a densely populated region of genes, or a convergence site between two genes transcribed in opposite directions; maintaining distance to minimize the possibility of long-range interactions between the vector-encoded transcription activator and adjacent genes, particularly cancer-related genes and microRNA gene promoters; and possessing clearly ubiquitous transcriptional activity, as reflected in broad spatial and temporal expression sequence tag (EST) expression patterns exhibiting ubiquitous transcriptional activity. This latter characteristic is particularly important in stem cells where chromatin remodeling during differentiation typically results in the silencing of some loci and the potential activation of others. Within regions favorable for exogenous insertion, the precise locus selected for insertion should lack repeating elements and conserved sequences, allowing for the easy design of primers for homologous arm amplification.

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

[0228] In the case of targeted in / dels, the editing site is often located in an endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted in / del is related to the regulation and adjustment of the immune response. In several other embodiments, the endogenous gene containing the targeted in / del is related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and adjustment, or proteins that suppress the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells, as well as cells from which they originate.

[0229] Accordingly, one aspect of the present invention provides a method for targeted integration at selected loci, including genome-safe harbors, pre-selected loci known or proven to be safe and well-controlled for continuous or transient gene expression, such as AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, or other loci that meet the criteria for genome-safe harbors. In some embodiments, the targeted integration is at one of the loci where knockdown or knockout of the gene as a result of integration is desired, such loci include, but are 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, a targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a construct containing a pair of homologous arms specific to a desired integration site and one or more exogenous sequences to enable site-directed homologous recombination by a cell-host enzyme mechanism, 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 targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a ZFN expression cassette containing a DNA-binding domain specific to a desired integration site into cells 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, 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 targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a TALEN expression cassette containing a DNA-binding domain specific to a desired integration site into cells 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, 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 targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a gRNA containing a CRISPR nuclease expression cassette and a guide sequence specific to a desired integration site into cells to enable CRISPR nuclease-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, 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 targeted integration method in cells comprises introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in a cell, introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing a DICE recombinase expression cassette to enable DICE-mediated targeted integration, 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, 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 containing 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. Thus, 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 operablely linked exogenous promoter contained in a construct for targeted incorporation. The promoter may be inductive or constitutive, and may be time-specific, tissue-specific, or cell-type-specific. Suitable constitutive promoters for the methods of the present invention include, but are not limited to, cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0234] Exogenous polynucleotides incorporated by the methods described herein may be driven at the integration site by an endogenous promoter of the host genome. In one embodiment, the method of the present invention is used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the method of the present invention is used for targeted integration at the ROSA26 locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the method of the present invention is used for targeted integration at the H11 locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous H11 promoter. In yet another embodiment, the method of the present invention is used for targeted integration at the collagen locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method of the present invention is used for targeted integration at the HTRP locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by an endogenous HTRP promoter. Theoretically, only correct insertion at the desired site would enable gene expression of the exogenous gene driven by the endogenous promoter.

[0235] In some embodiments, one or more exogenous polynucleotides included in a construct for a targeted integration method are driven by a single promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to broaden the physical separation between the parts and maximize access to the enzymatic mechanism. The linker peptide of the linker sequence may consist of amino acids selected to make the physical separation between the parts (exogenous polynucleotides, and / or proteins or peptides encoded therefrom) more flexible or more rigid, depending on the relevant function. The linker sequence may be cleavable by proteases or chemically cleavable to produce distinct parts. Examples of enzymatic cleavage sites in the linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is either naturally produced by the host or exogenously introduced. Alternatively, cleavage sites in the linker may be sites that are cleavable upon exposure to selected chemicals, such as cyanide bromide, hydroxylamine, or low pH. Optional linker sequences may serve purposes other than providing cleavage sites. The linker sequence should allow for the effective positioning of a part relative to another adjacent part for the part to function properly. The linker may also be a simple amino acid sequence of sufficient length to prevent steric hindrance between parts. In addition, the linker sequence may provide post-translational modifications, including but not limited to phosphorylation sites, biotinylation sites, sulfated sites, and γ-carboxylation sites. In some embodiments, the linker sequence is flexible so as not to hold a biologically active peptide into a single undesirable conformation. To provide flexibility, the linker may be composed primarily of amino acids with small side chains, such as glycine, alanine, and serine. In some embodiments, about 80 or 90 percent or more of the linker sequence consists of glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, the G4S linker peptide separates the terminal processing domain and endonuclease domain of the fusion protein. In other embodiments, the 2A linker sequence allows two distinct proteins to be produced from a single translation. A suitable linker sequence can be easily identified empirically. In addition, a 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 distinct.

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

[0237] III. Methods for obtaining and maintaining genetically engineered iPSCs The present invention provides a method for obtaining and maintaining a genome-engineered iPSC comprising one or more targeted edits at one or more desired sites, wherein the targeted edits remain intact and functional at their respective selected editing sites in the expanded genome-engineered iPSC or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions, i.e., targeted integrations and / or in / dels, at selected sites into the genome of the iPSC and its derived cells. Compared to directly manipulating primary effector cells derived from patient peripheral blood, the many advantages of obtaining genome-engineered derived cells by editing and differentiating iPSCs as provided herein include, but are not limited to: an unlimited source of designed effector cells; no need to repeatedly manipulate effector cells, especially when multiple designed modalities are involved; the obtained effector cells are rejuvenated due to elongated telomeres and less depletion; and the effector cell population is homogeneous in terms of editing site, copy number, and lack of allelic deformation, random mutation, and expression diversity, primarily due to the possibility of clonal selection in the manipulated iPSCs provided herein.

[0238] In certain embodiments, genome-engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells for extended periods in cell culture media shown in Table 3 as Fate Maintenance Medium (FMM), and the iPSCs retain the targeted edits and functional modifications at the selected sites. The composition of the medium may be present in the optimal range of amounts shown in Table 3. iPSCs cultured in FMM have been shown to remain undifferentiated, with a basal or naive profile, maintain genomic stability without the need to wash or select the culture, and readily induce in vitro differentiation via all three somatic cell lineages, embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation via teratoma formation. See, for example, International Publication WO2015 / 134652, the disclosure of which is incorporated herein by reference.

[0239] [Table 4]

[0240] In some embodiments, genome-engineered iPSCs containing one or more targeted embedded and / or in / del are maintained, passaged, and expanded in a medium containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not containing or essentially not containing a TGFβ receptor / ALK5 inhibitor, and the iPSCs retain intact and functional targeted edits at selected sites.

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

[0242] In some embodiments, to simultaneously manipulate and reprogram non-pluripotent cells genomes, targeted embeddings and / or in / dels may also be introduced into non-pluripotent cells after a multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, before the construct-carrying vector is introduced into the reprogrammed cells before they exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181, and CD30.

[0243] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor, as well as 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 above methods 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 a method for generating genetically engineered iPSCs, the method comprises genetically engineering an iPSC by introducing one or more targeted embeddings and / or in / dels into the iPSC to obtain a genetically engineered iPSC having at least one genotype listed in Table 2. Alternatively, a method for generating a genetically engineered iPSC comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genetically engineered non-pluripotent cell containing targeted embeddings and / or in / dels at a selected site, and (b) contacting the genetically engineered non-pluripotent cell with a small molecule composition containing 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 a genetically engineered iPSC containing targeted embeddings and / or in / dels at a selected site. Alternatively, a method for generating genome-engineered iPSCs comprises (a) initiating the reprogramming of non-pluripotent cells by contacting them 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; (b) introducing one or more targeted embeddings and / or in / dels into the reprogrammed non-pluripotent cells for genome engineering; and (c) obtaining clone genome-engineered iPSCs containing targeted embeddings and / or in / dels at selected sites.

[0245] 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 (these disclosures are incorporated herein by reference). One or more reprogramming factors may be in the form of polypeptides. Reprogramming factors may also be in the form of polynucleotides and are therefore 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 virus vector. In some embodiments, one or more polynucleotides are introduced by a combination of plasmids that take into account the stoichiometry of 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 translocated with multiple constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted incorporation at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding targeted 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 RNA, including but not limited to siRNA, shRNA, miRNA, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue or cell type-specific promoters. Thus, the polynucleotides can be expressed under conditions that activate the promoters, for example, in the presence of an inducer, or in a particular differentiated cell type. In some embodiments, the polynucleotides are expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, one or more suicide genes are driven by a constitutive promoter, e.g., capase-9 driven by CAG. These constructs, comprising different exogenous polynucleotides and / or different promoters, can be transferred into non-pluripotent cells either simultaneously or sequentially. Non-pluripotent cells subjected to targeted integration of multiple constructs can be simultaneously exposed to one or more reprogramming factors to initiate reprogramming concurrently with genomic manipulation, thereby obtaining genomically engineered iPSCs containing multiple targeted integrations in the same pool of cells. Thus, this robust method enables simultaneous reprogramming and manipulation strategies to lead to clonally engineered hiPSCs with multiple modalities integrated into one or more selected target sites.In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions described herein include at least one genotype listed in Table 2.

[0247] IV. Method for differentiating genome-modified iPSCs and obtaining genetically modified effector cells by CAR endodomain screening using an iPSC differentiation platform. Further embodiments of the present invention provide a method for in vivo differentiation of genomically engineered iPSCs by teratoma formation, wherein in vivo differentiated cells derived from genomically engineered iPSCs retain intact and functional targeted edits, including targeted integration and / or in / del at desired sites. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma 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 that meet the criteria for a genome-safe harbor. In some other embodiments, in vivo differentiated cells derived from genomically engineered iPSCs via teratoma contain polynucleotides encoding a targeted modality, or polynucleotides encoding proteins that promote the transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some embodiments, in vivo differentiated cells derived from genomically engineered iPSCs via teratomas containing one or more inducible suicide genes further include one or more in / dels of endogenous genes associated with the regulation and mediation of immune responses. In some embodiments, the in / dels include one or more endogenous checkpoint genes. In some embodiments, the in / dels include one or more endogenous T cell receptor genes. In some embodiments, the in / dels include one or more endogenous MHC class I suppressor genes. In some embodiments, the in / dels include one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the in / dels include one or more endogenous genes including, but not limited to, B2M, PD1, TAP1, TAP2, tapasin, and TCR genes. In one embodiment, a genomically engineered iPSC containing one or more exogenous polynucleotides at a selected site further includes targeted editing in the gene encoding B2M (beta-2-microglobulin).

[0248] In certain embodiments, genome-engineered iPSCs containing one or more genetic modifications provided herein are used to derive hematopoietic cell lines or any other specific cell types in vitro, and the derived non-pluripotent cells retain functional genetic modifications, including targeted editing at selected sites. In one embodiment, cells derived from genome-engineered iPSCs include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), 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, and these cells derived from genome-engineered iPSCs retain functional genetic modifications, including targeted editing at desired sites.

[0249] Applicable differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lines include, for example, those shown in International Publication No. WO2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, the methods and compositions for generating hematopoietic cell lines are mediated through pluripotent stem cell-derived secondary hematopoietic endothelium (HE), including hiPSCs, under serum-free, feeder-free, and / or stroma-free conditions, and on a scalable, monolayer EB-free culture platform. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated and transdifferentiated cells, as well as cells of various lineages that have directly transitioned to hematopoietic fate without passing through pluripotent intermediates. Similarly, cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells, and all intervening hematopoietic cell lineages.

[0250] A method for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture involves contacting pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from pluripotent stem cells are obtained from the pluripotent stem cells without embryoid body formation and are expanded. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with secondary hematopoietic endothelial (HE) potential from the pluripotent stem cells without embryoid body formation. Subsequent contact with bFGF, and optionally with a ROCK inhibitor and / or a WNT pathway activator, allows the mesodermal cells with secondary HE potential to differentiate into secondary HE cells, which are also expanded during differentiation.

[0251] The method provided herein for obtaining hematopoietic cells is superior to pluripotent stem cell differentiation via EB because EB formation results in moderate to minimal cell expansion, which is important for many applications requiring homogeneous expansion, and does not allow for homogeneous differentiation of cells within a population, which is difficult and inefficient.

[0252] The provided monolayer differentiation platform facilitates differentiation into secondary hematopoietic endothelium, resulting in the derivation of hematopoietic stem cells and differentiated offspring 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 therapeutically appropriate numbers of pluripotent stem cell-derived effector cells for various therapeutic applications. Furthermore, monolayer cultures using the methods provided herein result in functional hematopoietic lineage cells that enable the entire range of in vitro differentiation, ex vivo regulation, and in vivo long-term hematopoietic autoregeneration, reconstitution, and engraftment. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, secondary hematopoietic endothelium, hematopoietic pluripotent progenitor cells, hematopoietic stem cells and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0253] A method for initiating the differentiation of pluripotent stem cells into cells of a secondary hematopoietic lineage, comprising: (i) contacting 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 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 secondary hematopoietic endothelial potential from the mesodermal cells; and (iii) contacting mesodermal cells with secondary hematopoietic endothelial potential with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally comprising 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 pluripotent stem cell-derived mesodermal cells with secondary hematopoietic endothelial potential.

[0254] In some embodiments, the method further comprises contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (the composition does not contain 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 containing one or more gene imprints, the one or more gene imprints contained in the iPSCs being retained in effector cells differentiated therefrom. In some embodiments of the method directed toward the differentiation of pluripotent stem cells into hematopoietic lineage cells, the differentiation of pluripotent stem cells into hematopoietic lineage cells lacks embryoid body formation and is in a monolayer culture form.

[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 secondary hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, thereby initiating the 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 one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, thereby initiating the differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells. In some embodiments of the method, the T cell progenitor cells derived from pluripotent stem cells are CD34+CD45+CD7+. In some embodiments of the method, the T cell progenitor cells derived from pluripotent stem cells are CD45+CD7+.

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

[0258] Therefore, using the differentiation method described above, one or more populations of 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) iMPP-A, iTC-A2, iTC-B2, iNK-A2, and (iv) Secondary HSCs using one or more culture media selected from 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 media are: a.iCD34-C contains one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, as well as optionally a Wnt pathway activator, and does not contain a TGFβ receptor / ALK inhibitor. b.iMPP-A comprises 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 is a ROCK inhibitor; it contains 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, as well as 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-manipulated iPSC-derived cells obtained by the above method 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, genome-engineered iPSC-derived cells include polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some embodiments, genome-engineered iPSC-derived cells containing one or more suicide genes further include one or more in / dels contained in one or more endogenous genes associated with the regulation and mediation of immune responses, including but not limited to checkpoint genes, endogenous T cell receptor genes, and MHC class I repressor genes. In one embodiment, genome-engineered iPSC-derived cells containing one or more suicide genes further include an in / del in the B2M gene, where B2M is knocked out.

[0260] In addition, applicable dedifferentiation methods and compositions for obtaining second-fate genome-modified hematopoietic cells from first-fate genome-modified hematopoietic cells 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 herein partially reprogram initiating 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 differentiation of intermediate cells into desired cell types. In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions herein include at least one genotype listed in Table 2.

[0261] V. Therapeutic applications of derived immune cells with exogenous functional modalities differentiated from genetically engineered iPSCs The present invention provides compositions comprising, in some embodiments, an isolated population or subpopulation of functionally enhanced derived immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSC comprises one or more targeted gene edits retainable in the iPSC-derived immune cells, and the genetically engineered iPSCs and their derived cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered 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 immunoregulatory cells or bone marrow-derived suppressor cells (MDSCs). In some embodiments, genetically engineered immune cells derived from iPSCs are further modified ex vivo for improved therapeutic potential. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In another embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of type I NKT cells. In yet another embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of adaptive NK cells. In some embodiments, an isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cell lineage cells, NK cell lineage cells, or bone marrow-derived suppressor cells derived from iPSCs is allogeneic. In some other embodiments, an isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, NKT cells, or MDSCs derived from iPSCs is autologous.

[0262] In some embodiments, iPSCs for differentiation include selected gene imprints to convey desired therapeutic attributes in effector cells, provided that the cell developmental biology during differentiation is not disrupted and the gene imprints are retained and functional in differentiated effector cells derived from the iPSCs.

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

[0264] In some embodiments, the genetically modified modality includes one or more safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, 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 several other embodiments, genetically modified iPSCs and their derived cells, including the genotypes listed in Table 2, further include additional genetically modified modalities, including (1) deletion or reduction of expression of one or more genes in the chromosome 6p21 region, such as TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and (2) introduced or increased expression of surface trigger receptors for binding to HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, antigen-specific TCR, Fc receptor, or bispecific, multispecific, or universal engagers.

[0265] In some other embodiments, the hematopoietic lineage cells include therapeutic attributes of source-specific immune cells relating 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 immunomodulation of bystander immune cells, (iv) improved target specificity by reducing extratumor effects, and (v) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0266] In some embodiments, iPSC-derived effector cells include the genotypes listed in Table 2, and these 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 CAR. In some embodiments, the engineered expression of cytokines and CAR is NK cell-specific. In some other embodiments, the engineered expression of cytokines and CAR is T cell-specific. In one embodiment, the CAR includes 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 humoral 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 escapes.

[0267] By introducing the immune cells of the present invention into subjects suitable for adoptive cell therapy, various diseases can be cured. In some embodiments, the iPSC-derived effector cells provided are for allogeneic adoptive cell therapy. In addition, in some embodiments, the present invention provides therapeutic uses of the above therapeutic compositions by introducing the compositions into subjects suitable for adoptive cell therapy, the subjects having autoimmune disorders, hematological malignancies, solid tumors, or infections 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 tumors include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), several forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, several forms of myocarditis, and multiple Examples of viral infections include, but are not limited to, HIV-(human immunodeficiency virus), HSV-(herpes simplex virus), KSHV-(herpes zoster virus), RSV-(respiratory syncytial virus), EBV-(Epstein-Barr virus), CMV-(cytomegalovirus), VZV-(varicella-zoster virus), adenovirus-(adenovirus), lentivirus-(adenovirus), and BK-polyomavirus-associated disorders).

[0268] Therapies using hematopoietic lineage cells derived from embodiments disclosed herein may be performed symptomatically or to prevent recurrence. Terms such as “to treat” and “therapy” are used herein in general to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. Where used herein, “therapy” encompasses all interventions for the disease in a subject and includes: preventing the onset of the disease in a subject susceptible to the disease but not yet diagnosed as having it; inhibiting the disease, i.e., preventing its onset; or alleviating the disease, i.e., regressing the disease. Therapeutic agents or compositions may be administered before, during, or after the onset of the disease or injury. The treatment of ongoing diseases in which the therapy stabilizes or reduces undesirable clinical symptoms in a patient is also of particular interest. In certain embodiments, the subject requiring therapy has a disease, condition, and / or injury in which at least one related symptom can be suppressed, restored, and / or improved by cell therapy. Certain embodiments are intended to include, but are not limited to, subjects requiring cell therapy, including, candidates for bone marrow or stem cell transplantation, subjects who have received chemotherapy or radiotherapy, subjects with or at risk of having hyperproliferative disorders or cancer, such as hematopoietic hyperproliferative disorders or cancer, subjects with or at risk of having tumors, such as solid tumors, or subjects with or at risk of having viral infections or diseases associated with viral infections.

[0269] When evaluating the response to a therapy involving hematopoietic lineage cells derived from embodiments disclosed herein, the response may be measured by criteria including at least one of the following: clinical benefit rate, survival to death, complete pathological response, semi-quantitative measurement of pathological response, complete clinical remission, partial clinical remission, clinically stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Responsive Reference Indicators for Solid Tumors) criteria.

[0270] Therapeutic compositions comprising the disclosed hematopoietic lineage cells may be administered to a subject before, during, and / or after other therapies. Thus, combination therapy methods may involve the administration or preparation of iPSC-derived immune cells before, during, and / or after the use of additional therapeutic agents. As provided above, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors containing one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory agents (IMiDs). The administration of iPSC-derived immune cells may be separated from the administration of additional therapeutic agents by time units of hours, days, or even weeks. In addition, or alternatively, the administration may be combined with other bioactive agents or modalities, such as antitumor agents, non-pharmacological therapies such as surgery, etc., but are not limited to these.

[0271] In some embodiments of combination cell therapy, the therapeutic combination comprises iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent, which is an antibody or antibody fragment. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody may be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability. In some embodiments, suitable antibodies for combination therapy as additional therapeutic agents to administered iPSC-derived hematopoietic lineage cells include CD20 antibodies (e.g., rituximab, bertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), HER2 antibodies (e.g., trastuzumab, bertuzumab), CD52 antibodies (e.g., alemtuzumab), EGFR antibodies (e.g., cerutuximab), GD2 antibodies (e.g., Examples include, but are not limited to, 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.

[0272] In some embodiments, additional therapeutic agents include one or more checkpoint inhibitors. Checkpoints are cellular molecules, often cell surface molecules, that, if not inhibited, can suppress or downregulate the immune response. Checkpoint inhibitors are antagonists that can reduce checkpoint gene expression or gene product, or decrease the activity of checkpoint molecules. Checkpoint inhibitors provided herein that are suitable for combination therapy with derived effector cells, including NK cells or T cells, include 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, and CD73. This list includes, but is not limited to, antagonists of retinoic acid receptor alpha (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 KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0273] Some embodiments of the combination therapy, including the provided derived effector cells, further include at least one inhibitor that targets a checkpoint molecule. Some other embodiments of the combination therapy with the provided derived effector cells include two or more inhibitors so that two or 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 or more checkpoint inhibitors may 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 inhibitor antibody may be a mouse antibody, a human antibody, a humanized antibody, a camel Ig antibody, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or a 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 three or more checkpoint inhibitors include at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents.

[0275] Combination therapy involving derived effector cells and one or more check inhibitors is effective for cutaneous T-cell lymphoma, non-Hodgkin lymphoma (NHL), mycosis fungoides, Paget's reticular disease, Sézary syndrome, granulomatous flaccid skin, lymphomatoid papulosis, chronic lichenoid pityriasis, acute lichenoid plaque, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Renat lymphoma, subcutaneous T-cell lymphoma, vascular central lymphoma, blastic NK-cell lymphoma, and B-cell lymphoma. This treatment is applicable to the treatment of humoral and solid tumors, including but not limited to Hodgkins lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyosarcoma, Lewis lung cancer (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, small cell neuroendocrine carcinoma of the prostate (SCNC), liver cancer, glioblastoma, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal cancer.

[0276] In some embodiments, in addition to the derived effector cells provided herein, the combination of therapeutic uses includes one or more additional therapeutic agents comprising chemotherapeutic agents or radioactive portions. Chemotherapeutic agents refer to cytotoxic antitumor agents, i.e., chemical agents 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 are also sometimes 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 drugs, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, mechloretamine, mephalin, chlorambucil, hairmethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), animitabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisanthren, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, meitansine, and amsacrine.Additional medications include amine glutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, carsterone, capecitabine, celecoxib, and cetonia. Simab, cladribine, cloflavin, cytarabine, dacarbazine, denileukin difutitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinylestradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, i Darbicin, Ifosfamide, Imatinib, Interferon alfa (2a, 2b), Irinotecan, Letrozole, Leucovorin, Leuprolide, Lebamisol, Mechloretamine, Megestrol, Melphalin, Mercaptopurine, Methotrexate, Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone, Nofetumomab, Oxaliplatin, Paclitaxel, Pamidronate, Pemetrexed, Pegademase, Pe These include guasparagaze, pentostatin, pipobromane, plicamycin, polyfeprosan, porfimer, procarbazine, quinacrine, rituximab, salglamostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those known in the art, that are approved as chemotherapeutic or radiotherapeutic agents.Such drugs can be found in one of several standard physician and oncologist references (for example, Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Ninth Edition, McGraw-Hill, NY, 1995) or through the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated regularly.

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

[0279] In addition to the isolated population of iPSC-derived hematopoietic lineage cells contained in the therapeutic composition, a composition suitable for administration to a patient may further include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable media, e.g., cell culture media), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition administered and by the specific method used to administer the therapeutic composition. Thus, a wide variety of suitable formulations of the therapeutic composition of the present invention exist (e.g., Remington's Pharmaceutical Sciences, 17) th See ed. 1985, the disclosure thereof (which is incorporated herein in its entirety by reference).

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

[0281] These pharmaceutically acceptable carriers and / or diluents may be present in amounts sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffer may be about 5% by weight relative to the total composition. Electrolytes, but not limited to sodium chloride and potassium chloride, may also be included in the therapeutic composition. In one embodiment, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition includes a buffer having a pH within one of these pH ranges. In yet another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of 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 pharmaceutically acceptable cell culture media in certain compositions and / or cultures of the present invention. Such compositions are suitable for administration to human subjects. Generally speaking, any medium supporting 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 may be free of animal matter and optionally free of protein. Optionally, the medium may contain recombinant proteins acceptable for biological formulations. A medium free of animal matter refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in a medium free of animal matter, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a protein-free medium is defined as substantially protein-free. 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 that are known to those skilled in the art.

[0283] Isolated pluripotent stem cell-derived hematopoietic cell lines may contain 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, bone marrow-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, isolated pluripotent stem cell-derived hematopoietic cell lines contain approximately 95% to approximately 100% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or bone marrow-derived suppressor cells (MDSCs). In some embodiments, the present invention provides therapeutic compositions having purified T cells or NK cells, such as compositions having an isolated population of about 95% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or bone marrow-derived suppressor cells (MDSCs), for treating subjects requiring cell therapy.

[0284] In one embodiment, the combination cell therapy comprises a therapeutic protein or peptide and a population of NK cells derived from a genome-modified iPSC containing the genotypes listed in Table 2, wherein the derived NK cells contain a CAR having the provided endodomain. In another embodiment, the combination cell therapy comprises an antigen-specific therapeutic protein or peptide and a population of T cells derived from a genome-modified iPSC containing the genotypes listed in Table 2, wherein the derived T cells contain a CD38 null and a CAR having the provided endodomain. In some embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202 and a population of NK cells or T cells derived from a genome-modified iPSC containing the genotypes enumerated in Table 2, wherein the derived NK cells or T cells contain a CAR having the provided endodomain, a CD38 null, and hnCD16. In several other embodiments, the combination cell therapy comprises daratumumab and a population of NK or T cells derived from genome-engineered iPSCs including the genotypes listed in Table 2, wherein the derived NK or T cells comprise a first CAR, CD38 null, hnCD16, and a second CAR having a provided endodomain, the first and / or second CARs targeting 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 additional embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202, and a population of NK or T cells derived from genome-engineered iPSCs including the genotypes listed in Table 2, wherein the derived NK or T cells comprise a first CAR having a provided endodomain, CD38 null, hnCD16, a second CAR, and one or more exogenous cytokines.In yet another embodiment, the combination cell therapy comprises a therapeutic protein or peptide and a population of NK cells derived from genome-engineered iPSCs comprising the genotypes listed in Table 2, wherein the derived NK cells comprise a first CAR having the provided endodomain, CD38 null, hnCD16, a second CAR, and one or more exogenous cytokines, as well as at least one of HLA-G overexpression or CD58 knockout and CD54 knockout. - / - CIITA - / - Includes.

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

[0286] In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is at least 0.1 × 10⁶ per dose. 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 These are cells. In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1 × 10⁶ per dose. 5 cells ~ approx. 1×10 6 Cells, approximately 0.5 × 10⁶ per dose 6 cells ~ approx. 1×10 7 Cells, approximately 0.5 × 10⁶ per dose 7cells ~ approx. 1×10 8 Cells, approximately 0.5 × 10⁶ per dose 8 cells ~ approx. 1×10 9 Cells, approximately 1 x 10⁶ per dose 9 Cells ~ approx. 5 x 10 9 Cells, approximately 0.5 × 10⁶ per dose 9 cells ~ approx. 8 x 10 9 Cells, approximately 3 x 10 per dose 9 cells ~ approx. 3 x 10 10 It is a cell, or any range between them. Generally, for a 60kg patient, it is 1 × 10⁻⁶ 8 The cell / dose ratio is 1.67 × 10⁶. 6 Converted to cells / kg.

[0287] In one embodiment, the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a portion or a single umbilical cord of blood, or at least 0.1 × 10⁶ 5 Cells / kg body weight, at least 0.5 × 10⁻⁶ 5 Cells / kg body weight, at least 1 × 10⁻⁶ 5 Cells / kg body weight, at least 5 × 10⁻⁶ 5 Cells / kg body weight, at least 10 × 10 5 Cells / kg body weight, at least 0.75 × 10⁻⁶ 6 Cells / kg body weight, at least 1.25 × 10⁻⁶ 6 Cells / kg body weight, at least 1.5 × 10⁻⁶ 6 Cells / kg body weight, at least 1.75 × 10⁻⁶ 6 Cells / kg body weight, at least 2 × 10⁻⁶ 6 Cells / kg body weight, at least 2.5 × 10⁻⁶ 6 Cells / kg body weight, at least 3 × 10⁻⁶ 6 Cells / kg body weight, at least 4 × 10⁻⁶ 6 Cells / kg body weight, at least 5 × 10⁻⁶ 6 Cells / kg body weight, at least 10 × 10 6 Cells / kg body weight, at least 15 × 10⁻⁶ 6 Cells / kg body weight, at least 20 × 10⁻⁶ 6 Cells / kg body weight, at least 25 × 10⁻⁶ 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 × 10⁶ 9 It is expressed as cells per kg of body weight.

[0288] In one embodiment, a certain dose of derived hematopoietic lineage cells is delivered to the subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2 × 10⁶ 6 Cells / kg, at least 3 × 10⁻⁶ 6 cells / kg, at least 4 × 10⁶ 6 Cells / kg, at least 5 × 10⁻⁶ 6 Cells / kg, at least 6 × 10⁶ 6 Cells / kg, at least 7 × 10⁶ 6 Cells / kg, at least 8 × 10⁻⁶ 6 Cells / kg, at least 9 × 10⁶ 6 cells / kg, or at least 10 × 10 6 This is cells / kg or more, and includes all intervening cell doses.

[0289] In another exemplary embodiment, the effective amount of cells provided to the subject is approximately 2 × 10⁻⁶ 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, or approximately 10 x 10 6 This is 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 approximately 2 × 10⁻⁶ 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 4×10 6 cells / kg ~ approx. 10×10 6cells / kg, about 5×10 6 cells / kg to about 10×10 6 cells / kg, 2×10 6 cells / kg to about 6×10 6 cells / kg, 2×10 6 cells / kg to about 7×10 6 cells / kg, 2×10 6 cells / kg to about 8×10 6 cells / kg, 3×10 6 cells / kg to about 6×10 6 cells / kg, 3×10 6 cells / kg to about 7×10 6 cells / kg, 3×10 6 cells / kg to about 8×10 6 cells / kg, 4×10 6 cells / kg to about 6×10 6 cells / kg, 4×10 6 cells / kg to about 7×10 6 cells / kg, 4×10 6 cells / kg to about 8×10 6 cells / kg, 5×10 6 cells / kg to about 6×10 6 cells / kg, 5×10 6 cells / kg to about 7×10 6 cells / kg, 5×10 6 cells / kg to about 8×10 6 cells / kg, or 6×10 6 cells / kg to about 8×10 6 cells / kg (including all 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 multiple-dose treatment. In some embodiments, the multiple-dose treatment is one administration per day, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days, every 45 days, or every 50 days, or one administration at any number of days in between.

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

[0293] In certain embodiments, the primary stimulatory and co-stimulatory signals of the derived hematopoietic lineage cells may be provided by different protocols. For example, the agents providing each signal may be in solution or conjugated to a surface. If conjugated to a surface, the agents may be conjugated to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent may be conjugated to a surface while the other is present in solution. In one embodiment, the agent providing the co-stimulatory signal may be conjugated to the cell surface, and the agent providing the primary activation signal may be in solution or conjugated to a surface. In certain embodiments, both agents may be in solution. In another embodiment, the agent is in a soluble form and can then be crosslinked to a surface such as an antibody or other binder that conjugates to an agent disclosed in U.S. Patent Application Publications 2004 / 0101519 and 2006 / 0034810, etc., for cells expressing Fc receptors or artificial antigen-presenting cells (aAPCs) intended for use in activating and expanding T lymphocytes in embodiments of the present invention.

[0294] Some variation in dosage, frequency, and protocol is inevitable depending on the condition of the patient being treated. The person responsible for administration will, in any case, determine the appropriate dosage, frequency, and protocol for each individual patient. [Examples]

[0295] The following examples are provided for illustrative purposes only and are not limiting.

[0296] Example 1 - Materials and Methods Using the applicant's proprietary hiPSC platform, which enables single-cell passage and high-throughput 96-well plate-based flow cytometry sorting to effectively select and test suicide systems in combination with different safe harbor locus integration strategies under the control of various promoters, it enables the induction of clonal hiPSCs by single or multiple gene regulation.

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

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

[0299] Bulk and clonal sorting of genome-edited iPSCs: iPSCs with genome-targeted editing using ZFN or CRISPR-Cas9 were bulk and clonal sorted as GFP+SSEA4+TRA181+iPSCs 20 days after puromycin selection. The single-cell dissociated targeted iPSC pool was resuspended in a newly prepared chilled staining buffer containing Hanks equilibrium salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1x penicillin / streptomycin (MediaTech), and 10 mM Hepes (MediaTech) for optimal performance. 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 a rate of 7 μL per 1 million cells in 100 μL of staining buffer. The solution was washed once with staining buffer, spun down at 225g for 4 minutes, resuspended in staining buffer containing 10 μM thiazovibone, and maintained on ice for flow cytometry sorting. Flow cytometry sorting was performed using FACS Aria II (BD Biosciences). For bulk sorting, GFP+SSEA4+TRA181+ cells were gated and sorted into 15 ml standard tubes filled with 7 ml of FMM. For clonal sorting, sorted cells were dispensed directly 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 involves adding one aliquot of Matrigel to 5 mL of DMEM / F12, then incubating overnight at 4°C to allow proper resuspension, and finally adding it to a 96-well plate at 50 μL per well, followed by incubation overnight at 37°C. The 5x Matrigel is aspirated immediately before adding the medium to each well. Once sorting is 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 day 7, 150 μL of medium was removed from each well and replaced with 100 μL of FMM. On day 10 after sorting, an additional 100 μL of FMM was resupplied to the wells. Colony formation was detected as early as day 2, and most colonies expanded within 7–10 days after sorting. For the first passaging, the wells were washed with PBS and dissociated with 30 μL of acetylene at 37°C for approximately 10 minutes. The need for extended acetylene treatment reflects the compactness of colonies that had been idling in long-term culture. After the cells were confirmed to be dissociated, 200 μL of FMM was added to each well and the colonies were disrupted by several pipettings. The dissociated colonies were transferred to another well in a 96-well plate pre-coated with 5x Matrigel and then centrifuged at 225 g for 2 minutes before incubation. This 1:1 passaging is performed to expand the initial colonies before they expand. Subsequent passaging was routinely performed with 3–5 minutes of accutase treatment and 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 level and TRA1-81 expression level. Clonal lines with near 100% GFP+ and TRA1-81+ were selected for further PCR screening and analysis. Flow cytometry analysis was performed using Guava EasyCyte 8 HT (Millipore) and analyzed using Flowjo (FlowJo, LLC).

[0300] Example 2 - Functional profiling of candidate CARs and derived NK or T cells expressing CARs containing novel endodomains To screen for functional chimeric antigen receptors (CARs), candidate CARs have the same antigen specificity but differ in their endodomain and / or transmembrane domains. ネオA group of these CAR constructs were expressed in primary NK cells and T cells to examine their cell-specific surface expression profiles. As shown in Figures 2A–C, these 29 constructs have identical scFv and CD8 hinge regions, differing only in their signaling components, including the endodomain. This assay was performed by comparing the expression profiles of these 29 different CAR constructs targeting the same specific antigen to determine which construct, more specifically, which endodomain component, provides efficient and detectable CAR expression on the cell surface. In one example, all candidate CARs were constructed to be specific to MICA / B. Similarly, functional screening can also utilize CD19 scFV for CAR specificity, for example. Derived NK cell lines were transduced with lentiviruses carrying their respective CAR constructs. Each CAR construct in Figures 2A–C contains the Thy1.1 marker at its C-terminus, which was isolated from the construct by a P2A peptide (not shown). Approximately 10 days after transduction, transduced cells were assayed for CAR and Thy1.1 expression by FACS. Successfully transduced cells were sorted based on Thy1.1 expression, and CAR staining was performed using antibodies specific to the scFv region of CAR. As shown in Figures 3A-I, the results show a clear but changing CAR expression pattern at the time of assay, with certain transmembrane regions (i.e., CD28, CD8) appearing 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 cellular stage and / or biology of the constructs.

[0301] To demonstrate antigen-specific killing mediated by CAR candidates, MICA / B-CAR ネオNK or T cells are co-cultured with tumor cells that express MICA / B or have null or low MICA / B expression. T cells expressing MICA / B-CD28-CD3z1XXCAR and NK cells expressing MICA / B-NKG2D-2B4-CD3zCAR, as well as CAR-free T cells and NK cells, are used as positive and negative controls. Each MICA / B-CAR exhibiting specific killing ability is then evaluated. ネオ However, it is transduced into iPSCs. All CARs ネオ -iPSC strains are tested for CAR expression, karyotype abnormalities, and genomic stability. Each CAR is examined regardless of whether it is expressed in the iPSC. ネオ -iPSC lines are continued for differentiation of both T cells and NK cells according to the method described herein. Intermediate cells at day 10, day 20, and other time points during differentiation are characterized for marker expression profiles and cell proliferation. Cell proliferation at key 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 candidate-expressing derived NK or T cells, MICA / B-CAR ネオ The stabilization of MICA / B on the cell surface is examined.

[0303] MICA / B-CAR ネオ iPSC-derived NK cells expressing (MICA / B-CAR ネオ A co-culture system is used that includes tumor cell lines (target cells) expressing iNK and MICA / B. The resulting MICA / B-CAR ネオ iNK activation and enhancement of function will also be tested using this co-culture system. MICA / B-positive tumors and MICA / B-CAR ネオ iNK co-cultures are examined using ELISA to assess the level of soluble MICA / B released into the culture supernatant. Compared to co-cultures with unmodified NK cells, target cells are examined for MICA / B-CAR ネオCo-culturing with iNK leads to a reduction in soluble MICA / B released into the culture supernatant, supporting the discovery of MICA / B stabilization on the tumor cell surface. A positive control for this test is used, co-culturing target cells with mAb7C6.

[0304] Under the same co-culture conditions, MICA / B-CAR ネオ iNK cell activation is investigated by the 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 to co-culture with MICA / B-negative cells, there was no observed difference in activity, and increased levels of cytokines and degranulation were observed, as well as MICA / B-CARA negative response to unmodified NK cells against MICA / B-positive target cells. ネオ Increased direct killing by iNK cells is observed in the presence of MICA / B-cell surface antigens in MICA / B-cell surface antigens. ネオ This indicates activation of iNK cells.

[0305] MICA / B-CAR ネオ To investigate whether the expression of MICA / B increases the surface density of MICA / B on target cell lines, we used MICA / B-CAR ネオ MICA / B was expressed in non-NK cell lines that could not kill target cells, and the resulting cells were co-cultured with MICA / B-positive targets. After co-culture, the level of MICA / B on target cells was evaluated by flow cytometry. MICA / B-CAR was compared to co-culture with unmodified NK cells. ネオ The increase in MICA / B levels in target cells after co-culture with non-NK cells expressing MICA / B-CAR ネオ This study demonstrates a positive effect on the surface density of MICA / B in target cell lines.

[0306] Increased levels of gene expression related to NK cell activity in response to increased surface MICA / B levels indicate MICA / B-positive target cells and MICA / B-CAR ネオThe study is conducted by single-cell RNA sequencing of sample NK cells derived from either in vitro co-culture with iNK, or from tissue samples derived from in vivo experiments, spheroids, organoids, or 3D co-culture experiments. Elevated control of perforin, granzyme A and B, and downregulation of immature markers such as CD62L in the co-culture or tissue-derived samples are evaluated by cellular MICA / B-CAR ネオ It shows an increase in NK cell activity related to expression.

[0307] MICA / B-CAR ネオ The in vivo function is evaluated using mouse melanoma cells expressing human MICA as a tumor cell target, or using human cell lines expressing endogenous MICA / B. In in vivo evaluation, MICA / B-CAR is inj...

Claims

[Claim 1] A chimeric antigen receptor comprising an ectodomain containing at least one antigen recognition domain, a transmembrane domain, and an endodomain containing at least one signal transduction domain, The at least one signaling domain is derived from the cytoplasmic domain of a signaling protein specific to the activation or function of T and / or NK cells. When the chimeric antigen receptor is contained in derived effector cells differentiated from induced pluripotent stem cells (iPSCs) containing the chimeric antigen receptor, it exhibits increased cytotoxicity compared to primary immune cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue. The chimeric antigen receptor comprises a transmembrane domain and an endodomain (TM-(endodomain)) containing any of the following forms: CD28H-(CD28H-2B4), CD28H-(CD28H-2B4-CD3ζ), DNAM1-(DNAM1-CS1), KIR2DS2-(KIR2DS2-DAP10-CD3ζ), NKG2D-(2B4-CS1), and NKG2D-(2B4-CS1-CD3ζ). (a) The signal transduction proteins are 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- It comprises any one of the following: 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 (native cytotoxicity-inducing receptor 3), NKp44 (native cytotoxicity-inducing receptor 2), NKp46 (native cytotoxicity-inducing receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain), and / or (b) The at least one signaling domain comprises an amino acid sequence that has at least 90% 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, as represented by SEQ ID NOs. 21-41, 54, and 56, and / or (c) A chimeric antigen receptor in which at least one signaling domain contains an amino acid sequence having at least 90% (except in the case of 90%) identity with the cytoplasmic domains of 2B4, CD28H, CD3ζ, DAP10, FcERIγ, KIR2DS2, NKG2D, CD3ζ, CD3ζ1XX, DNAM1, CS1, or combinations thereof.

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