Multi-targeted effector cells and uses thereof

Abstract

Methods and compositions are provided for obtaining functionally enhanced derivative effector cells obtained from the directed differentiation of genomically engineered iPSCs. [Solution] A cell or a population thereof is provided, wherein (i) the cell is an induced pluripotent cell (iPSC) or a derived cell obtained by iPSC differentiation, and (ii) the cell contains a polynucleotide encoding at least a BCMA-CAR (chimeric antigen receptor).

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 944,187, filed December 5, 2019, and U.S. Provisional Application No. 62 / 906,046, filed September 25, 2019, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Incorporation by Reference of Sequence Listing The entire sequence listing entitled "056932-519001WO_SL_ST25.TXT", created on September 25, 2020, and having a size of 73,729 bytes, is incorporated herein by reference.

[0003] The present disclosure relates generally to the field of off-the-shelf immune cell products. More specifically, the present disclosure relates to strategies for developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. The cell products developed under the present disclosure address significant limitations of patient-derived cell therapy. [Background technology]

[0004] The field of adoptive cell therapy currently focuses on the use of patient- and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapies and provide therapy to all patients who could benefit. There is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote favorable patient outcomes. Lymphocytes, such as T cells and natural killer (NK) cells, are potent antitumor effectors that play a key 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, significant opportunities remain to fully utilize the potential of T cells and NK cells, or other lymphocytes, in adoptive immunotherapy. Summary of the Invention

[0005] Functionally improved effector cells are needed that address issues ranging from response rates, cell depletion, loss of transfused cells (survival and / or persistence), tumor escape due to target loss or lineage switching, precision of tumor targeting, off-target toxicity, extratumoral effects, to efficacy against solid tumors, i.e., the tumor microenvironment and associated immune suppression, recruitment, trafficking, and infiltration.

[0006] The object of the present invention is to provide methods and compositions for generating differentiated derivative non-pluripotent cells from a single-cell-derived clonal iPSC (induced pluripotent stem cell) line, where the iPSC line contains one or more genetic modifications in its genome, including DNA insertions, deletions, and substitutions, that remain retained and functional in subsequent derived cells after differentiation, expansion, passaging, and / or transplantation.

[0007] The iPSC-derived non-pluripotent cells of the present application include, but are not limited to, CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells. The iPSC-derived non-pluripotent cells of the present application contain one or several genetic modifications in their genomes through differentiation from iPSCs containing the same genetic modifications. The engineered clonal iPSC differentiation strategy to obtain genetically engineered derivative cells also requires that the developmental potential of the iPSCs in directed differentiation is not adversely affected by the engineered modality of the iPSCs and that the engineered modality functions as intended in the derivative cells. Furthermore, this strategy overcomes the current barriers to engineering primary lymphocytes, such as T cells or NK cells, obtained from peripheral blood, which often lack reproducibility and homogeneity, resulting in poor cell persistence with high cell death and low cell proliferation, and is difficult to engineer. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations that are otherwise obtained using initially heterogeneous primary cell sources.

[0008] Some embodiments of the present invention provide genomically engineered iPSCs obtained using methods including (I), (II), or (III), which reflect strategies of genomic engineering after, simultaneously with, and before the reprogramming process, respectively.

[0009] (I): iPSCs are genetically engineered by one or both of (i) and (ii) in any order: (i) introducing one or more constructs into iPSCs to enable targeted integration at selected site(s); (ii)(a) introducing one or more double-strand breaks into iPSCs at selected site(s) using one or more endonucleases capable of recognizing the selected sites; (b) culturing the iPSCs from step (I)(ii)(a) to allow endogenous DNA repair to generate targeted in / dels at the selected site(s); thereby obtaining genomically engineered iPSCs capable of differentiation into partially or fully differentiated cells.

[0010] (II): Genetically engineering the reprogrammed non-pluripotent cells to obtain genomically engineered iPSCs, which includes (i) contacting the non-pluripotent cells with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells; and (ii) introducing into the reprogrammed non-pluripotent cells of step (II)(i) one or both of (a) and (b): (a) one or more constructs enabling targeted integration at a selected site; (b) one or more double-strand breaks at the selected site using at least one endonuclease capable of recognizing the selected site, in any order; and then culturing the cells of step (II)(ii)(b) to allow endogenous DNA repair to generate targeted in / dels at the selected site; thus, the resulting genomically engineered iPSCs contain at least one functional targeted genome edit, and the genomically engineered iPSCs are capable of differentiating into partially or fully differentiated cells.

[0011] (III): Genetically engineering non-pluripotent cells for reprogramming to obtain genomically engineered iPSCs. This involves (i) introducing into the non-pluripotent cells one or both of (a) and (b), in any order: (a) one or more constructs that allow targeted integration at selected sites, and (b) one or more double-strand breaks at selected sites using at least one endonuclease capable of recognizing the selected sites. The cells of step (III)(i)(b) are then cultured to allow for the generation of targeted indels at the selected sites by endogenous DNA repair. and (ii) contacting the cells of step (III)(i) with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, to obtain genomically engineered iPSCs comprising a targeted edit at the selected site, thereby obtaining genomically engineered iPSCs comprising at least one functional targeted genome edit, wherein the genomically engineered iPSCs are capable of differentiating into partially or fully differentiated cells.

[0012] In one embodiment of the above-described method, at least one targeted genome edit at one or more selected sites comprises the insertion of one or more exogenous polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of the genomically engineered iPSCs or their derivatives. In some embodiments, the exogenous polynucleotides for insertion are operably linked to one or more exogenous promoters, including 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 within the selected sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for genomic safe harbor. In some embodiments, the genomically engineered iPSCs generated using the above-described methods comprise one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B-cell CD20, and where, if the genomically engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated into different safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, H11, beta2 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 the exogenous polynucleotide are in the form of a fusion protein.

[0013] In some other embodiments, the genomically engineered iPSCs generated using the methods provided herein comprise in / dels in one or more endogenous genes associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response regulation and modulation, or proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derivatives. In some embodiments, the endogenous genes for disruption include at least one of B2M, TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, and any gene in the chromosome 6p21 region.

[0014] In yet some other embodiments, the genomically engineered iPSCs generated using the methods provided herein comprise an exogenous polynucleotide encoding a caspase at the AAVS1 locus and an exogenous polynucleotide encoding a thymidine kinase at the H11 locus.

[0015] In some other embodiments, approaches (I), (II), and / or (III) contact the genomically engineered iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain the pluripotency of the genomically engineered iPSCs. In one embodiment, the resulting genomically engineered iPSCs containing at least one targeted genome edit are functional, differentiation-competent, and capable of differentiating into non-pluripotent cells containing the same functional genome edit.

[0016] Thus, in one aspect, the invention provides a cell or population thereof, wherein (i) the cell is an induced pluripotent cell (iPSC), or a derivative cell obtained from iPSC differentiation, and the cell comprises a polynucleotide encoding at least a BCMA-CAR (chimeric antigen receptor). In some embodiments, the derivative cell obtained from iPSC differentiation is a hematopoietic cell, and the hematopoietic cell comprises longer telomeres compared to its native counterpart obtained from peripheral blood, umbilical cord blood, or any other donor tissue, or the BCMA-CAR exhibits the following characteristics: (i) being T cell-specific, (ii) being NK cell-specific, (iii) binding to surface BCMA, (iv) expressing a gene encoding AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1. or (v) inserted at one of the loci B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, wherein the insertion optionally knocks out expression of a gene at the locus.

[0017] In some embodiments of the above-described cells or populations thereof, the cells further comprise one or more of the following: (i) a CD38 knockout; (ii) B2M null or low, optionally CIITA null or low, compared to their native counterparts; (iii) introduced expression of HLA-G or uncleavable HLA-G, or knockout of one or both of CD58 and CD54; (iv) exogenous CD16 or a variant thereof; (v) a chimeric antigen receptor (CAR) with target specificity other than BCMA; (vi) a cell surface-expressed protein complex comprising a partial or full length cytokine, cytokine receptor, or any combination thereof; (vii) at least one of the genotypes listed in Table 1; (viii) TAP1, TAP2, or TAP3, compared to their native counterparts; (ix) deleted or reduced expression of at least one of tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, and (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, checkpoint inhibitor, antibody or functional fragment and variant thereof, engager, and surface triggering receptor for binding with bi- or multispecific or universal engager, compared to the native counterpart cell.In some embodiments, the BCMA-CAR comprises at least (a) a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37; (b) a light chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38; or (c) an scFV represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39-44. In some embodiments, the cells are derived NK or derived T cells and have at least one of the following characteristics compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue: (i) improved persistence and / or survival, (ii) increased tolerance to natural immune cells, (iii) increased cytotoxicity, improved tumor infiltration, enhanced or acquired ADCC, enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability to rescue tumor antigen escape, (ix) ability to stabilize tumor antigens, and (x) ability to avoid fratricide.

[0018] In some embodiments of the above-described cells or populations thereof, the cells further comprise high-affinity non-cleavable CD16 (hnCD16) or a variant thereof. In various embodiments, the exogenous CD16 or variant thereof comprises at least one of the following: (a) F176V and S197P in the ectodomain of CD16, (b) a complete or partial ectodomain derived from CD64, (c) a non-native (or non-CD16) transmembrane domain, (d) a non-native (or non-CD16) intracellular domain, (e) a non-native (or non-CD16) signaling domain, (f) a non-native stimulatory domain, and (g) transmembrane, signaling, and stimulatory domains not derived from CD16 but derived from the same or a different polypeptide. In some embodiments, (a) the non-native transmembrane domain is derived from a 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, or T cell receptor (TCR) polypeptide; and (b) the non-native stimulatory domain is derived from a CD27, CD28, 4-1BB, (c) the non-native signaling domain is derived from an OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide; and (d) the non-native transmembrane domain is derived from NKG2D, the non-native stimulatory domain is derived from 2B4, and the non-native signaling domain is derived from CD3ζ.

[0019] In some embodiments of the above-described cells or populations thereof, the cells further comprise a CAR with a target specificity other than BCMA, wherein the CAR is (i) T cell-specific or NK cell-specific, (ii) a bispecific antigen-binding CAR, (iii) a switchable CAR, (iv) a dimerized CAR, (v) a split CAR, (vi) a multi-chain CAR, (vii) an inducible CAR, or (viii) a cell surface-expressed protein comprising a partial or full length of a cytokine, cytokine receptor, or any combination thereof. (xi) co-expressed with an antibody or functional fragment or variant thereof, or a checkpoint inhibitor, optionally in a separate construct or a bicistronic construct; (xii) specific for at least one of CD19, MICA / B, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1; and / or (xiii) ADGRE2, carbonic anhydrase Protein IX (CAlX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, cytomegalovirus (CMV)-infected cell antigen, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor Tyrosine protein kinases erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19).9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal 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. In some embodiments, the cells contain at least a CAR inserted into the TRAC locus and / or driven by the endogenous promoter of the TCR and / or the TCR is knocked out by the CAR insertion.

[0020] In some embodiments of the above-described cells or populations thereof, the cells comprise a surface-expressed protein complex comprising a partial or full length of a cytokine, cytokine receptor, or any combination thereof, wherein the cell surface-expressed protein complex comprises at least one of (a) IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or variants thereof, and their respective receptors or variants; or (b) (i) co-expression of IL15 and IL15Rα by using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein having a truncated intracellular domain of IL15Rα; (iv) a fusion protein of IL15 and the membrane-binding Sushi domain of IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and common receptor γC (wherein common receptor γC is native or modified); and (vii) a homodimer of IL15Rβ, wherein any one of (i) to (vii) can be co-expressed with the CAR in a separate or bicistronic construct; and optionally (c) transiently expressed.

[0021] In some embodiments of the above-described cells or populations thereof, the cells are derived NK cells or derived T cells, wherein the derived NK cells are capable of recruiting and / or migrating T cells to tumor sites, and wherein the derived NK cells or derived T cells are capable of reducing tumor immunosuppression in the presence of one or more checkpoint inhibitors.

[0022] In some embodiments of the above-described cells or populations thereof, the cells comprise introduced or increased expression of at least one checkpoint inhibitor, which may be an antagonist to one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some embodiments, the checkpoint inhibitor comprises (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0023] In some embodiments of the above-described cells or populations thereof, wherein the cells are derivative cells obtained from iPSC differentiation, the derivative cells comprise derivative CD34 cells, derivative hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell progenitors, derived NK cell progenitors, derivative T cells, derived NKT cells, derived NK cells, or derivative B cells.

[0024] In some embodiments of the above-described cells or populations thereof, the cells comprise (i) one or more exogenous polynucleotides integrated into one safe harbor locus or selected locus, or (ii) more than two exogenous polynucleotides integrated into different safe harbor loci or two or more selected loci, or (iii) a polynucleotide encoding IL15Δ comprising an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21. In some embodiments, the safe harbor locus includes at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, and the selected locus is one of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and integration of the exogenous polynucleotide optionally knocks out expression of a gene at the locus. In some embodiments, the TCR locus is the constant region of TCR alpha or TCR beta.

[0025] In another aspect, the present invention provides a cell or population thereof comprising a BCMA-CAR and one or more of: (i) a CD38 knockout; (ii) exogenous CD16 or a variant thereof; and (iii) a cell surface expressed protein complex comprising a partial or full length of a cytokine, cytokine receptor, or any combination thereof, wherein the cell is an immune effector cell. In some embodiments, the BCMA-CAR comprises at least (a) a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37; (b) a light chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38; or (c) an scFV represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39-44. In some embodiments, the cells or populations thereof have at least one of the following characteristics compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue: (i) improved persistence and / or survival, (ii) increased resistance to natural immune cells, (iii) increased cytotoxicity, (iv) improved tumor infiltration, (v) enhanced or acquired ADCC, (vi) enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site, (vii) enhanced ability to reduce tumor immunosuppression, (viii) improved ability to rescue tumor antigen escape, (ix) ability to stabilize tumor antigens, and (x) ability to avoid fratricide. In some embodiments, the immune cells are T-lineage or NK-lineage cells.

[0026] In yet another aspect, the present invention provides a composition comprising the above-described cells or populations thereof. In yet another aspect, the present invention provides a composition for therapeutic use comprising the above-described cells and one or more therapeutic agents. In some embodiments, the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector comprising one or more polynucleic acids of interest, an antibody or functional variant or fragment thereof, a chemotherapeutic agent or radioactive moiety, or an immunomodulatory drug (IMiD). In embodiments where the composition comprises a checkpoint inhibitor, the checkpoint inhibitor is selected from the group consisting of: (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, Ra (b) one or more antagonists of checkpoint molecules, including ra (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab. In various embodiments of the composition, the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide. In various embodiments of the composition, the therapeutic agent comprises a gamma secretase inhibitor (GSI). Generally, GSIs are inhibitors of gamma secretase, a protease complex involved in the processing of certain type I integral membrane proteins, such as Notch.In various embodiments of the composition, the antibody comprises one or more of: (a) an anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibody; (b) rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, celtuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof; or (c) daratumumab.

[0027] In yet another aspect, the present invention provides a therapeutic use of any of the compositions described above for adoptive cell therapy, wherein such use is by introducing the composition into a subject having (i) an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a viral infection, (ii) an inflammatory autoimmune disease, a cancer of plasma cells, or a cancer of B lymphocytes, (iii) an autoimmune disease associated with autoreactive plasma cells and / or autoreactive memory B cells, (iv) systemic lupus erythematosus (SLE), or rheumatoid arthritis, multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, plasma cell leukemia, or Hodgkin's disease, or (v) multiple myeloma. Thus, the present invention provides the use of any of the compositions described above in a medicine for treating: (i) an autoimmune disorder, a hematological malignancy, a solid tumor, a cancer, or a viral infection; (ii) an inflammatory autoimmune disease, a cancer of plasma cells, or a cancer of B lymphocytes; (iii) an autoimmune disease associated with autoreactive plasma cells and / or autoreactive memory B cells; (iv) systemic lupus erythematosus (SLE), or rheumatoid arthritis, multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, plasma cell leukemia, or Hodgkin's disease; or (v) multiple myeloma.

[0028] In yet another aspect, the present invention provides a method of producing a derivative cell as described above, wherein the method of producing comprises differentiating iPSCs, wherein the iPSCs comprise a polynucleotide encoding a BCMA-CAR and optionally one or more of the following: (i) CD38 knockout, (ii) B2M null or low, and optionally CIITA null or low compared to their native counterparts, (iii) introduced expression of HLA-G or uncleavable HLA-G, or knockout of one or both of CD58 and CD54, (iv) high affinity uncleavable CD16 (hnCD16) or a variant thereof, (v) a chimeric antigen receptor (CAR) with target specificity other than BCMA, (vi) a cell surface expressed protein complex comprising partial or full length of a cytokine, cytokine receptor, or any combination thereof, (vii) a protein listed in Table 1 provided herein. (viii) deleted or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT compared to the native counterpart, and (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, checkpoint inhibitor, antibody or functional fragment or variant thereof, engager, and surface triggering receptor for binding with bi- or multispecific or universal engager compared to the native counterpart.

[0029] In some embodiments of the method of producing derivative cells, the method further comprises genomic engineering of clonal iPSCs to knock in a polynucleotide encoding a BCMA-CAR, and optionally (i) knock out CD38, or (ii) knock out B2M and CIITA, knock out one or both of CD58 and CD54, or introduce expression of a cell surface-expressed protein complex comprising partial or full-length HLA-G or uncleavable HLA-G, exogenous CD16 or a variant thereof, a second CAR, and / or a cytokine, cytokine receptor, or any combination thereof. In some embodiments of the method of producing derivative cells, the genomic engineering comprises targeted editing. In some embodiments, the targeted editing comprises a deletion, insertion, or in / del, and the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

[0030] In yet another aspect, the present invention provides CRISPR-mediated editing of clonal iPSCs, wherein the editing includes knocking in a polynucleotide encoding a BCMA-CAR, and wherein the edited clonal iPSCs comprise at least one of the genotypes listed in Table 1 provided herein. In some embodiments, the editing of the clonal iPSCs further includes knocking out CD38, or the BCMA-CAR has at least one of the following characteristics: (i) being T cell-specific, (ii) being NK cell-specific, (iii) binding to surface BCMA, (iv) comprising a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37, (vi) an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38. (vii) comprising an scFV represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39, 40, 41, 42, 43, or 44; and (viii) an insertion at one of the following loci: B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, where the insertion knocks out expression of a gene at the locus. In some embodiments, the editing further comprises insertion of a BCMA-CAR or a second CAR in the constant region of the TCR locus, and / or the CAR is driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by insertion of the CAR.

[0031] In yet another aspect, the present invention provides a method of improving the treatment of multiple myeloma, the method comprising administering to a subject receiving treatment effector cells comprising a BCMA-CAR, a CD38 knockout, and a high-affinity non-cleavable CD16 or variant thereof, wherein the BCMA-CAR has at least one of the following characteristics: (i) is T cell-specific, (ii) is NK cell-specific, (iii) binds to and stabilizes surface BCMA, (iv) comprises a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37, (vi) is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38, (vii) comprising a light chain variable region represented by an amino acid sequence that is 8%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39, 40, 41, 42, 43, or 44; and (viii) an insertion at one of the following loci: B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, where the insertion knocks out expression of the gene at the locus.In some embodiments, the effector cells comprise derived hematopoietic cells, including derived NK cells or derived T cells, wherein the derived NK cells or derived T cells further comprise one or more of the following: (i) B2M and CIITA knockout, (ii) introduction of expression of HLA-G or uncleavable HLA-G, or knockout of one or both of CD58 and CD54, (iii) introduction of expression of a second CAR, and / or a cell surface-expressed protein complex comprising partial or full-length cytokines, cytokine receptors, or any combination thereof, and / or (iv) at least one of the phenotypes listed in Table 1 provided herein. In some embodiments, the method further comprises administering an anti-CD38 antibody and / or a gamma secretase inhibitor (GSI). In some embodiments, the effector cells comprise a BCMA-CAR, a CD38 knockout, high-affinity uncleavable CD16 or a variant thereof, and have been in contact with or are in contact with a GSI. In some embodiments, the multiple myeloma is a relapsed or refractory form of multiple myeloma.

[0032] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description, taken in conjunction with the accompanying drawings, in which are set forth, by way of illustration and example, certain embodiments of the invention. [Brief explanation of the drawings]

[0033] [Figure 1] 1 is a diagram of several construct designs for cell surface expression of cytokines in iPSC-derived cells. IL15 is used as an illustrative example and can be substituted with other desired cytokines. [Figure 2A] Various compositions of CD38-targeted transgene knock-in constructs are shown (A and B vs. C and D) with one or more transgenes driven by an exogenous promoter or the CD38 endogenous promoter (B and D vs. A and C) for generating CD38- / - transgene+ pluripotent stem cells and their derived effector cells. [Figure 2B]Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 3-1] 1 shows exemplary nucleic acid sequences included in an exogenous promoter-driven CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct for generating CD38- / -CD16 IL15 effector cells derived from engineered pluripotent stem cells using the construct and variants thereof. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 4-1] FIG. 1 shows exemplary nucleic acid sequences contained in a CD38-targeted IL15 / IL15ra-2A-hnCD16 knock-in construct driven by the CD38 endogenous promoter to generate CD38- / -CD16+ IL15+ effector cells derived from engineered pluripotent stem cells using the construct and variants thereof. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5] Figure 1 shows that DAP10 promotes BCMA-CAR surface expression in 293T cells. [Figure 6] 1 shows that no BCMA-CAR surface expression was detected in engineered iPS cells, regardless of the presence or absence of DAP10 expression. [Figure 7A] Shown is surface expression of BCMA-CAR and IL15Δ in each of the indicated iNK cell lines, and DAP10 does not affect BCMA-CAR surface expression in NK cells differentiated from iPSCs that contain the same CAR but lack its detectable surface expression. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8] Graphical representation of telomere length determined by flow cytometry, showing that mature derived NK cells from iPSCs maintain longer telomeres compared to adult peripheral blood NK cells. [Figure 9] BCMA-CAR surface expression on iNK cell lines is depicted as indicated. [Figure 10] Figure 1 shows that T cells expressing BCMA-CAR have specific cytotoxicity in a 4-hour killing assay. [Figure 11] We show that BCMA-specific CAR and daratumumab synergize in a 48-hour killing assay to effectively eliminate multiple myeloma cancer cells by multi-antigen-targeted iNK cells containing BCMA-CAR, IL15Δ, CD38 null, and hnCD16. [Figure 12A] Figure 12 shows that the combination of daratumumab and BCMA-CAR / IL15Δ / CD38null / hnCD16 iNK cells eliminates tumor burden in a disseminated mouse model of multiple myeloma. 5x10 MM.1S-luciferase (MM.1S-luc) cells were intravenously injected into immunodeficient NSG mice. Two days later, mice were left untreated or treated with 200 μg of daratumumab (Dara) alone or in combination with 1x10 iNK cells. Mice were intravenously injected with hIL-15 (5 μg) three times during the first week to support NK cell persistence. Figure 12A shows representative bioluminescence images, and Figure 12B shows cumulative data from n=10 mice per group, demonstrating the in vivo efficacy of BCMA-CAR / IL15Δ / CD38null / hnCD16 iNK cells. [Figure 12B] Same as above. [Figure 13]Figure 1 shows that iNK cells effectively target BCMA+ multiple myeloma tumor cell lines via both CAR-mediated and ADCC mechanisms. Cytotoxicity of FT576 against (A) RPMI-8266 and (B) U266 multiple myeloma tumor lines at an effector:target ratio of 3:1, with or without 2 μg / ml daratumumab. Samples were incubated for 4 hours at 37°C, and target cell apoptosis was assessed by flow cytometry. Compared to iNK cells lacking CAR expression, BCMA-CAR / IL15Δ / CD38null / hnCD16 iNK cells showed enhanced cytotoxicity in the absence of daratumumab, and additional ADCC was observed for both cell types in the presence of daratumumab. In each pair of bars, the left bar is the non-CAR control. [Figure 14]

[0023] Figure 1 shows exemplary results of a restimulation cytotoxicity assay performed on MM.1S cells in the presence or absence of daratumumab using BCMA-CAR / IL15Δ / CD38null / hnCD16 iNK cells as effector cells at a 1:1 E:T ratio compared to CAR-negative parental cells. The X-axis is shown in 12-unit increments. The Y-axis is shown in 0.5-unit increments. [Figure 15] 1 shows cytokine production of BCMA-CAR / IL15Δ / CD38null / hnCD16iNK cells in response to CAR and CD16 stimulation. [Figure 16] Shown are exemplary BLI images of NSG mice transplanted with MM.1S-Luc cells and treated with multiple doses of BCMA-CAR / IL15Δ / CD38null / hnCD16 effector cells, in the presence or absence of exogenous cytokine support, compared to CAR T cells with or without the same cytokine treatment. [Figure 17] 1 shows that gamma secretase inhibitors increase the in vivo efficacy of BCMA-CAR / IL15Δ / CD38null / hnCD16 effector cells. Naive mice are shown in open square symbols with dashed lines, and non-transplanted control mice are shown with open symbols and dashed lines. DETAILED DESCRIPTION OF THE INVENTION

[0034] Genome modifications of iPSCs (induced pluripotent stem cells) include polynucleotide insertion, deletion, and substitution. Exogenous gene expression in genomically engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genomically engineered iPSCs, after cell differentiation, and in dedifferentiated cell types derived from the genomically engineered iPSCs. Meanwhile, directly manipulating primary immune cells such as T cells or NK cells is difficult, posing obstacles to the preparation and delivery of engineered immune cells for adoptive cell therapy. The present invention provides an efficient and reliable targeted approach for stably integrating one or more exogenous genes, including suicide genes and other functional modalities, which confer improved therapeutic properties regarding engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, longevity, self-renewal, persistence, and / or survival rate to iPSC-derived cells, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T cells, NKT cells, and NK cells.

[0035] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular.

[0036] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0037] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0038] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0039] The term "and / or" should be understood to mean either one or both of the alternatives.

[0040] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0041] As used herein, the term "substantially" or "essentially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about the same as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0042] As used herein, the terms "substantially free" and "essentially free" are used interchangeably 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 source thereof, e.g., is 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance (1) is not included in the composition at any concentration, or (2) is functionally inactive but is included in the composition at low concentrations. A similar meaning can be applied to the term "absence," which refers to the absence of a particular substance or source of a composition.

[0043] Throughout this specification, unless the context requires otherwise, the terms "comprise," "comprises," and "comprising" are understood to mean the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include," "having," "containing," and "comprise" are used synonymously.

[0044] "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0045] "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or operation specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but other elements are not optional and may or may not be present depending on whether they affect the activity or operation of the listed elements.

[0046] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of these phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0047] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed 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 live cells or tissues taken from an organism and cultured in a laboratory setting, usually under sterile conditions, typically for a few hours or up to about 24 hours (although up to 48 or 72 hours or longer, depending on the circumstances). In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures using live cells or tissues lasting longer than a few days are typically considered to be "in vitro," although in certain embodiments, the term may be used interchangeably with ex vivo.

[0048] The term "in vivo" generally refers to activities that take place inside a living organism.

[0049] As used herein, the term "reprogramming" or "dedifferentiation" or "increased differentiation potential" or "increased developmental potential" refers to a method of increasing the differentiation potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased differentiation potential has greater developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in an unreprogrammed state. In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in an unreprogrammed state.

[0050] As used herein, the term "differentiation" refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or differentiation-induced cells are cells that have adopted a more specialized ("committed") position within a cellular lineage. The term "committed," when applied to the process of differentiation, refers to cells that, under normal circumstances, have progressed along a differentiation pathway to the point where they continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, are unable to differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells that cannot give rise to a complete organism (e.g., epiblast stem cells or EpiSCs), to more primitive, more pluripotent cells that can give rise to a complete organism (e.g., embryonic stem cells).

[0051] As used herein, the term "induced pluripotent stem cells" or iPSCs means stem cells produced from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed into cells that can differentiate into all tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells found in nature.

[0052] As used herein, the term "embryonic stem cells" refers to naturally occurring pluripotent stem cells in the inner cell mass of blastocysts. Embryonic stem cells are pluripotent and give rise to all derivatives of the three major germ layers of ectoderm, endoderm, and mesoderm during development. They do not contribute to extraembryonic membranes or placenta, i.e., they are not totipotent.

[0053] As used herein, the term "multipotent stem cells" refers to cells that have the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Therefore, 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 has the potential to form many cell types in a given lineage but not cells of other lineages. For example, multipotent hematopoietic cells can form many different blood cell types (red, white, platelets, etc.), but cannot form neurons. Therefore, the term "multipotency" refers to a state of a cell that has a degree of developmental potential lower than totipotency and pluripotency.

[0054] Pluripotency can be determined, in part, by assessing cellular pluripotency characteristics, including, but not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers, including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) the formation of teratomas composed of the three somatic cell lineages, and (vi) the formation of embryoid bodies composed of cells from the three somatic cell lineages.

[0055] Two types of pluripotency have previously been described: a pluripotent "primed" or "metastable" state, similar to the epiblast stem cells (EpiSCs) of late blastocysts, and a pluripotent "naive" or "ground" state, similar to the cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or ground state additionally exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival during single-cell culture, (iii) globally reduced DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmentally regulated gene promoters, and (v) reduced expression of differentiation markers compared to primed pluripotent cells. Standard cell reprogramming methodologies, 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 possess the characteristics of the primed pluripotent state. Under standard pluripotent cell culture conditions, such cells remain in a primed state and exhibit ground state characteristics unless expression of an exogenous transgene is maintained, at which point ground state characteristics are observed.

[0056] As used herein, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a small, round shape with a high nucleus-to-cytoplasm ratio, prominent nucleoli, and typical intercellular spacing.

[0057] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domestic animal.

[0058] "Pluripotency factors" or "reprogramming factors" refer to agents that can increase the developmental potential of cells, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of cells. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0059] "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 singular "medium"), "supplement," and "medium supplement" refer to nutritional compositions in which cell cultures are cultivated.

[0060] "Culturing" or "maintaining" refers to maintaining, expanding (growing), and / or differentiating cells outside of a tissue or outside the body, for example, in sterile plastic (or coated plastic) cell culture dishes or flasks. "Culturing" or "maintaining" may utilize culture medium as a source of nutrients, hormones, and / or other factors that help to grow and / or maintain the cells.

[0061] As used herein, the term "mesoderm" refers to one of three germ layers that emerge during early embryonic development and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0062] As used herein, the terms "secondary hemogenic endothelial cells" (HE) or "pluripotent stem cell-derived secondary hemogenic 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 conversion. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm to hemangioblasts to secondary hemogenic endothelial cells and hematopoietic progenitor cells.

[0063] The terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including multipotent hematopoietic stem cells (hemocyte blasts), myeloid progenitors, megakaryocytic progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). As used herein, the term "secondary hematopoietic stem cells" refers to CD34+ hematopoietic cells that can give rise to both mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0064] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major type of white blood cell that completes maturation in the thymus and has various roles in the immune system, including identifying specific foreign antigens in the body and activating and deactivating other immune cells. T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any stage of development, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), etc. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem or progenitor cells.

[0065] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are involved in cell-mediated immune responses. They are characterized by a secretory profile after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. "CD4" is a 55 kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as the relevant recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, they define helper / inducer subsets.

[0066] "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 on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the relevant recognition element in major histocompatibility complex class I restricted interactions.

[0067] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of T cell receptor (CD3). As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56+, express at least one of NKG2C and CD57, and optionally CD16, but lack expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, an isolated subpopulation of CD56+ NK cells contains expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. CD56+ may be dim or bright.

[0068] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the non-classical MHC molecule, CD1d. Two types of NKT cells have been recognized. Invariant or type I NKT cells express a very limited TCR repertoire, i.e., a standard α chain (Vα24-Jα18 in humans) associated with a limited spectrum of β chains (Vβ11 in humans). A second population of NKT cells, termed non-classical or non-invariant type II NKT cells, exhibits more heterogeneous TCRαβ usage. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0069] As used herein, the term "isolated" or similar refers to a cell or population of cells that has been separated from its original environment, i.e., the environment of the isolated cell is substantially free of at least one component found in the environment of the "non-isolated" reference cell. This term includes cells that have been removed from some or all components found in their natural environment, e.g., isolated from a tissue or biopsy sample. This term also includes cells that have been removed from at least one, some, or all components found in a non-naturally occurring environment, e.g., isolated from a cell culture or cell suspension. Thus, an isolated cell is partially or completely separated from at least one component, including other substances, cells, or cell populations, as found in nature or when grown, stored, or subsisting in a non-naturally occurring environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a non-naturally occurring medium. Isolated cells can be obtained by separating a desired cell or population of cells from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0070] As used herein, the term "purify" or the like refers to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0071] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to 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 resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0072] A "construct" refers to a macromolecule or complex of molecules containing a polynucleotide that is delivered to a host cell either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell, where it can replicate and / or express in the target cell. As used herein, the term "vector" includes the delivered construct. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. 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, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, etc.

[0073] "Integration" means that one or more nucleotides of a construct are stably inserted into a cell genome, i.e., covalently linked to a nucleic acid sequence within the chromosomal DNA of the cell. "Targeted integration" means that nucleotides of a construct are inserted into the chromosome or mitochondrial DNA of a cell at a preselected site or "integration site." As used herein, the term "integration" also refers to a process involving the insertion of one or more exogenous sequences or nucleotides of a construct, with or without deletion of the endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" can further include replacing the deleted endogenous sequence or nucleotide with one or more inserted nucleotides.

[0074] As used herein, the term "exogenous" is intended to mean that the referenced molecule or referenced activity is introduced into the host cell or is non-native to the host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the host's genetic material, e.g., by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in reference to expression of an encoding nucleic acid, the term refers to introducing the encoding nucleic acid into a cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity that is present in the host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0075] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of an appropriate regulatory sequence, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo.A gene of interest or polynucleotide may include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence.For example, a gene of interest may encode miRNA, shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof; a variant polypeptide (i.e., a variant of a natural polypeptide that has less than 100% sequence identity with the natural polypeptide) or a fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, etc.

[0076] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) for thymine. Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotide also refers to both double-stranded and single-stranded molecules.

[0077] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably 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, generally referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as polypeptides or proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0078] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it can affect the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operably linked to a regulatory sequence in sense or antisense orientation.

[0079] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to preferential therapeutic attributes in source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" refers to 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 lineage cell. iPSCs derived from source cells and cells differentiated therefrom are sometimes collectively referred to as "derived" or "derived" cells, depending on the context. For example, as used throughout this specification, derived effector cells, or derived NK cells or derived T cells are cells differentiated from iPSCs, compared to their primary counterparts obtained from a natural / natural source, such as peripheral blood, umbilical cord blood, or other donor tissue. As used herein, genetic imprints that confer preferential therapeutic attributes are incorporated into iPSCs either by reprogramming donor-, disease-, or treatment-response-specific selected source cells, or by using genome editing to introduce genetically modified modalities into iPSCs. In embodiments of source cells obtained from specifically selected donors, diseases, or treatment situations, genetic imprints that contribute to preferential therapeutic attributes can include any situation-specific genetic or epigenetic modifications that exhibit a retainable phenotype, i.e., preferential therapeutic attributes, that are transmitted to descendants of the selected source cells, regardless of whether the underlying molecular events have been identified. Donor-specific, disease-specific, or treatment response-specific source cells may contain genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including, but not limited to, pre-positioned monospecific TCRs, for example, from virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable genetic polymorphisms, for example, homozygosity for a point mutation encoding the high-affinity CD16 receptor of the selected donor; predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting population-enriched haplotypes.As used herein, preferential therapeutic attributes include improved engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity of the derived cells. Preferential therapeutic attributes may also be related to the expression of antigen-targeting receptors, HLA presentation or lack thereof, tolerance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity due to reduced extratumoral effects, and resistance to treatments such as chemotherapy.

[0080] As used herein, the term "enhanced therapeutic properties" refers to enhanced therapeutic properties of cells compared to typical immune cells of the same general 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, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity. Therapeutic properties of immune cells may also be demonstrated by the expression of antigen-targeting receptors, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity due to reduced extratumoral effects, and resistance to treatments such as chemotherapy.

[0081] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between immune cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, neutrophils, and tumor cells, and activate the immune cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers, or multispecific killer cell engagers, or universal engagers that are compatible with multiple immune cell types.

[0082] As used herein, the term "surface triggering receptor" refers to a receptor that can induce or initiate an immune response, e.g., a cytotoxic response. Surface triggering receptors can be engineered and expressed on effector cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils. In some embodiments, the surface triggering receptor facilitates bispecific or multispecific antibody binding between an effector cell and a specific target cell, e.g., a tumor cell, regardless of the effector cell's native receptor and cell type. This approach can be used to generate iPSCs containing a universal surface triggering receptor, which can then be differentiated into a population of various effector cell types that express the universal surface triggering receptor. "Universal" means that the surface triggering receptor can be expressed and activated on any effector cell, regardless of cell type, and all effector cells that express the universal receptor can bind or ligate to an engager that has the same epitope recognizable by the surface triggering receptor, regardless of the engager's tumor-binding specificity. In some embodiments, an engager with the same tumor-targeting specificity is used to bind to the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to a universal surface triggering receptor. Thus, one or more effector cell types may be used to kill a specific type of tumor cell, or two or more types of tumors. Surface triggering receptors generally contain a costimulatory domain for effector cell activation and an epitope-binding region specific for an epitope of the engager. Bispecific engagers are specific for the epitope-binding region of a surface triggering receptor at one end and for a tumor antigen at the other end.

[0083] 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 that have permanently incorporated a gene encoding the safety switch protein into their genome. This conditional regulation can be variable and can include post-translational activation via small molecules and tissue-specific and / or temporal transcriptional regulation. Safety switches can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some cases, safety switch proteins are activated by exogenous molecules, e.g., prodrugs, and upon activation, trigger apoptosis and / or cell death of 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 the suicide gene product and kills the transduced cells.

[0084] As used herein, the term "pharmaceutically active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. Pharmaceutically active proteins have curative, curative, or palliative properties for disease and can be administered to ameliorate, alleviate, reduce, reverse, or mitigate the severity of disease. Pharmaceutically active proteins also have prophylactic properties and are used to prevent the onset of disease or to lessen the severity of such disease or pathological conditions once they appear. Pharmaceutically active proteins include whole proteins or peptides or pharmaceutically active fragments thereof. They also include pharmaceutically active analogs of proteins or peptides or analogs of fragments of proteins or peptides. The term pharmaceutically active protein also refers to multiple proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0085] As used herein, the term "signaling molecule" refers to any molecule that regulates, is involved in, inhibits, activates, reduces, or increases cell signaling. Signal transduction refers to the transmission of a molecular signal in the form of a chemical modification through the recruitment of protein complexes along a pathway that ultimately leads to biochemical events within the cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Tollgate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0086] As used herein, the term "targeting modality" refers to molecules, e.g., polypeptides, that are genetically incorporated into cells to promote antigen and / or epitope specificity, including, but not limited to: i) antigen specificity when associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when associated with a monoclonal antibody or bispecific engager; iii) transformed cell targeting; iv) cancer stem cell targeting; and v) other targeting strategies in the absence of a particular antigen or surface molecule.

[0087] As used herein, the terms "specific" or "specificity" may be used to refer to the ability of a molecule, e.g., a receptor or engager, to selectively bind to a target molecule, as opposed to non-specific or non-selective binding.

[0088] As used herein, the term "adoptive cell therapy," as used herein, refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, specified as T cells or B cells, whether genetically modified or not, that have been expanded in vivo prior to infusion.

[0089] As used herein, a "therapeutically sufficient amount" includes, within its meaning, a non-toxic, but sufficient and / or effective amount of the particular treatment and / or pharmaceutical composition to which it refers to provide 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 certain embodiments, a therapeutically sufficient amount is sufficient and / or effective to ameliorate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0090] Differentiation of pluripotent stem cells requires changes in the culture system, such as the addition of stimuli 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 for initiating lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development because they give rise to multiple lineages within a three-dimensional area. Throughout the differentiation process, which typically takes hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and develop into cystic EBs, at which point (typically days to weeks) they are further processed to continue differentiation. EB formation is initiated by placing pluripotent stem cells in close proximity to each other in a three-dimensional, multilayered cell cluster; this is typically achieved by one of several methods, including sedimenting pluripotent cells into droplets, settling cells into "U"-bottom well plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require further differentiation cues to promote EB development. Therefore, pluripotent stem cell aggregates must be transferred to a differentiation medium that provides cues for induction toward a selected lineage. EB-based culture of pluripotent stem cells typically results in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm germ layers) with moderate proliferation within the EB cell clusters. Although proven to promote cell differentiation, EBs give rise to heterogeneous cells with different differentiation states because the three-dimensional structure of cells is not consistently exposed to differentiation cues from the environment. In addition, EBs are difficult to generate and maintain. Furthermore, EB-mediated cell differentiation involves moderate cell expansion, which also contributes to reduced differentiation efficiency.

[0091] In contrast, "aggregate formation," which differs from "EB formation," can be used to expand populations of pluripotent stem cell-derived cells. For example, during aggregate-based pluripotent stem cell expansion, a culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates to form larger aggregates, which can then be routinely mechanically or enzymatically dissociated into smaller aggregates to maintain cell growth and increase cell number in culture. Unlike EB culture, cells cultured within aggregates in maintenance culture maintain markers of pluripotency. Pluripotent stem cell aggregates require additional differentiation cues to induce differentiation.

[0092] As used herein, "monolayer differentiation" refers to a differentiation method that differs from differentiation through three-dimensional multilayered clusters 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 specific lineages is considered minimal compared to differentiation into all three germ layers in EBs.

[0093] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be enzymatically or mechanically dissociated from each other, such as by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture plate or other surface. Thus, dissociation can involve disruption of cellular interactions with the extracellular matrix (ECM) and the substrate (e.g., the culture surface), or disruption of the ECM between cells.

[0094] As used herein, "feeder cells" or "feeders" are terms used to describe cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimuli, growth factors, and nutrients to support the second cell type. Feeder cells are optionally from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with mitotic antagonists such as mitomycin to prevent them from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type can be human feeder, such as human skin fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEF). Generally, various feeder cells can be partially used to maintain pluripotency, direct differentiation into a specific lineage, enhance proliferation capacity, and promote maturation into specialized cell types, such as effector cells.

[0095] As used herein, a "feeder-free" (FF) environment refers to an environment, such as a culture condition, cell culture, or culture medium, that is essentially free of feeder cells or stromal cells and / or is not preconditioned by culturing feeder cells. A "preconditioned" medium refers to a medium that is harvested after feeder cells have been cultured in the medium for a period of time, such as at least one day. A preconditioned medium contains many mediator substances, including growth factors and cytokines, secreted by feeder cells cultured in the medium. In some embodiments, the feeder-free environment does not contain either feeder cells or stromal cells and is not preconditioned by culturing feeder cells.

[0096] 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" refers to (1) successful knock-in, knock-out, knock-down gene expression, transgenic or controlled gene expression at the genetic level, e.g., inducible or transient expression at a desired developmental stage of the cell, achieved by direct genome editing or modification, or by "transfer" via differentiation or reprogramming from an initially genomically engineered starting cell, or (2) (i) the genetic modification or modification of the resulting gene in the cell by direct genome editing. (ii) modifications of gene expression that are maintained in a cell by "transfer" from an initially genomically engineered starting cell through differentiation or reprogramming thereof; (iii) downstream gene regulation in a cell as a result of modifications of gene expression that are only apparent in the cell's early developmental stages or only in the starting cell that gave rise to the cell through differentiation or reprogramming; or (iv) removal, addition, or alteration of favorable cellular functions / properties at the cellular level through enhanced or newly acquired cellular functions or attributes that are exhibited in the mature cell product originally derived from genome editing or modifications performed on iPSC, progenitor, or dedifferentiated cellular sources.

[0097] "HLA-deficient," including HLA class I-deficient, HLA class II-deficient, or both, refers to any cell that lacks or no longer maintains surface expression of complete MHC complexes comprising HLA class I protein heterodimers and / or HLA class II heterodimers, or has reduced or diminished levels that are lower than those naturally detectable by other cells or synthetic methods.

[0098] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that have been further modified by introducing genes expressing proteins related to, but not limited to, 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, CD3z, 41BBL, CD47, CD113, and PDL1, to improve differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity. "Modified HLA-deficient" cells also include cells other than iPSCs.

[0099] 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 IgA are called Fc-alpha receptors (FcαR), and those that bind IgE are called Fc-epsilon receptors (FcεR). FcR classes are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells, and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγR) include several members: FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b), which have different molecular structures and therefore different antibody affinities.

[0100] "Chimeric Fc receptor," abbreviated as CFcR, is a term used to describe engineered Fc receptors in which their native transmembrane and / or intracellular signaling domains have been modified or replaced with non-native transmembrane and / or intracellular signaling domains. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane and signaling domains being non-native, one or more stimulatory domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance receptor-induced cell activation, expansion, and function. Unlike chimeric antigen receptors (CARs), which contain an antigen-binding domain for a target antigen, chimeric Fc receptors bind to an Fc fragment, or the Fc region of an antibody, or the Fc region contained in an engager or binding molecule, and activate cells regardless of whether the targeted cell is in close proximity. For example, Fcγ receptors can be engineered to contain selected transmembrane, stimulatory, and / or signaling domains within the intracellular region, which respond to IgG binding at the extracellular domain, thereby generating CFcR. In one example, CFcR is generated by engineering the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domain. To further improve the binding affinity of CD16-based CFcR, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcR 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 so that it is not cleavable, i.e., is not susceptible to shedding, thereby resulting in a CD16-based CFcR.

[0101] The FcγR receptor CD16 has been identified as having 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 cellular cytotoxicity (ADCC). As used herein, "high-affinity CD16," "non-cleavable CD16," or "high-affinity non-cleavable CD16 (hnCD16)" refers to naturally occurring or non-naturally occurring variants of CD16. Wild-type CD16 has low affinity, and upon NK cell activation, it undergoes ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary high-affinity CD16 polymorphic variants. CD16 variants in which the cleavage site (positions 195-198) in the membrane-proximal region (positions 189-212) has been modified or eliminated are not subject to shedding. The cleavage site and membrane-proximal region are described in detail in WO2015 / 148926, the complete disclosure of which is incorporated herein by reference. The CD16 S197P variant is an engineered, non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high-affinity and non-cleavable. Another exemplary high-affinity, non-cleavable CD16 (hnCD16) variant is an engineered CD16 containing an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0102] I. Cells and Compositions Useful for Adoptive Cell Therapy with Enhanced Properties Provided herein is a strategy for systematically manipulating the regulatory circuitry of clonal iPSCs while enhancing the therapeutic properties of derived cells without affecting the differentiation potential of iPSCs or the cellular developmental biology of iPSCs and their derived cells. The derived cells are functionally improved and suitable for adoptive cell therapy after a combination of selective modalities is introduced into cells at the iPSC level through genomic manipulation. Prior to the present invention, it was unclear whether modified iPSCs containing one or more provided gene edits would still have the ability to enter cellular development and / or mature and generate functional differentiated cells while retaining regulated activity. Unexpected failures during directed cell differentiation from iPSCs have been attributed to factors including, but not limited to, developmental stage-specific gene expression or lack thereof, the requirement for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need to reconfigure the differentiation protocol to allow for changes in cell phenotype and / or function. The present application has demonstrated that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from the individual or combined genomic modifications retained in the effector cells following iPSC differentiation.

[0103] 1. BCMA-CAR One or more CAR designs may be applied to genetically engineered iPSCs and their derived effector cells. A chimeric antigen receptor (CAR) is a fusion protein that generally includes an ectodomain, including an antigen recognition region, a transmembrane domain, and an endodomain. In some embodiments, the ectodomain may further include a signal peptide or leader sequence and / or a spacer. In some embodiments, the endodomain may further include a signaling peptide that activates effector cells expressing the CAR. In some embodiments, the antigen recognition domain is capable of specifically binding to an antigen. In some embodiments, the antigen recognition domain is capable of specifically binding to an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, and the tumor may be a liquid or solid tumor. In some embodiments, the CAR is suitable for activating T cells, NK cells, or NKT cells expressing the CAR. In some embodiments, CARs containing NK-specific signaling components are NK cell-specific. In some embodiments, CARs containing NKT-specific signaling components are NKT cell-specific. In certain embodiments, the T cells are derived from CAR-expressing iPSCs, and the derived T cells can include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof. In certain embodiments, the NK cells are derived from CAR-expressing iPSCs. In certain embodiments, the NKT cells are derived from CAR-expressing iPSCs.

[0104] In certain embodiments, the antigen recognition region comprises a mouse antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, antigen-binding single-chain variable fragments (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen-binding fragments (sdAb, nanobodies), recombinant heavy chain-only antibodies (VHH), and other antibody fragments that maintain the binding specificity of a whole antibody.

[0105] In one example, the present disclosure provides a CAR comprising an antigen recognition region that targets the tumor antigen BCMA (B-cell maturation antigen). BCMA is a transmembrane glycoprotein of the tumor necrosis factor receptor superfamily 17 (TNFRSF17 or CD269) that is expressed at significantly higher levels in multiple myeloma cells of all patients, but not in other normal tissues except normal plasma cells. In some embodiments of the BCMA targeting CAR, the antigen recognition region is an scFV that specifically binds to the extracellular domain of CD269. In one embodiment, the scFV comprises a heavy chain (V) represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NOs: 33, 35, and 37. H ) and a light chain (V) represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NOs: 34, 36, and 38. L In one embodiment of a BCMA scFV for a CAR construct, the scFV comprises a V represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO:33 and SEQ ID NO:34, respectively, or to SEQ ID NO:35 and SEQ ID NO:36, or to SEQ ID NO:37 and SEQ ID NO:38, respectively. H and V LIn one embodiment of a MICA / B scFV, the scFV is represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to any one of SEQ ID NOs: 39, 40, 41, 42, 43, or 44. Another aspect herein provides genetically engineered iPSCs and derivative cells thereof, wherein the cells comprise an exogenous polynucleotide encoding at least a BCMA-CAR. In some embodiments, the iPSC-derived effector cells comprising an exogenous polynucleotide encoding at least a BCMA-CAR are T cells. In some other embodiments, the iPSC-derived effector cells comprising an exogenous polynucleotide encoding at least a BCMA-CAR are NK cells. In some other embodiments, the iPSC-derived effector cells comprising an exogenous polynucleotide encoding at least a BCMA-CAR are NKT cells. In some other embodiments, iPSC-derived effector cells comprising an exogenous polynucleotide encoding at least a BCMA-CAR possess functional or structural characteristics that are absent or atypical of T, NK, or NKT cells or any other immune cells of natural origin. In one example, the present specification provides a CAR that comprises an antigen recognition region that targets the tumor antigen BCMA. SEQ ID NO: 33 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVRQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS (BCMA scFV heavy chain-1 (VH)) SEQ ID NO: 34 EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELK (BCMA scFv light chain-1 (VL)) SEQ ID NO: 35 QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQKFTGRVTMTRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSS (BCMA scFV heavy chain-2 (VH)) SEQ ID NO: 36 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCSQSSIYPWTFGQGTKLEIK (BCMA scFv light chain-2 (VL)) SEQ ID NO: 37 QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQKFTGRVTMTRDTSSSTAYMELSSLRSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSS (BCMA scFV heavy chain-3 (VH)) SEQ ID NO: 38 DIVMTQTPLSLSVTPGEPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGADFTLKISRVEAEDVGVYYCAETSHVPWTFGQGTKLEIK (BCMA scFv light chain-3 (VL)) SEQ ID NO: 39 JPEG2026041849000001.jpg34164 (BCMA scFV-1; VH-linker-VL; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible) SEQ ID NO: 40 JPEG2026041849000002.jpg34163 (BCMA scFV-2; VL-linker-VH; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible) SEQ ID NO: 41 JPEG2026041849000003.jpg34166 (BCMA scFV-3; VH-linker-VL; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible) SEQ ID NO: 42 JPEG2026041849000004.jpg35166 (BCMA scFV-4; VL-linker-VH; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible) SEQ ID NO: 43 JPEG2026041849000005.jpg35164 (BCMA scFV-5; VH-linker-VL; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible) SEQ ID NO: 44 JPEG2026041849000006.jpg34163 (BCMA scFV-6; VL-linker-VH; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible)

[0106] In another aspect herein, engineered iPSCs and their derivative cells comprising at least an exogenous polynucleotide encoding a BCMA-CAR further comprise additional CAR cells that target an antigen other than BCMA. In some embodiments, the additional CAR targets the tumor antigens MICA and MICB (MICA / B). In some embodiments of a MICA / B-targeting CAR, the antigen recognition region is an scFV that specifically binds to the conserved α3 ​​domain of MICA and MICB. In one embodiment, the scFV comprises a heavy chain variable region represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO:45, and a light chain variable region represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO:46. In one embodiment of the MICA / B scFV, the scFV is represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 47. In another embodiment of the MICA / B scFV, the scFV is represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 48. SEQ ID NO: 45 QIQLVQSGPELKKPGETVKVSCKASGYMFTNYAMNWVKQAPEKGLKWMGWINTHTGDPTYADDFKGRIAFSLETSASTAYLQINNLKNEDTATYFCVRTYGNYAMDYWGQGTSVTVSS (118AA.MICA / B scFV heavy chain (HC)) SEQ ID NO: 46 DIQMTQTTSSLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVKLLIYDTSILHLGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKFPRTFGGGTTLEIK (107AA.MICA / B scFV light chain (LC)) SEQ ID NO: 47 JPEG2026041849000007.jpg19165 (MICA / B scFV; HC-linker-LC; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible) SEQ ID NO: 48 JPEG2026041849000008.jpg19164 (MICA / B scFV; LC-linker-HC; signal peptide / leader—other signal peptides are possible; linker—other linkers are possible)

[0107] As shown herein, targeting the MICA / B tumor antigen with the provided MICA / B-CARs inhibits surface MICA / B shedding observed in many human and mouse tumor cell lines, resulting in increased MICA / B cell surface density, a decrease in soluble shed MICA / B, and enhanced NK cell-mediated tumor killing. The provided MICA / B-CARs, which can target and stabilize tumor cell surface MICA / B, do not interfere with the binding of NKG2D to tumor MICA and MICB.

[0108] Non-limiting examples of antigens that may be targeted by additional CAR(s) other than MICA / B contained 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, CDS, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), and the like. epidermal cell adhesion molecule (EpCAM), epidermal growth factor receptor (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), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A 1 (MAGE-A1), mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal 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.In one embodiment of iPSCs comprising a BCMA-CAR and derived effector cells therefrom, the cells further comprise a MICA / B-CAR. In another embodiment of iPSCs comprising a BCMA-CAR and derived effector cells therefrom, the cells further comprise a CD19-CAR. In yet another embodiment of iPSCs comprising a BCMA-CAR and derived effector cells therefrom, the cells further comprise a HER2 CAR. In yet another embodiment of iPSCs comprising a BCMA-CAR and derived effector cells therefrom, the cells further comprise an MSLN CAR. In a further embodiment of iPSCs comprising a BCMA-CAR and derived effector cells therefrom, the cells also comprise a PSMA CAR. In yet another embodiment of iPSCs comprising a BCMA-CAR and derived effector cells therefrom, the cells also comprise a VEGF-R2 CAR.

[0109] In some embodiments, the transmembrane domain of the CAR comprises the full length or at least a portion of a native or modified transmembrane region of CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T-cell receptor polypeptide. In one embodiment, the BCMA-CAR and / or additional CAR (target antigen other than BCMA) comprises a transmembrane domain derived from CD28. In one embodiment, the BCMA-CAR and / or additional CAR comprises a transmembrane domain derived from NKG2D.

[0110] In some embodiments, the signaling domain of the endodomain (or intracellular domain) comprises the full length or at least a portion of a polypeptide of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the signaling peptide of a CAR disclosed herein comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to at least one ITAM (immunoreceptor tyrosine-based activation motif) of CD3ζ.

[0111] In certain embodiments, the endodomain further comprises at least one costimulatory signaling region, which may comprise the full length or at least a portion of a polynucleotide of CD27, CD28, 4-1BB, OX40, ICOS, PD1, LAG3, 2B4, BTLA, DAP10, DAP12, CTLA4, or NKG2D, or any combination thereof.

[0112] In one embodiment, the BCMA-CAR provided in the present application comprises a signaling domain comprising native or modified ITAM1 of CD3ζ and is represented by an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 13. In a further embodiment, the CAR comprising a costimulatory domain derived from CD28 and native or modified ITAM1 of CD3ζ also comprises a hinge domain derived from CD28 and a transmembrane domain, wherein the scFv may be connected to the transmembrane domain via the hinge, and the CAR comprises an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 14. SEQ ID NO: 13 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGE RRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (153 a.a. CD28 costimulation + CD3ζ ITAM) SEQ ID NO: 14 TIFF2026041849000009.tif21164 (219 a.a. CD28 hinge + TM of CD28 + CD28 costimulation + CD3ζ ITAM)

[0113] In another embodiment, the BCMA-CAR provided in the present application includes a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain including natural or modified CD3ζ, and is represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 15. The CAR including a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain including natural or modified CD3ζ may further include a CD8 hinge, and the amino acid sequence of such a structure has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 16. SEQ ID NO: 15 JPEG2026041849000010.jpg26164 (263 a.a NKG2D TM + 2B4 + CD3ζ) SEQ ID NO: 16 JPEG2026041849000011.jpg31165 (308 a.a CD8 hinge + NKG2D TM + 2B4 + CD3ζ)

[0114] Non-limiting CAR strategies include heterodimeric, conditionally activated CARs through dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9,587,020), split CARs, which are homologous recombination of antigen-binding, hinge, and endodomains to generate a CAR (see, e.g., U.S. Patent Publication No. 2017 / 0183407), multi-chain CARs that allow for a non-covalent link between two transmembrane domains connected to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Patent Publication No. 2014 / 0134142), CARs with bispecific antigen-binding domains (see, e.g., U.S. Patent No. 9,447,194), or with a pair of antigen-binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Patent No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin Invest. 2016;126(8):3036-3052), inducible CARs (see, e.g., U.S. Patent Publication Nos. 2016 / 0046700, 2016 / 0058857, 2017 / 0166877), switchable CARs (see, e.g., U.S. Patent Publication No. 2014 / 0219975), and other designs known in the art.

[0115] Genomic loci suitable for insertion of a BCMA CAR and / or additional CARs (target antigens other than BCMA) include loci that meet the genomic safe harbor criteria provided herein and loci where gene knockdown or knockout at the selected locus is desired as a result of recombination. In some embodiments, suitable genomic loci for BCMA CAR insertion include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0116] In one embodiment, iPSCs and their derived cells comprising BCMA-CARs have a CAR inserted into the TCR constant region, resulting in TCR knockout, and optionally placing CAR expression under the control of an endogenous TCR promoter. In one specific embodiment of iPSC-derived cells comprising TCR null and BCMA CARs, the derived cells are T cells. In another embodiment, iPSCs and their derived cells comprising a CAR have a CAR inserted into the NKG2A locus 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 specific embodiment of iPSC-derived cells comprising NKG2A or NKG2D null and BCMA CARs, the derived cells are NK cells. In a further embodiment, iPSCs and their derived cells comprising BCMA-CARs 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, iPSCs and their derived cells comprising a BCMA-CAR have a CAR inserted into the CD58 coding region, resulting in CD58 knockout. In one embodiment, iPSCs and their derived cells comprising a BCMA-CAR have a CAR inserted into the CD54 coding region, resulting in CD54 knockout. In one embodiment, iPSCs and their derived cells comprising a BCMA-CAR have a CAR inserted into the CIS (cytokine-inducible SH2-containing protein) coding region, resulting in CIS knockout. In one embodiment, iPSCs and their derived cells comprising a BCMA-CAR have a CAR inserted into the CBL-B (E3 ubiquitin protein ligase CBL-B) coding region, resulting in CBL-B knockout. In one embodiment, iPSCs and their derived cells comprising a BCMA-CAR have a CAR inserted into the SOCS2 (E3 ubiquitin protein ligase CBL-B) coding region, resulting in SOCS2 knockout. In one embodiment, iPSCs and their derived cells comprising a BCMA-CAR have a CAR inserted into the CD56 (NCAM1) coding region.In another embodiment, iPSCs and derived cells comprising a BCMA-CAR have the CAR inserted into the coding region of any one of PD1, CTLA4, LAG3, and TIM3, resulting in gene knockout at the insertion site. In a further embodiment, iPSCs and derived cells comprising a BCMA-CAR have the CAR inserted into the coding region of TIGIT, resulting in TIGIT knockout.

[0117] Thus, provided herein are derivative cells obtained from differentiating, genomically engineered iPSCs, wherein both the iPSCs and the derivative cells comprise at least a BCMA-CAR. Also provided are iPSCs and derivative cells that comprise a BCMA-CAR and one or more additional modification modalities, including, but not limited to, a second CAR specific for a target other than BCMA; CD38 knockout; hnCD16; exogenous cytokine signaling components; HLA-I and / or HLA-II deficiency with overexpression of at least one of HLA-G, CD58, and CD54; TCR null; surface presenting CD3; antigen-specific TCR; NKG2C; DAP10 / 12; NKG2C-IL15-CD33 ("2C1533"), as further described herein.

[0118] 2.CD38 knockout The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated cancer cell depletion typically results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). In addition to ADCC, immune effector mechanisms that cooperate with therapeutic antibodies may also include phagocytosis (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0119] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells and NK cells, as well as activated T and B cells. During hematopoiesis, CD38 is expressed in association with CD34 + It is expressed on stem cells and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages during the final stages of maturation, which continue through 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 mediates the synthesis of NAD + CD38 catalyzes the synthesis and hydrolysis of ribosomal ATP to ADP-ribose, thereby producing the second messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which are important in the process of cell adhesion (this process is calcium-dependent). As a receptor, CD38 recognizes CD31 and regulates cytokine release and cytotoxicity in activated NK cells. CD38 associates with cell surface proteins in lipid rafts and mediates the release of cytosolic Ca. 2+ It has also been reported to regulate bundles and mediate signaling in lymphoid and myeloid cells.

[0120] In cancer treatment, the systemic use of T cells transduced with the CD38 antigen-binding receptor has been shown to lyse the CD38+ fraction of CD34+ hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells, resulting in incomplete therapeutic responses and reduced or eliminated efficacy due to impaired recipient immune effector cell function. Additionally, in multiple myeloma patients treated with the CD38-specific antibody daratumumab, a reduction in NK cells was observed in both bone marrow and peripheral blood, whereas 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, the CD38-null effector cells provided by BCMA-CARs can overcome CD38-mediated fratricide and avoid effector cell depletion or reduction induced by certain antibodies and / or CD38 antigen-binding domains. Additionally, because CD38 is upregulated on 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 provided CD38-null, adaptive allogeneic effector cells, thereby reducing and / or preventing allorejection of these effector cells by host lymphocytes and increasing the survival and persistence of these effector cells despite the presence of CD38 antibodies used for lymphodepletion. Thus, the present application also provides strategies for reducing or preventing allorejection by using CD38-specific antibodies, secreted CD38-specific engagers, or CD38 CARs (chimeric antigen receptors) that inhibit the activation of and / or eliminate activated recipient T and B cells while enhancing the persistence and / or survival of effector cells. Specifically, the strategies provided include generating iPSC lines with BCMA-CAR and CD38 knockout and producing clonal cell banks of iPSCs, as well as generating BCMA-CAR-expressing and CD38-null iPSCs (BCMA-CAR CD38 - / -) derived effector cells by inducing differentiation of engineered iPSC lines. Prior to this application, it was unclear whether editing in iPSCs, including BCMA-CAR and / or CD38 knockout, would disrupt aspects including iPSC differentiation, derived cell phenotype, and effector cell function, given that CD38 plays many important roles in cell developmental biology and cell function, as discussed above.

[0121] In one embodiment provided herein, the CD38 knockout in the iPSC line is a biallelic knockout. As disclosed herein, the provided CD38-null iPSC line can be induced to differentiate to produce functional derived hematopoietic effector cells. In some embodiments, the derived hematopoietic effector cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, the derived hematopoietic effector cells have functional or structural characteristics that are absent or atypical of T, NK, or NKT cells, or any other immune cells from natural sources. In some embodiments, when CD38 antibodies are used to induce ADCC or CD38 CARs are used for targeted cell killing, CD38 - / -The iPSCs and / or their derived effector cells are not eliminated by the CD38 antibody or CD38 CAR, thereby increasing the persistence and / or viability of the iPSCs and their effector cells in the presence and / or after exposure to such therapeutic agents. In some embodiments, the effector cells have increased in vivo persistence and / or viability in the presence and / or after exposure to such therapeutic agents. In some embodiments, the CD38 null effector cells are NK cells derived from the iPSCs. In some embodiments, the CD38 null effector cells are T cells derived from the iPSCs. In some embodiments, the CD38 null iPSCs and derived cells comprise one or more additional genome edits described herein, including, but not limited to, expression of exogenous CD16 or a variant thereof, CAR expression, cytokine / cytokine receptor expression, HLA I and / or HLA II knockout, and additional modalities provided.

[0122] In another embodiment, inserting one or more transgenes, including a BCMA-CAR provided herein, into a selected location in CD38 while simultaneously knocking out CD38 can be achieved, for example, by a CD38-targeted knock-in / knock-out (CD38-KI / KO) construct (FIGS. 2A-D). In some embodiments of the construct, the construct comprises a pair of CD38-targeting homology arms for site-selective insertion within the CD38 locus. In some embodiments, the preselected targeting site is within an exon of CD38. The CD38-KI / KO constructs provided herein allow the transgene(s) to be expressed either under the CD38 endogenous promoter or under an exogenous promoter included in the construct. When two or more transgenes are inserted into a selected location in 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 for the production of distinct proteins from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter included in the CD38-KI / KO construct can be CAG or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, including, but not limited to, CMV, EF1α, PGK, and UBC. Figures 3 and 4 show exemplary sequences of constructs designed to knock out CD38 expression by inserting hnCD16 and IL15RF (in this particular example, truncated IL15RF) at selected locations in the CD38 locus, driven by the CAG promoter (Figure 3) or the CD38 endogenous promoter (Figure 4).As presented in the figures and understood by one of skill in the art, some of the components included in the constructs shown in Figures 3 and 4 are not essential as they are optional, and the nucleic acid sequences for some of the included components may vary and may have less than about 95%, 90%, 85%, 80%, 75%, 70%, but more than 50% sequence identity to the exemplary nucleic acid sequence of each component or the entire construct presented in the figures. In one embodiment, BCMA-CAR was inserted into the CD38 locus to simultaneously knock out CD38 in iPSCs. Thus, the present invention further provides iPSCs and derived cells therefrom comprising BCMA-CAR and CD38 knockout.

[0123] 3. Exogenously introduced CD16 or its variants CD16 has been identified as two isoforms of the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and facilitating antibody-dependent cellular cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high-affinity CD16," "uncleavable CD16," or "high-affinity uncleavable CD16" refer to various CD16 variants. Wild-type CD16 has low affinity and undergoes ectodomain shedding, a proteolytic cleavage process that regulates 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 an exemplary genetically engineered, non-cleavable version of CD16. Engineered CD16 variants containing both F176V and S197P have high affinity and are non-cleavable, as described in more detail in WO2015 / 148926, the full disclosure of which is incorporated herein by reference. In addition, a chimeric CD16 receptor in which the ectodomain of CD16 is essentially replaced with at least a portion of the CD64 ectodomain can also achieve the desired high affinity and non-cleavable characteristics of a CD16 receptor capable of performing ADCC. In some embodiments, the replaced ectodomain of the chimeric CD16 comprises one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoforms or polymorphic variants).

[0124] Thus, in some embodiments, the high-affinity non-cleavable CD16 receptor (hnCD16) comprises both F176V and S197P, and in some embodiments, F176V and the cleavage region is eliminated. In some other embodiments, hnCD16 comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, identity when compared to any of the exemplary sequences SEQ ID NOS: 7, 8, and 9, each of which comprises at least a portion of the CD64 ectodomain. SEQ ID NOS: 7, 8, and 9 are encoded by exemplary sequences SEQ ID NOS: 10-12, respectively. As used herein and throughout this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using an art-recognized mathematical algorithm. SEQ ID NO:7: JPEG2026041849000012.jpg29160 (340 aa CD64 domain-based construct; CD16TM; CD16ICD) SEQ ID NO:8 JPEG2026041849000013.jpg29159 (336 aa CD64 exon-based construct; CD16TM; CD16ICD) SEQ ID NO:9 JPEG2026041849000014.jpg30157 (335 aa CD64 exon-based construct; CD16TM; CD16ICD) SEQ ID NO: 10 cttggagaca acatgtggtt cttgacaact ctgctccttt gggttccagt tgatgggcaa gtggacacca caaaggcagt gatcactttg cagcctccat gggtcagcgt gttccaagag gaaaccgtaa ccttgcattg tgaggtgctc catctgcctg ggagcagctc tacacagtgg tttctcaatg gcacagccac tcagacctcg acccccagct acagaatcac ctctgccagt gtcaatgaca gtggtgaata caggtgccag agaggtctct cagggcgaag tgaccccata cagctggaaa tccacagagg ctggctacta ctgcaggtct ccagcagagt cttcacggaa ggagaacctc tggccttgag gtgtcatgcg tggaaggata agctggtgta caatgtgctt tactatcgaa atggcaaagc ctttaagttt ttccactgga attctaacct caccattctg aaaaccaaca taagtcacaa tggcacctac cattgctcag gcatgggaaa gcatcgctac acatcagcag gaatatctgt cactgtgaaa gagctatttc cagctccagt gctgaatgca tctgtgacat ccccactcct ggaggggaat ctggtcaccc tgagctgtga aacaaagttg ctcttgcaga ggcctggttt gcagctttac ttctccttct acatgggcag caagaccctg cgaggcagga acacatcctc tgaataccaa atactaactg ctagaagaga agactctggg ttatactggt gcgaggctgc cacagaggat ggaaatgtcc ttaagcgcag ccctgagttg gagcttcaag tgcttggcct ccagttacca actcctgtct ggtttcatta ccaagtctct ttctgcttgg tgatggtact cctttttgca gtggacacag gactatattt ctctgtgaag acaaacattcgaagctcaac aagagactgg aaggaccata aatttaaatg gagaaaggac cctcaagaca aa Sequence number 11 cttggagaca acatgtggtt cttgacaact ctgctccttt gggttccagt tgatgggcaa gtggacacca caaaggcagt gatcactttg cagcctccat gggtcagcgt gttccaagag gaaaccgtaa ccttgcattg tgaggtgctc catctgcctg ggagcagctc tacacagtgg tttctcaatg gcacagccac tcagacctcg acccccagct acagaatcac ctctgccagt gtcaatgaca gtggtgaata caggtgccag agaggtctct cagggcgaag tgaccccata cagctggaaa tccacagagg ctggctacta ctgcaggtct ccagcagagt cttcacggaa ggagaacctc tggccttgag gtgtcatgcg tggaaggata agctggtgta caatgtgctt tactatcgaa atggcaaagc ctttaagttt ttccactgga attctaacct caccattctg aaaaccaaca taagtcacaa tggcacctac cattgctcag gcatgggaaa gcatcgctac acatcagcag gaatatctgt cactgtgaaa gagctatttc cagctccagt gctgaatgca tctgtgacat ccccactcct ggaggggaat ctggtcaccc tgagctgtga aacaaagttg ctcttgcaga ggcctggttt gcagctttac ttctccttct acatgggcag caagaccctg cgaggcagga acacatcctc tgaataccaa atactaactg ctagaagaga agactctggg ttatactggt gcgaggctgc cacagaggat ggaaatgtcc ttaagcgcag ccctgagttg gagcttcaag tgcttggttt gttctttcca cctgggtacc aagtctcttt ctgcttggtg atggtactcc tttttgcagt ggacacagga ctatatttct ctgtgaagac aaacattcga agctcaacaa gagactggaa ggaccataaa tttaaatgga gaaaggaccc tcaagacaaa sequence number 12 atgtggttct tgacaactct gctcctttgg gttccagttg atgggcaagt ggacaccaca aaggcagtga tcactttgca gcctccatgg gtcagcgtgt tccaagagga aaccgtaacc ttgcactgtg aggtgctcca tctgcctggg agcagctcta cacagtggtt tctcaatggc acagccactc agacctcgac ccccagctac agaatcacct ctgccagtgt caatgacagt ggtgaataca ggtgccagag aggtctctca gggcgaagtg accccataca gctggaaatc cacagaggct ggctactact gcaggtctcc agcagagtct tcacggaagg agaacctctg gccttgaggt gtcatgcgtg gaaggataag ctggtgtaca atgtgcttta ctatcgaaat ggcaaagcct ttaagttttt ccactggaac tctaacctca ccattctgaa aaccaacata agtcacaatg gcacctacca ttgctcaggc atgggaaagc atcgctacac atcagcagga atatctgtca ctgtgaaaga gctatttcca gctccagtgc tgaatgcatc tgtgacatcc ccactcctgg aggggaatct ggtcaccctg agctgtgaaa caaagttgct cttgcagagg cctggttgc agctttactt ctccttctac atgggcagca agaccctgcg aggcaggaac acatcctctg aataccaaat actaactgct agaagagaag actctgggtt atactggtgc gaggctgcca cagaggatgg aaatgtcctt aagcgcagcc ctgagttgga gcttcaagtg cttggcttct ttccacctgg gtaccaagtc tctttctgct tggtgatggt actcctttt gcagtggaca caggactata tttctgtg aagacaaaca ttcgaagctc aacaagagac tggaaggacc ataaatttaa atggagaaag gaccctcaag acaaa

[0125] Thus, among other editing techniques contemplated and described herein, provided herein are clonal iPSCs engineered to contain exogenous CD16 or a variant thereof, which can differentiate into effector cells containing the same exogenous CD16 introduced into the iPSCs. In some embodiments, the exogenous CD16 is a high-affinity, non-cleavable CD16 receptor (hnCD16). The exogenous hnCD16 expressed in iPSCs or their derivatives exhibits high affinity for binding not only to ADCC antibodies or fragments thereof, but also to bispecific, trispecific, or multispecific engagers or binders that recognize the CD16 or CD64 extracellular binding domains of the hnCD16. Bispecific, trispecific, or multispecific engagers or binders are further described below in this application (see Section I.7). Thus, the present application provides derived effector cells, or populations thereof, pre-loaded with one or more pre-selected ADCC antibodies via high affinity binding to the extracellular domain of hnCD16 expressed on the derived effector cells, in an amount sufficient for therapeutic use in the treatment of conditions, diseases, or infections as further detailed in Section V below, wherein the hnCD16 comprises the extracellular binding domain of CD64 or of CD16 having F176V and S197P.

[0126] In some other embodiments, the native CD16 transmembrane and / or intracellular domains of exogenous CD16 are further modified or replaced to produce chimeric Fc receptors (CFcRs) that contain a non-native transmembrane domain, a non-native stimulatory domain, and / or a non-native signaling domain. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain, or signaling domain is derived from a different receptor than the receptor that provides the extracellular domain. In the present illustration, a CFcR based on CD16 or a variant thereof does not have a transmembrane domain, stimulatory domain, or signaling domain derived from CD16. In some embodiments, the CD16-based CFcR comprises a non-native transmembrane domain derived from a CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, T cell receptor polypeptide. In some embodiments, the CD16-based CFcR comprises a non-native stimulatory / inhibitory domain derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD1, LAG3, 2B4, BTLA, DAP10, DAP12, CTLA4, or NKG2D polypeptide. In some embodiments, the CD16-based CFcR comprises a non-native signaling domain derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of exogenous CD16, a chimeric receptor is provided comprising a transmembrane domain and a signaling domain, both derived from one of IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides.One specific embodiment of the CD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, wherein the extracellular domain of the CD16-based chimeric Fc receptor is derived from the full-length or partial extracellular domain of CD64 or CD16, wherein the extracellular domain of CD16 comprises F176V and S197P. Another embodiment of the CD16-based chimeric Fc receptor comprises the transmembrane domain and signaling domain of CD3ζ, wherein the extracellular domain of the CD16-based chimeric Fc receptor is derived from the full-length or partial extracellular domain of CD64 or CD16, wherein the extracellular domain of CD16 comprises F176V and S197P.

[0127] Various embodiments of the CD16-based chimeric Fc receptor described above can bind with high affinity to the Fc region of an antibody or fragment thereof, or to the Fc region of a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory and / or signaling domains of the chimeric receptor enable effector cell activation and cytokine secretion, as well as killing of tumor cells targeted by the antibody or bispecific, trispecific, or multispecific engager or binder having a tumor antigen-binding component and an Fc region. Without being limited by theory, through the non-native transmembrane, stimulatory, and / or signaling domains of the CD16-based chimeric Fc receptor, or through binding of the engager to the ectodomain, the CFcR contributes to the killing ability of the effector cell and increases the proliferation and / or growth potential of the effector cell. Antibodies and engagers can bring antigen-expressing tumor cells and CFcR-expressing effector cells into close proximity, also contributing to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, 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, trispecific, multispecific engagers or binders suitable for binding effector cells expressing CD16-based CFcRs in attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0128] Unlike endogenous CD16 expressed by primary NK cells, which is cleaved from the cell surface after NK cell activation, the various non-cleavable versions of CD16 in derived NK cells avoid CD16 shedding and maintain constant expression. In derived NK lineage cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicating improved cell function. Non-cleavable CD16 also enhances antibody-dependent cellular cytotoxicity (ADCC) and the binding of bispecific, trispecific, or multispecific engagers. ADCC is an NK cell-mediated lysis mechanism via CD16 binding to antibody-coated target cells. The additional high-affinity feature of the introduced hnCD16 in derived NK cells also enables in vitro loading of ADCC antibodies via hnCD16 prior to administering the cells to subjects requiring cell therapy. As provided, hnCD16, in some embodiments, may comprise F176V and S197P, or may comprise a complete or partial ectodomain derived from CD64, as exemplified by SEQ ID NO: 7, 8, or 9, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides derived effector cells, or cell populations thereof, pre-loaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed below. In some embodiments, the derived effector cells comprising hnCD16 further comprise a BCMA-CAR provided herein. In some embodiments, the derived effector cells comprising a BCMA-CAR, hnCD16, and a CD38 knockout further comprise a CD38 knockout. In some embodiments, the derived effector cells comprising a BCMA-CAR, hnCD16, and a CD38 knockout are pre-loaded with a CD38 antibody. In some embodiments, the pre-loaded CD38 antibody is daratumumab.

[0129] Unlike primary NK cells, mature T cells from primary sources (i.e., native / 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 CD16 could differentiate into functional derived T lineage cells that not only express exogenous CD16 but also can perform functions via an adaptive CDCC mechanism without compromising T cell developmental biology. This adaptive ADCC in derived T lineage cells can further be used as an approach for dual targeting and / or to rescue antigen escape, which often occurs with CAR-T cell therapy, where tumors recur with reduced or lost expression of the antigen targeted by the CAR-T or with mutated antigens that evade recognition by the CAR (chimeric antigen receptor). When the derived T lineage cells have adaptive 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 recurrence of the targeted tumor, which is common with CAR-T therapy. Such a strategy of achieving dual targeting while reducing and / or preventing antigen escape applies equally to NK lineage cells or any engineered effector cells expressing one or more CARs. Various CARs that can be used in this antigen escape reduction and prevention strategy are described in more detail below.

[0130] Thus, the present invention provides derived T lineage cells comprising exogenous CD16. In one embodiment, the derived T lineage cells obtained herein comprise a BCMA-CAR and exogenous CD16. In further provided embodiments, the derived T lineage cells obtained herein comprise a CD38 knockout in addition to expression of hnCD16 and a BCMA-CAR. In some embodiments, the hnCD16 contained in the derived T lineage cells comprises F176V and S197P. In some other embodiments, the hnCD16 contained in the derived T lineage cells comprises a complete or partial ectodomain derived from CD64, as exemplified by SEQ ID NO: 7, 8, or 9, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As described, such derived T lineage cells possess an adaptive mechanism for targeting tumors with monoclonal antibodies medicated by ADCC to enhance the therapeutic effect of the antibody. As disclosed, the present application also provides derived T lineage cells or cell populations thereof pre-loaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in treating a condition, disease, or infection, as further detailed below. In some other embodiments, the derived T lineage cells expressing hnCD16 and BCMA CARs are also CD38 null, such that the cells can avoid elimination in the presence of a therapeutic agent targeting the tumor antigen CD38. In one embodiment, the therapeutic agent targeting the tumor antigen CD38 is a CD38 antibody. In another embodiment, the therapeutic agent targeting the tumor antigen CD38 is a CAR comprising a CD38 binding region, e.g., an anti-CD38 scFV.

[0131] 4. Exogenously Introduced Cytokines and / or Cytokine Signaling Avoiding systemic administration of high doses of clinically relevant cytokines reduces the risk of dose-limiting toxicity associated with such practice while establishing cytokine-mediated cell autonomy. To achieve lymphocyte autonomy without the need for additional soluble cytokine administration, polynucleotides encoding protein complexes containing partial or full-length peptides of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, partial or full-length peptides of one or more of their receptors, and any combination thereof, are introduced into cells to enable cytokine signaling, thereby maintaining or improving cell growth, proliferation, expansion, and / or effector function while reducing the risk of cytokine toxicity. 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 temporary.

[0132] Figure 1 presents several construct designs using IL15 as an illustrative example. The transmembrane (TM) domain of any of the designs in Figure 1 may be native to the IL15 receptor or may be modified with or replaced by the transmembrane domain of any other membrane-bound protein.

[0133] Design 1: IL15 and IL15Rα are co-expressed by using a self-cleaving peptide, mimicking the trans-presentation of IL15 without eliminating the cis-presentation of IL15.

[0134] Design 2: IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without eliminating cis-presentation of IL15 and ensuring membrane binding of IL15.

[0135] Design 3: IL15Rα with a truncated intracellular domain is fused to IL15 at its C-terminus via a linker, mimicking IL15 trans-presentation, maintaining IL15 membrane binding, and eliminating cis-presentation and / or any other possible signaling pathways mediated by normal IL15R via its intracellular domain. The intracellular domain of IL15Rα is believed to be important for the receptor to be expressed in IL15-responsive cells and for the expansion and function of responding cells. Such truncated constructs comprise an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, which can be encoded by the exemplary nucleic acid sequence represented by SEQ ID NO: 18. In one embodiment of a truncated IL15 / IL15Rα, the construct does not include the last four amino acids "KSRQ" of SEQ ID NO: 17 and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 21. SEQ ID NO: 17 JPEG2026041849000015.jpg36163 (379 aa; signal and linker peptides are underlined) SEQ ID NO: 18 ATGGACTGGACCTGGATTCTGTTCCTGGTCGCGGCTGCAACGCGAGGTCCATAGCGGTATC CATGTTTTTATTCTTGGGTGTTTTTCTGCTGGGCTGCCTAAGACCGAGGCCAACTGGGTA AATGTCATCAGTGACCTCAAGAAAATAGAAGACCTTATACAAAGCATGCACATTGATGCT ACTCTCTACACTGAGTCAGATGTACATCCCTCATGCAAAGTGACGGCCATGAAATGTTTC CTCCTCGAACTTCAAGTCATATCTCTGGAAAGTGGCGACGCGTCCATCCACGACACGGTC GAAAACCTGATAATACTCGCTAATAATAGTCTCTCTTCAAATGGTAACGTAACCGAGTCA GGTTGCAAAGAGTGCGAAGAGTTGGAAGAAAAAAACATAAAGGAGTTCCTGCAAAGTTTC GTGCACATTGTGCAGATGTTCATTAATACCTCTAGCGGCGGAGGATCAGGTGGCGGTGGA AGCGGAGGTGGAGGCTCCGGTGGAGGAGGTAGTGGCGGAGGTTCTCTTCAAATAACTTGT CCTCCACCGATGTCCGTAGAACATGCGGATATTTGGGTAAAATCCTATAGCTTGTACAGC CGAGAGCGGTATATCTGCAACAGCGGCTTCAAGCGGAAGGCCGGCACAAGCAGCCTGACC GAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTAGCCTGAAGTGC ATCAGAGATCCCGCCCTGGTGCATCAGCGGCCTGCCCCTCCAAGCACAGTGACAACAGCT GGCGTGACCCCCCAGCCTGAGAGCCTGAGCCCTTCTGGAAAAGAGCCTGCCGCCAGCAGC CCCAGCAGCAACAATACTGCCGCCACCACAGCCGCCATCGTGCCTGGATCTCAGCTGATG CCCAGCAAGAGCCCTAGCACCGGCACCACCGAGATCAGCAGCCACGAGTCTAGCCACGGC ACCCCATCTCAGACCACCGCCAAGAACTGGGAGCTGACAGCCAGCGCCTCTCACCAGCCT CCAGGCGTGTACCCTCAGGGCCACAGCGATACCACAGTGGCCATCAGCACCTCCACCGTG CTGCTGTGTGGACTGAGCGCCGTGTCACTGCTGGCCTGCTACCTGAAGTCCAGACAGTGA (1140 n.a.) Sequence No. 21 JPEG2026041849000016.jpg36162 (375 aa; signal and linker peptides are underlined)

[0136] Those skilled in the art will understand that the above signal peptide and linker sequences are exemplary and in no way limit the variations thereof suitable for use as signal peptides or linkers. Many suitable signal peptide or linker sequences are known 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.

[0137] Design 4: Because the constructs in Design 3 were shown to function in promoting effector cell survival and expansion, and because the cytoplasmic domain of IL15Rα could be omitted without adversely affecting the effector cell-autonomous features of the IL15 signaling complex, Design 4 offers another practical alternative to Design 3. Essentially, the entire IL15Rα is removed, except for the Sushi domain, which is fused to IL15 on one end and a transmembrane domain on the other (mb-Sushi), with an optional linker between the Sushi and transmembrane domains. 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 through IL15Rα, including cis-presentation, is eliminated while only the desired trans-presentation of IL15 is retained. In some embodiments, the component comprising IL15 fused to a Sushi domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 19, which may be encoded by the exemplary nucleic acid sequence represented by SEQ ID NO: 20. SEQ ID NO: 19 JPEG2026041849000017.jpg26162 (242 aa; signal and linker peptides are underlined) SEQ ID NO: 20 ATGGACTGGACCTGGATTCTGTTCCTGGTCGCGGCTGCAACGCGAGGTCCATAGCGGTATC CATGTTTTTATTCTTGGGTGTTTTTCTGCTGGGCTGCCTAAGACCGAGGCCAACTGGGTA AATGTCATCAGTGACCTCAAGAAAATAGAAGACCTTATACAAAGCATGCACATTGATGCT ACTCTCTACACTGAGTCAGATGTACATCCCTCATGCAAAGTGACGGCCATGAAATGTTTC CTCCTCGAACTTCAAGTCATATCTCTGGAAAGTGGCGACGCGTCCATCCACGACACGGTC GAAAACCTGATAATACTCGCTAATAATAGTCTCTCTTCAAATGGTAACGTAACCGAGTCA GGTTGCAAAGAGTGCGAAGAGTTGGAAGAAAAAAACATAAAGGAGTTCCTGCAAAGTTTC GTGCACATTGTGCAGATGTTCATTAATACCTCTAGCGGCGGAGGATCAGGTGGCGGTGGA AGCGGAGGTGGAGGCTCCGGTGGAGGAGGTAGTGGCGGAGGTTCTCTTCAAATAACTTGT CCTCCACCGATGTCCGTAGAACATGCGGATATTTGGGTAAAATCCTATAGCTTGTACAGC CGAGAGCGGTATATCTGCAACAGCGGCTTCAAGCGGAAGGCCGGCACAAGCAGCCTGACC GAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTAGCCTGAAGTGC ATCAGA (726 na)

[0138] Those skilled in the art will understand that the above signal peptide and linker sequences are exemplary and in no way limit the variations thereof suitable for use as signal peptides or linkers. Many suitable signal peptide or linker sequences are known 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.

[0139] Design 5: Native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker, allowing constitutive signaling and maintaining membrane-bound and trans-expression of IL15.

[0140] Design 6: 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 the cytokine. Common receptor γC is also known as common gamma chain or CD132, and 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, IL2, IL4, IL7, IL9, IL15, and IL21 receptors.

[0141] Design 7: An engineered IL15Rβ that forms homodimers in the absence of IL15 is useful for producing constitutive cytokine signaling.

[0142] In some embodiments, one or more of the cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or their receptors, can be introduced into iPSCs and their derivative cells upon iPSC differentiation using one or more of the designs in Figure 1. In some embodiments, IL2 or IL15 cell surface expression and signaling is via the constructs illustrated in any one of Designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling is via the constructs illustrated in Designs 5, 6, or 7, using either a common receptor or a cytokine-specific receptor (such as the IL4 receptor). The transmembrane (TM) domains of any of the designs in Figure 1 may be native to the respective cytokine receptor or may be modified or replaced with the transmembrane domain of any other membrane-bound protein.

[0143] In iPSCs and derived cells containing both CAR and exogenous cytokine and / or cytokine receptor signaling, the CAR and IL may be expressed in separate constructs or co-expressed in a bicistronic construct containing both the CAR and IL, where the IL can be any cytokine, cytokine receptor, variant thereof, or combination thereof. In some further embodiments, IL15 in a form represented by any of the construct designs in Figure 1 may be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence, for example, as illustrated as CAR-2A-IL15 or IL15-2A-CAR. Thus, the IL15 and CAR are in a single open reading frame (ORF). In one embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises the IL15 of Design 3 in Figure 1. In another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises the IL15 of Design 3 in Figure 1. In yet another embodiment, the CAR-2A-IL15 or IL15-2A-CAR construct comprises IL15 of design 7 in Figure 1. Upon expression of CAR-2A-IL15 or IL15-2A-CAR, the self-cleaving 2A peptide dissociates the expressed CAR and IL15, and the dissociated IL15 is presented on the cell surface. The CAR-2A-IL15 or IL15-2A-CAR bicistronic design allows for coordinated expression of CAR and IL15 in both timing and amount, and under the same regulatory mechanism, which may be selected, for example, to incorporate an inducible promoter for 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 teschovirus-1 (PTV-I) (Donnelly, ML, et al., J. Gen. Virol., 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732 (2001)), as well as members of the cardioviruses such as theiloviruses (e.g., Theiler's murine encephalomyelitis virus) and encephalomyocarditis virus.The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are also referred to as "F2A," "E2A," "P2A," and "T2A," respectively.

[0144] The bicistronic CAR-2A-IL15 or IL15-2A-CAR embodiments disclosed herein for IL15 are also applicable to the expression of any other cytokine provided herein, e.g., IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, IL2 cell surface expression and signaling is via a construct depicted in any of Designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling is via a construct depicted in Designs 5, 6, or 7, using either a common receptor and / or cytokine-specific receptors.

[0145] 5. HLA-I and HLA-II deficiency To avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins must be matched for histocompatibility with the allogeneic recipient. Provided herein are iPSC cell lines and their differentiated derivatives in which expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced. 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 reduced expression levels of HLA class I-associated 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 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-associated 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, iPSC lines and their derivatives that are both HLA-I and HLA-II deficient due to the lack of both B2M and CIITA expression are provided herein, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoiding recognition and killing by host (allogeneic) T cells.

[0146] However, in some cell types, lack of class I expression leads to lysis by NK cells. To overcome this "loss of self" response, HLA-G can optionally be knocked in to prevent NK cell recognition and killing of HLA-I-deficient effector cells derived from engineered iPSCs. In one embodiment, the provided HLA-I-deficient iPSCs and their derived cells further comprise an HLA-G knockin. Alternatively, in some embodiments, the provided HLA-I-deficient iPSCs and their derived cells further comprise one or both of a CD58 knockout and a CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and facilitate the migration of cells, including immune cells. Prior to the present invention, it was unknown whether and how disruption of CD58 and / or CD54 in iPSCs would affect pluripotency and developmental biology in iPSC differentiation toward functional immune effector cells, including T cells and NK cells. It was also previously unclear whether CD58 and / or CD54 knockout could effectively and / or sufficiently reduce the susceptibility of effector cells derived from HLA-I-deficient iPSCs to allogeneic NK cell killing. Here, we show that CD58 knockout is more efficient at reducing allogeneic NK cell activation than CD54 knockout, while double knockout of both CD58 and CD54 most effectively reduces NK cell activation. Some observations suggest that CD58 and CD54 double knockout is even more effective than HLA-G overexpression in HLA-I-deficient cells in overcoming the "loss of self" effect.

[0147] 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 yet some other embodiments, HLA-I and HLA-II-deficient iPSCs and their derived cells are CD54-null and CD54-null. Furthermore, in some embodiments of iPSCs and their derived cells comprising a BCMA-CAR, the cells are HLA-I and HLA-II-deficient and have an exogenous polynucleotide encoding HLA-G. In some embodiments of iPSCs and their derived cells comprising a BCMA-CAR, the cells are HLA-I and HLA-II-deficient and CD58-null. In some embodiments of iPSCs and derived cells thereof comprising a BCMA-CAR, the cells are HLA-I and HLA-II deficient and CD54 null. In yet some other embodiments of iPSCs and derived cells thereof comprising a BCMA-CAR, the cells are HLA-I and HLA-II deficient and both CD58 null and CD54 null.

[0148] 6. Genetically engineered iPSC lines and derived cells provided herein In light of the above, the present application provides iPSCs, iPS cell line cells, or populations thereof, and derivative functional effector cells obtained by differentiating the iPSCs, each comprising a BCMA-CAR. In some embodiments, the application provides iPSCs, iPS cell line cells, or populations thereof, and derivative effector cells obtained by differentiating the iPSCs, each comprising at least an exogenous polynucleotide encoding a BCMA-CAR. In some embodiments, the functional derivative cells are hematopoietic cells. In some embodiments, functional derivative cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell precursors, NK cell precursors, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, functional derivative hematopoietic cells include effector cells such as T cells, NK cells, and regulatory cells, hi some embodiments, functional derivative hematopoietic cells include effector cells that have functional or structural characteristics that are not present in or typical of naturally occurring T, NK, or NKT cells, or any other immune cells.

[0149] CD38 - / - iPSCs, iPS cell line cells, or populations thereof, and CD38 (also referred to herein as "CD38 null" or CD38 knockout) - / - Also provided herein are induced functional derivative cells comprising a CD38 knockout obtained from differentiation of iPSCs. In some embodiments, CD38 - / -The iPSCs, iPS cell line cells, or populations thereof, and the derived functional derivative cells further comprise an exogenous polynucleotide encoding a BCMA-CAR. In some embodiments, the polynucleotide encoding the BCMA-CAR is in the CD38 locus. In some embodiments, the functional derivative cells comprising a BCMA-CAR and a CD38 knockout are hematopoietic cells. In some embodiments, the functional derivative cells comprising a BCMA-CAR and a CD38 knockout are mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell precursors, NK cell precursors, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, the functional derivative hematopoietic cells include effector cells such as T cells, NK cells, and regulatory cells. In some embodiments, functional derivative hematopoietic cells are effector cells with enhanced functional or structural characteristics that are not present in or typical of T, NK, or NKT cells, or any other immune cells, from natural sources.

[0150] Further provided herein are iPSCs comprising a polynucleotide encoding a BCMA-CAR and a polynucleotide encoding high-affinity, non-cleavable CD16 (hnCD16), wherein the iPSCs are capable of directed differentiation to produce functional hematopoietic cells. Cells comprising both a BCMA-CAR and hnCD16 are suitable for dual targeting by CAR binding and CD16-mediated ADCC, enhancing the precision of tumor targeting, facilitating tumor killing, and minimizing the effects of tumor antigen escape. Furthermore, in some embodiments, iPSCs and / or their derived effector cells comprising a BCMA-CAR and hnCD16 are also CD38 null, such that when a CD38 antibody is used to induce hnCD16-mediated enhanced ADCC, iPSCs and / or their derived effector cells comprising a CD38 knockout, a MICA / B-CAR, and hnCD16 can target CD38-expressing (tumor) cells without causing effector cell elimination, i.e., reduction or depletion of CD38-expressing effector cells, thereby increasing the persistence and / or viability of iPSCs and their effector cells. iPSC-derived effector cells comprising a BCMA-CAR, CD38 null, and exogenous CD16 experience reduced cell depletion in the presence of a CD38 antibody or CD38 CAR, providing multiple mechanisms for tumor killing.

[0151] Provided herein are iPSCs comprising a polynucleotide encoding a BCMA-CAR and a polynucleotide encoding a second chimeric antigen receptor (CAR) with target specificity other than BCMA, which can be differentiated to generate functional derived effector cells bearing two CARs targeting two different tumor antigens. In one embodiment, the second CAR contained in the iPSC and its derivative effector cells comprising the BCMA-CAR target tumor cell surface proteins CD19, MICA / B, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1. In one embodiment, the iPSCs and / or their derived effector cells have a second CAR targeting CD38, and the cells are also CD38 null. Therefore, the CD38-CAR cannot eliminate the iPSCs and / or their derived effector cells by CD38-mediated fratricide. In some embodiments, the CAR contained in iPSCs and their derived effector cells comprising a CD38 knockout does not target CD38.

[0152] Further, the iPSC line comprises a polynucleotide encoding a BCMA-CAR and a protein complex comprising at least one exogenous cytokine and / or cytokine receptor (IL) or variant thereof that enables cytokine signaling that contributes to cell viability, persistence, and / or proliferation, and can be induced to differentiate to produce functional derived hematopoietic cells with improved viability, persistence, proliferation, and effector cell function. The exogenously introduced cytokine signaling comprises signaling for any one or two or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments, the introduced partial or complete peptide of the cytokine and / or its respective receptor for cytokine signaling is expressed on the cell surface. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary. In some embodiments, the transient / transient expression of cell surface cytokines / cytokine receptors is via retrovirus, Sendai virus, adenovirus, episomes, minicircles, or RNA, including mRNA. In some embodiments, the exogenous cell surface cytokine and / or receptor comprised in the BCMA-CAR iPSCs or their derived cells allows for IL7 signaling. In some embodiments, the exogenous cell surface cytokine and / or receptor comprised in the BCMA-CAR iPSCs or their derived cells allows for IL10 signaling. In some embodiments, the exogenous cell surface cytokine and / or receptor comprised in the BCMA-CAR iPSCs or their derived cells allows for IL15 signaling. In some embodiments of the BCMA-CAR IL iPSCs, IL15 expression is via construct 3 of Figure 1. In some embodiments of the BCMA-CAR IL iPSCs, IL15 expression is via construct 4 of Figure 1.The BCMA-CAR IL iPSCs and their derived cells of the above-described embodiments are able to autonomously maintain or improve cell growth, proliferation, proliferation, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo. In some embodiments of the BCMA-CAR IL iPSCs and their derived effector cells, the cells are CD38 null and can be used with CD38 antibodies to induce ADCC without causing effector cell elimination, thereby synergistically increasing the persistence and / or viability of the iPSCs and their effector cells.

[0153] Also provided are iPSCs comprising a BCMA-CAR, a B2M knockout, and a CIITA knockout, and optionally one of HLA-G overexpression, a CD58 knockout, and a CD54 knockout, wherein the iPSCs are capable of producing functional derivative hematopoietic cells upon directed differentiation. - / - CIITA - / - The iPSCs and their derived effector cells are deficient in both HLA-I and HLA-II. In further embodiments, the HLA-I and HLA-II deficient BCMA-CAR iPSCs and their derived effector cells are also CD38 null and can be used with a CD38 antibody to induce ADCC without causing effector cell elimination, thereby increasing the persistence and / or survival of the iPSCs and their effector cells. In some embodiments, the effector cells have increased in vivo persistence and / or survival.

[0154] In view of the above, provided herein are iPSCs comprising a BCMA-CAR and optionally one, two, three, or more of CD38 knockout, hnCD16, a second CAR, an exogenous cytokine / receptor, and a B2M / CIITA knockout, wherein if B2M is knocked out, a polynucleotide encoding HLA-G or at least one of CD58 and CD54 knockouts is optionally introduced, and the iPSCs can be induced to differentiate to produce functional derived hematopoietic cells. Also included in the present application are functional iPSC-derived hematopoietic cells comprising a BCMA-CAR and optionally one, two, three, or more of CD38 knockout, exogenous CD16, a B2M / CIITA knockout, a second CAR, and an exogenous cytokine / receptor protein complex, wherein if B2M is knocked out, a polynucleotide encoding HLA-G or at least one of CD58 and CD54 knockouts is optionally introduced. In some embodiments, derived hematopoietic cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some other embodiments, derived hematopoietic cells are effector cells with functional or structural characteristics that are absent or atypical of naturally occurring NK, T, or NKT cells, or any other immune cells.

[0155] Another aspect provided herein includes iPSCs or iPSC-derived cells comprising a truncated fusion protein of IL15 and IL15Rα, wherein the fusion protein does not comprise an intracellular domain. As shown in FIG. 1 as "IL15Rα(ΔICD) fusion" and "IL5 / mb-Sushi," these embodiments are further collectively abbreviated as IL15Δ throughout the application and are one of the "IL" embodiments shown in Table 1. In some "IL" embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21. In some "IL" embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 17. In some "IL" embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments of "IL," the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments of iPSCs or iPSC-derived cells comprising a truncated IL15 / IL15Rα fusion protein lacking the intracellular domain (IL15Δ), the cells further comprise a BCMA-CAR, and optionally one or more of a CD38 knockout, hnCD16, a second CAR, an exogenous cytokine / receptor, and a B2M / CIITA knockout, where if B2M is knocked out, a polynucleotide encoding HLA-G, or one of a CD58 and CD54 knockout is optionally introduced, and the iPSCs can be induced to differentiate to produce functional derivative hematopoietic cells. In some embodiments, derived hematopoietic cells include, but are not limited to, mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.In some other embodiments, the derived hematopoietic cells are effector cells that have functional or structural characteristics that are not present in or typical of naturally occurring NK, T, or NKT cells, or any other immune cells.

[0156] Thus, the present application provides iPSCs and their functionally derived hematopoietic cells, comprising any one of the following genotypes in Table 1. (第2) " represents a CAR with a different target specificity than a BCMA-CAR, and non-limiting examples include CARs targeting at least one of CD19, MICA / B, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1. As provided in Table 1, "IL" represents one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which specific cytokine / receptor expression is selected. Additionally, "IL" also encompasses the embodiment of IL15Δ, which is detailed above as a truncated fusion protein of IL15 and IL15Rα, but does not include the intracellular domain. Furthermore, when iPSCs and their functionally derived hematopoietic cells have a genotype that includes both a CAR (BCMA-CAR, or a second CAR) and an IL, in one embodiment of the cells, the CAR and IL are contained in a bicistronic expression cassette that includes a 2A sequence. By comparison, in some other embodiments, the CAR and IL are contained in separate expression cassettes contained in the iPSCs and their functionally derived hematopoietic cells. In a specific embodiment, the iPSCs and their functionally derived effector cells that express both a CAR and an IL include IL15 in construct 3 or 4 of Figure 1, where the IL15 construct is contained in an expression cassette together with or separate from the CAR. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0157] 7. Additional Modifications In some embodiments, iPSCs and their derived effector cells comprising any one of the genotypes in Table 1 may further comprise a deletion or reduced expression of 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 The present invention may further comprise introduced or increased expression in at least one of a surface triggering receptor for binding to an R, an antigen-specific TCR, an Fc receptor, a checkpoint inhibitor, an antibody or functional fragment or variant thereof, an engager, and a bispecific, multispecific, or universal engager.

[0158] Bispecific or multispecific engagers are fusion proteins consisting of 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 binds to tumor cells via a tumor-specific surface molecule. Exemplary effector cell surface molecules or surface triggering receptors that can be used for bispecific or multispecific engager recognition or coupling 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 comprises the CD16 (including F176V and optionally S197P) or CD64 extracellular domain, as well as a native or non-native transmembrane domain, stimulatory domain, and / or signaling domain, as described in Section I.2. In some embodiments, the CD16 expressed on the surface of effector cells for engager recognition is a CD16-based chimeric Fc receptor (CFcR). In some embodiments, the CD16-based CFcR comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, and the extracellular domain of hnCD16 is derived from the full-length or partial extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and optionally S197P. Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. In one embodiment, the bispecific antibody is CD3-CD19.In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In another embodiment, the bispecific antibody is CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody is CD3-CD33. In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and tumor cell antigen-binding domains, e.g., modified IL15 (referred to in some publications as TriKE, or trispecific killer engager) as a linker for effector NK cells to 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").

[0159] In some embodiments, the surface triggering receptor for a bispecific or multispecific engager can be endogenous to the effector cell, sometimes depending on the cell type. In other embodiments, the methods and compositions provided herein can be used to further manipulate iPSCs containing the genotypes listed in Table 1, to induce differentiation of iPSCs into T cells, NK cells, or any other effector cells containing the same genotype and surface triggering receptor as the source iPSCs, and to introduce one or more exogenous surface triggering receptors into the effector cells.

[0160] 8. Antibodies for immunotherapy In some embodiments, in addition to the genomically engineered effector cells provided herein, additional therapeutic agents, including antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication, can be used with these effector cells in combination therapy. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, tumor- or virus-specific antigens activate the administered iPSC-derived effector cells, enhancing their killing capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include, but are not limited to, CD20 antibodies (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), HER2 antibodies (trastuzumab, pertuzumab), 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 humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof. In some embodiments, the iPSC-derived effector cells comprise hematopoietic lineage cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise NK cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise T cells comprising a genotype listed in Table 1.

[0161] In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises a preselected monoclonal antibody and iPSC-derived NK cells or T cells comprising at least a BCMA-CAR. In other embodiments of combinations useful for treating liquid or solid tumors, the combination comprises a preselected monoclonal antibody and iPSC-derived NK cells or T cells comprising at least a BCMA-CAR and hnCD16. In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises iPSC-derived NK cells or T cells comprising at least a BCMA-CAR, CD38 null, and a CD38 antibody. In one embodiment, the combination comprises iPSC-derived NK cells comprising a BCMA-CAR, CD38 null, and hnCD16, and one of the CD38 antibodies daratumumab, isatuximab, or MOR202. In one embodiment, the combination comprises iPSC-derived NK cells comprising a BCMA-CAR, CD38 null, and hnCD16, and daratumumab. In some further embodiments, the iPSC-derived NK cells included in combination with daratumumab comprise a BCMA-CAR, CD38 null, hnCD16, IL15, and a CAR targeting at least one of CD38 or CD19, MICA / B, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1, where IL15 is co-expressed or separately expressed with the CAR, and IL15 is in any one of the forms presented in constructs 1-7 in Figure 1. In some specific embodiments, IL15, when expressed simultaneously with or separately from the CAR, is in the form of construct 3, 4, or 7.

[0162] 9. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that, if uninhibited, can suppress or downregulate immune responses. It is now clear that tumors exploit certain immune checkpoint pathways as a primary mechanism of immune resistance, particularly to tumor antigen-specific T cells. Checkpoint inhibitors (CIs) are antagonists that reduce checkpoint gene expression or gene products or decrease the activity of checkpoint molecules, thereby blocking the inhibitory checkpoint and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, and these agents have led to long-term remissions in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a significant concern. One aspect of the present application provides a therapeutic approach to overcoming CI resistance by including genomically engineered functional derivative cells to be provided in combination with CIs. In one embodiment of the combination therapy, the derivative cells are NK cells. In another embodiment of the combination therapy, the derivative cells are T cells. In addition to exhibiting direct antitumor capabilities, the derived NK cells provided herein have been shown to have the ability to resist PDL1-PD1-mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at tumor sites. Thus, the tumor infiltration of T cells promoted by functionally potent, genome-engineered derived NK cells indicates that the NK cells can synergize with T cell-targeted immunotherapies, including checkpoint inhibitors, to alleviate local immunosuppression and reduce tumor burden.

[0163] In one embodiment, the derived NK cells for checkpoint inhibitor combination therapy comprise a BCMA-CAR, and optionally one, two, three, or more of CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and exogenous cell surface cytokine and / or receptor expression, where if B2M is knocked out, optionally at least one of a polynucleotide encoding HLA-G, or a knockout of CD58 or CD54 is included. In some embodiments, the derived NK cells comprise any one of the genotypes listed in Table 1. In some embodiments, the aforementioned derived NK cells further comprise deleted or reduced expression of 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, checkpoint inhibitor, antibody or functional fragment or variant thereof, engager, and further comprising introduced or increased expression in at least one of a surface triggering receptor for binding with a bispecific, multispecific or universal engager.

[0164] In another embodiment, the derived T cells for checkpoint inhibitor combination therapy comprise a BCMA-CAR, and optionally one, two, three, or more of CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and exogenous cell surface cytokine and / or receptor expression, where if B2M is knocked out, optionally one of a polynucleotide encoding HLA-G, or a knockout of CD58 or CD54 is included. In some embodiments, the derived T cells comprise any one of the genotypes listed in Table 1. In some embodiments, the derivative T cells described above further comprise deleted or reduced expression of 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, checkpoint inhibitor, antibody or functional fragment or variant thereof, Fc receptor, engager, and further comprising introduced or increased expression of a surface triggering receptor for binding to a bispecific, multispecific, or universal engager.

[0165] The aforementioned derived NK cells or derived T cells are obtained from differentiating an iPSC clonal line comprising a BCMA-CAR and optionally one, two, three or all four of CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and exogenous cell surface cytokine expression, wherein if B2M is knocked out, a polynucleotide encoding HLA-G, or at least one of knockout of CD58 and CD54 is optionally introduced. In some embodiments, the iPSC clonal line described above further comprises deleted or reduced expression of 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, checkpoint inhibitor, antibody or functional fragment or variant thereof, engager, and further comprising introduced or increased expression in at least one of a surface triggering receptor for binding with a bispecific, multispecific or universal engager.

[0166] 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, CD73 (NT5E), , CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2) antagonists.

[0167] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of whole antibodies and may be more cost-effective to produce, easier to use, or more sensitive than whole antibodies. In some embodiments, the one, two, or three or more checkpoint inhibitors comprise 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), lilimumab (KIR antibody), monalizumab (NKG2A antibody), nivolumab (PD1 mAb), pembrolizumab (PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0168] In some embodiments, antagonists that inhibit any of the above checkpoint molecules are microRNA-based, as many miRNAs are found to be regulators of immune checkpoint expression (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.

[0169] Some embodiments of the provided combination therapies with derived NK cells or derived T cells include at least one checkpoint inhibitor that targets at least one checkpoint molecule, wherein the derivative cells have a genotype listed in Table 1. Some other embodiments of the provided combination therapies with derived NK cells or T cells include two, three, or more checkpoint inhibitors, such that two, three, or more checkpoint molecules are targeted. In some embodiments of the combination therapies including at least one checkpoint inhibitor and derivative cells having a genotype listed in Table 1, 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 derivative cells by expressing an exogenous polynucleotide sequence encoding the antibody, or fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the checkpoint-inhibiting antibody, or fragment or variant thereof, is co-expressed with the CAR, either in a separate construct or in a bicistronic construct including both the CAR and sequences encoding the antibody or fragment thereof. In some further embodiments, the sequence encoding the antibody or fragment thereof can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence, depicted as, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences for the checkpoint inhibitor and CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered, expressed, and secreted as a payload by derived effector cells capable of infiltrating the tumor microenvironment (TME), binding to the TME counteracts inhibitory checkpoint molecules, allowing effector cell activation by activating modalities such as CARs or activating receptors.In some embodiments, the checkpoint inhibitor co-expressed with the CAR inhibits at least one of the 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 KIR. In some embodiments, the checkpoint inhibitor co-expressed with the CAR in the derivative cells having a genotype listed in Table 1 is selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is atezolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is nivolumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0170] In some other embodiments of the combination therapies comprising the derivative cells provided herein and at least one antibody that inhibits a checkpoint molecule, the antibody is not produced by or within the derivative cells and is further administered before, concurrently with, or after administration of the derivative cells having a genotype listed in Table 1. In some embodiments, the administration of one, two, three or more checkpoint inhibitors in the combination therapy with the provided derived NK cells or T cells is simultaneous or sequential. In one embodiment of a combination therapy comprising derived NK cells or T cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments of combination therapies comprising derived effector cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is atezolizumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising derived effector cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is nivolumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof. In some embodiments of combination therapies comprising derived effector cells having a genotype listed in Table 1, the checkpoint inhibitor included in the treatment is pembrolizumab, or a humanized or Fc-modified variant, fragment, and functional equivalent or biosimilar thereof.

[0171] II. Methods for targeted genome editing at selected loci in iPSCs Genome editing, or genomic editing, or gene editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and / or replaced in the genome of a targeted cell. Targeted genome editing (interchangeably referred to as "targeted genomic editing" or "targeted gene editing") allows for insertion, deletion, and / or replacement at preselected sites within the genome. When endogenous sequences are deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or down due to the deletion of the sequence. Therefore, targeted editing can also be used to precisely disrupt the expression of endogenous genes. The term "targeted integration" is similarly used herein to refer to a process involving the insertion of one or more exogenous sequences, with or without deletion of endogenous sequences at the insertion site. In comparison, randomly integrated genes are subject to position effects and silencing, and their expression is unreliable and unpredictable. For example, centromeric and subtelomeric regions are particularly susceptible to transgene silencing. Reciprocally, newly integrated genes can affect surrounding endogenous genes and chromatin, potentially altering cellular behavior or supporting cellular transformation. Therefore, inserting exogenous DNA into preselected loci, such as safe harbor loci or genomic safe harbors (GSH), is important for safety, efficiency, copy number control, and reliable gene response control. Alternatively, exogenous DNA can be inserted into preselected loci where disruption of gene expression, including knockdown and knockout, is intended.

[0172] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is induced by the host cell's enzymatic machinery via homologous sequences flanking the exogenous polynucleotide to be inserted.

[0173] Alternatively, targeted editing can be achieved at higher frequencies 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 genetic material, NHEJ often results in the random insertion or deletion (in / del) of a small number of endogenous nucleotides. In contrast, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homologous arms, the exogenous genetic material can be introduced into the genome during homology-directed repair (HDR) via homologous recombination, resulting in "targeted integration." In some cases, the targeted integration site is intended to be within the coding region of a selected gene, allowing targeted integration to disrupt gene expression and simultaneously result in knock-in and knock-out (KI / KO) in a single editing step.

[0174] Inserting one or more transgenes into a selected location of a locus of interest (GOI) and simultaneously knocking out the genes can be achieved by the construct designs illustrated in Figures 2A-D, using the CD38 locus as an example. Other loci suitable for simultaneous knock-in and knock-out (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. With their respective CD38-targeting homology arms for site-selective insertion, the constructs provided herein allow transgene(s) to be expressed under the CD38 endogenous or exogenous promoters included in the construct (compare Figure 2, A vs. B, and C vs. D). The selected insertion / knockout position within the CD38 locus is compatible with the sequences of the adjacent left and right homology arms (LHA / CD38 and RHA / CD38) included in the construct. LHA / CD38 and RHA / CD38 can have various lengths and sequences depending on the preselected targeting site within the CD38 locus. In some embodiments, the preselected targeting site is within a CD38 exon. When two or more transgenes are inserted into the selected position of the CD38 locus, a linker sequence, such as a 2A linker or IRES, is placed between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-I, and TaV (also referred to as "F2A," "E2A," "P2A," and "T2A," respectively), allowing for the production of distinct proteins from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter can be CAG or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, including, but not limited to, CMV, EF1α, PGK, and UBC.

[0175] 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 regularly interspaced palindromic repeats) systems. Additionally, the DICE (dual integrase cassette exchange) system, which utilizes phiC31 and Bxb1 integrases, is also a promising tool for targeted integration.

[0176] ZFN is a targeted nuclease containing a nuclease fused to a zinc finger DNA binding domain. "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 the zinc finger binding domain, and its structure is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A "designed" zinc finger domain is a domain that does not exist in nature, whose design / composition is primarily based on rational criteria, such as 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. Patent Nos. 6,140,081, 6,453,242, and 6,534,261, and also WO98 / 53058, WO98 / 53059, WO98 / 53060, WO02 / 016536, and WO03 / 016496. A "selected" zinc finger domain is a domain not found in nature, and its production primarily results from empirical processes such as phage display, interaction trapping, or hybrid selection. ZFNs are described in more detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of a FokI nuclease with a zinc finger DNA binding domain.

[0177] TALENs are targeted nucleases containing a nuclease fused to a TAL effector DNA-binding domain. "Transcription activator-like effector DNA-binding domain," "TAL effector DNA-binding domain," or "TALE DNA-binding domain" refers to the polypeptide domain of a TAL effector protein involved in binding the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA-binding domain, and activate gene transcription at these sequences via their transactivation domain. The specificity of the TAL effector DNA-binding domain depends on the effector's variable number of imperfect 34-amino acid repeats, which contain polymorphisms at select repeat positions called repeat variable dinucleotides (RVDs). TALENs are described in more detail in U.S. Patent Application Publication No. 2011 / 0145940, incorporated herein by reference. The most recognized example of a TALEN in the art is a polypeptide fusion of FokI nuclease to a TAL effector DNA binding domain.

[0178] Another example of a targeted nuclease that finds use in the subject methods is a targeted Spo11 nuclease, which is a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain having specificity for a DNA sequence of interest, e.g., a zinc finger DNA-binding domain, a TAL effector DNA-binding domain, etc. See, e.g., U.S. Patent Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0179] 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.

[0180] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases; and the like.

[0181] Using Cas9 as an example, CRISPR / Cas9 requires two key components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When coexpressed, the two components form a complex and are recruited to a target DNA sequence containing a PAM and a seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target a selected sequence. These two components can be delivered into mammalian cells via transfection or transduction.

[0182] DICE-mediated insertion uses a pair of recombinases, e.g., phiC31 and Bxb1, to provide unidirectional integration of exogenous DNA, strictly limited by each enzyme's own small attB and attP recognition sites. These target att sites do not naturally occur in mammalian genomes and must therefore first be introduced into the genome at the desired integration site. See, e.g., U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0183] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method for targeted integration comprises introducing the construct into a cell to allow site-specific homologous recombination by the cellular host enzyme machinery. In another embodiment, the method for targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a ZFN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow ZFN-mediated insertion. In yet another embodiment, the method for targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a TALEN expression cassette comprising a DNA binding domain specific to the desired integration site into the cell to allow TALEN-mediated insertion. In another embodiment, the method for targeted integration 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 the desired integration site into the cell to allow Cas9-mediated insertion. In yet another embodiment, a method for targeted integration in a cell comprises introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for the DICE recombinase to enable targeted integration via DICE.

[0184] Promising sites for targeted integration include, but are not limited to, safe harbor loci or genomic safe harbors (GSHs), which are intragenic or extragenic regions of the human genome that, theoretically, can accommodate predictable expression of the newly integrated DNA without adversely affecting the host cell or organism. A useful safe harbor must allow sufficient transgene expression to obtain the desired levels of vector-encoded protein or non-coding RNA. The safe harbor must also not predispose cells to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it should ideally meet several criteria, including but not limited to: no disruption of regulatory elements or genes as determined by sequence annotation; an intergenic region within a gene-dense region or a convergent position between two genes transcribed in opposite directions; distances between vector-encoded transcriptional activators and promoters of neighboring genes, particularly cancer-associated and microRNA genes, to minimize the possibility of long-range interactions; and apparent ubiquitous transcriptional activity, as reflected by widespread spatial and temporal expressed sequence tag (EST) expression patterns indicative of ubiquitous transcriptional activity. This latter feature 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 a region suitable for exogenous insertion, the precise locus selected for insertion should lack repetitive elements and conserved sequences, allowing for easy design of primers for homologous arm amplification.

[0185] Suitable sites for human genome editing, or specifically targeted integration, include, but are not limited to, the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the human ortholog of the mouse ROSA26 locus. Additionally, the human ortholog of the mouse H11 locus may also be suitable for insertion using the compositions and targeted integration methods disclosed herein. Furthermore, the 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 sequence and placement, and construct design, is often required.

[0186] In the case of targeted in / dels, the editing site is often contained in the endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted in / del is associated with the regulation and adjustment of immune response. In some other embodiments, the endogenous gene containing the targeted in / del is associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, the regulation and adjustment of immune response, or proteins that suppress the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells and their derived cells.

[0187] Thus, one aspect of the present invention provides methods for targeted integration at selected loci that include a genomic safe harbor, preselected loci known or proven to be safe and well-regulated for continuous or transient gene expression, such as AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, or other loci that meet the criteria of a genomic safe harbor. In some embodiments, the targeted integration is at one of the loci where gene knockdown or knockout is desired as a result of integration, including, but not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0188] In one embodiment, a method for targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing a construct comprising a pair of homologous arms specific for a desired integration site and one or more exogenous sequences to enable site-specific homologous recombination by the cellular host enzyme machinery, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0189] In another embodiment, a method for targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, to allow for ZFN-mediated insertion. In yet another embodiment, a method for targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site to enable TALEN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In another embodiment, a method for targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a gRNA comprising a Cas9 expression cassette and a guide sequence specific for a desired integration site to allow Cas9-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.In yet another embodiment, a method for targeted integration in a cell includes introducing a construct comprising one or more att sites for a pair of DICE recombinases into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable targeted integration via DICE, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0190] Furthermore, as provided herein, the above-described methods for targeted integration under safe harbor can be used to insert any polynucleotide of interest, such as polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and proteins that promote stem and / or progenitor cell engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival. In some other embodiments, the construct comprising one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the present invention is a suicide gene encoding a safety switch protein. Suitable suicide gene systems for induced cell death include, but are not limited to, caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV); cytosine deaminase (CD) and 5-fluorocytosine (5-FC). In addition, some suicide gene systems are cell type specific; for example, genetic modification of T lymphocytes with the B cell molecule CD20 allows them to be eliminated by administration of the mAb rituximab. Furthermore, modified EGFR-containing epitopes recognized by cetuximab can be used to deplete genetically engineered cells when exposed to cetuximab. Thus, one aspect of the present invention provides a method for targeted integration of one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0191] In some embodiments, one or more exogenous polynucleotides integrated by the methods herein are driven by an operably linked exogenous promoter included in a construct for targeted integration. The promoter may be inducible or constitutive, and may be time-specific, tissue-specific, or cell-type specific. Constitutive promoters suitable for the methods of the present invention include, but are not limited to, the cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0192] The exogenous polynucleotides integrated by the methods herein can be driven at the integration site by an endogenous promoter in the host genome. In one embodiment, the methods of the present invention are used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the methods of the present invention are used for targeted integration at the ROSA26 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the methods of the present invention are used for targeted integration at the H11 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the methods of the present invention are used for targeted integration at the collagen locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the methods of the present invention are used for targeted integration at the HTRP locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous HTRP promoter. In theory, only correct insertion at the desired location will allow gene expression of the exogenous gene driven by the endogenous promoter.

[0193] In some embodiments, one or more exogenous polynucleotides included in a construct for targeted integration methods 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 increase the physical separation between the portions and maximize access to enzymatic machinery. The linker peptide of the linker sequence can be composed of amino acids selected to make the physical separation between the portions (exogenous polynucleotides and / or the proteins or peptides encoded therefrom) more flexible or more rigid depending on the function involved. The linker sequence can be cleavable by a protease or chemically cleavable to generate separate portions. Examples of enzymatic cleavage sites in a linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is naturally produced by the host or is exogenously introduced. Alternatively, the cleavage site in the linker can be cleavable upon exposure to a selected chemical, such as cyanogen bromide, hydroxylamine, or low pH. The optional linker sequence may serve a purpose other than providing a cleavage site. The linker sequence should allow for effective positioning of a moiety relative to another adjacent moiety for proper function. The linker may also be a simple amino acid sequence of sufficient length to prevent steric hindrance between the moieties. In addition, the linker sequence may provide for post-translational modifications, including, but not limited to, phosphorylation sites, biotinylation sites, sulfation sites, gamma-carboxylation sites, etc. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single, undesired 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 contains glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, a G4S linker peptide separates the terminal processing domain and the endonuclease domain of the fusion protein. In other embodiments, a 2A linker sequence allows two separate proteins to be produced from a single translation. Suitable linker sequences can be easily identified empirically. In addition, the suitable size and sequence of the linker sequence can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some embodiments, any two consecutive linker sequences are different.

[0194] Introduction of a construct containing an exogenous polynucleotide into cells for targeted integration can be achieved using known methods for gene transfer into cells. In one embodiment, the construct comprises a viral vector backbone, such as an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a Sendai viral vector. In some embodiments, a plasmid vector is used to deliver and / or express the exogenous polynucleotide into target cells (e.g., pAl-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.). In other embodiments, an episomal vector is used to deliver the exogenous polynucleotide into target cells. In some embodiments, recombinant adeno-associated viruses (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV does not integrate into the host genome. In addition, episomal rAAV vectors mediate homology-directed gene targeting at a much higher rate than traditional targeting plasmid transfection. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions into target sites in the genome of iPSCs. In some embodiments, the genomically modified iPSCs and their derivatives obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0195] III. Methods for Obtaining and Maintaining Genome-Engineered iPSCs The present invention provides methods for obtaining and maintaining genomically engineered iPSCs containing one or more targeted edits at one or more desired sites, wherein the targeted edits remain intact and functional at each selected edit site in expanded genomically engineered iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions, i.e., targeted integrations and / or indels, at selected sites into the genome of iPSCs and derived cells. Compared to directly manipulating primary effector cells derived from patient-derived peripheral blood, the many advantages of obtaining genomically engineered derivative cells by editing and differentiating iPSCs as provided herein include, but are not limited to: an unlimited source of engineered effector cells; no need to repeatedly manipulate effector cells, especially when multiple engineered modalities are involved; the obtained effector cells have elongated telomeres and are younger due to less attrition; and, due primarily to the ability to perform clonal selection on the engineered iPSCs provided herein, the effector cell population is homogeneous in terms of editing site, copy number, and absence of allelic variation, random mutation, and expression diversity.

[0196] In certain embodiments, genomically engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and expanded long-term as single cells in a cell culture medium shown in Table 2 as fate-maintaining medium (FMM), where the iPSCs retain the targeted edits and functional modifications at the selected sites. The composition of the medium may be present in the medium in amounts within the optimal ranges shown in Table 2. iPSCs cultured in FMM have been shown to remain undifferentiated, maintaining a basal or naive profile, maintaining genomic stability without the need to wash or select the culture, and readily give rise to all three somatic lineages, in vitro differentiation via embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation via teratoma formation. See, e.g., U.S. Patent Application No. 61 / 947,979, the disclosure of which is incorporated herein by reference. [Table 2]

[0197] In some embodiments, genomically engineered iPSCs containing one or more targeted integrations and / or in / dels are maintained, passaged, and expanded in medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and free of or essentially free of a TGFβ receptor / ALK5 inhibitor, wherein the iPSCs retain intact and functional targeted edits at the selected sites.

[0198] Another aspect of the present invention provides methods for generating genomically engineered iPSCs through targeted editing of iPSCs, or by first generating genomically engineered non-pluripotent cells through targeted editing and then reprogramming the selected / isolated genomically engineered non-pluripotent cells to obtain iPSCs containing the same targeted edits as the non-pluripotent cells. A further aspect of the present invention provides genomically engineered non-pluripotent cells undergoing simultaneous reprogramming by introducing targeted integrations and / or targeted in / dels into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and the reprogramming conditions include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genomic engineering and reprogramming, the targeted integrations and / or targeted in / dels can be introduced into the non-pluripotent cells prior to or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and, optionally, small molecules.

[0199] In some embodiments, for simultaneous genome manipulation and reprogramming of non-pluripotent cells, targeted integrations and / or in / dels may also be introduced into non-pluripotent cells after the multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and the vector carrying the construct is introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181, and CD30.

[0200] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor, and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 2). In some embodiments, genomically engineered iPSCs by any of the methods described above are further maintained and expanded using a mixture comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 2).

[0201] In some embodiments of the method of generating genomically engineered iPSCs, the method comprises genomically engineering iPSCs by introducing one or more targeted integrations and / or in / dels into the iPSCs to obtain genomically engineered iPSCs having at least one genotype listed in Table 1. Alternatively, the method of generating genomically engineered iPSCs comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genomically engineered non-pluripotent cell comprising a targeted integration and / or in / del at a selected site, and (b) contacting the genomically engineered non-pluripotent cell 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 genomically engineered iPSC comprising a targeted integration and / or in / del at the selected site. Alternatively, a method for generating genomically engineered iPSCs includes: (a) contacting non-pluripotent cells with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, to initiate reprogramming of the non-pluripotent cells; (b) introducing one or more targeted integrations and / or in / dels into the reprogramming non-pluripotent cells for genomic engineering; and (c) obtaining clonal genomically engineered iPSCs comprising the targeted integrations and / or in / dels at selected sites.

[0202] The reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in PCT / US2015 / 018801 and PCT / US16 / 57136 (the disclosures of which are incorporated herein by reference). One or more reprogramming factors may be in the form of a polypeptide. Reprogramming factors may also be in the form of polynucleotides, and are introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced via a combination of plasmids that takes into account the stoichiometry of the various reprogramming factors. See, e.g., U.S. Patent Application No. 62 / 571,105, the disclosure of which is incorporated herein by reference.

[0203] In some embodiments, non-pluripotent cells are transfected with multiple constructs containing different exogenous polynucleotides and / or different promoters via multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides may include genes encoding suicide genes, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, potential drug targets, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNAs, including, but not limited to, siRNAs, shRNAs, miRNAs, 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 promoter, such as in the presence of an inducer, or in specific differentiated cell types. 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, containing different exogenous polynucleotides and / or different promoters, can be introduced into non-pluripotent cells either simultaneously or sequentially. Non-pluripotent cells subjected to targeted integration of multiple constructs can be simultaneously contacted with one or more reprogramming factors to initiate reprogramming simultaneously with genome manipulation, thereby obtaining genomically engineered iPSCs containing multiple targeted integrations in the same pool of cells. This robust method thus enables simultaneous reprogramming and manipulation strategies to lead to clonal genomically engineered hiPSCs with multiple modalities integrated into one or more selected target sites.In some embodiments, the genomically modified iPSCs and derived cells thereof obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0204] IV. Methods for Obtaining Genetically Engineered Effector Cells by Differentiating Genome-Engineered iPSCs Further aspects of the present invention provide methods for in vivo differentiation of genomically engineered iPSCs via teratoma formation, wherein the in vivo differentiated cells derived from the genomically engineered iPSCs retain intact and functional targeted edits, including targeted integrations and / or indels, at desired sites. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma formation 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 genomic safe harbor. In some other embodiments, the in vivo differentiated cells derived from genomically engineered iPSCs via teratoma formation contain polynucleotides encoding targeting modalities or proteins that promote stem and / or progenitor cell trafficking, homing, viability, self-renewal, persistence, and / or survival. In some embodiments, in vivo differentiated cells derived from iPSCs genomically engineered via teratomas containing one or more inducible suicide genes further comprise one or more in / dels in endogenous genes associated with regulating and mediating immune responses. In some embodiments, the in / dels are comprised in one or more endogenous checkpoint genes. In some embodiments, the in / dels are comprised in one or more endogenous T cell receptor genes. In some embodiments, the in / dels are comprised in one or more endogenous MHC class I suppressor genes. In some embodiments, the in / dels are comprised in one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the in / dels are comprised in one or more endogenous genes, including, but not limited to, B2M, PD1, TAP1, TAP2, tapasin, and TCR genes. In one embodiment, the genomically engineered iPSCs containing one or more exogenous polynucleotides at selected sites further comprise targeted editing in a gene encoding B2M (beta-2-microglobulin).

[0205] In certain embodiments, genomically engineered iPSCs containing one or more genetic modifications provided herein are used to direct hematopoietic lineages or any other specific cell type in vitro, and the derived non-pluripotent cells retain the functional genetic modification, including the targeted edit at the selected site. In one embodiment, genomically engineered iPSC-derived cells include, but are not limited to, mesodermal cells with secondary hemogenic endothelial (HE) potential, secondary HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, and these cells derived from genomically engineered iPSCs retain the functional genetic modification, including the targeted edit at the desired site.

[0206] Applicable differentiation methods and compositions for obtaining hematopoietic cell lineages from iPSCs include, for example, those set forth in International Application No. PCT / US2016 / 044122, the disclosure of which is incorporated herein by reference. As provided, methods and compositions for generating hematopoietic cell lineages via secondary hemogenic endothelium (HE) derived from pluripotent stem cells, including hiPSCs, under serum-free, feeder-free, and / or stroma-free conditions, and in a culture platform that is scalable and does not require monolayer EB formation. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated and transdifferentiated cells, as well as cells of various lineages that transition directly to hematopoietic fates without passing through a pluripotent intermediate. Similarly, cells produced by differentiating stem cells range from multipotent stem cells or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0207] A method for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture includes contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from the pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with secondary hemogenic endothelial (HE) potential without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF and, optionally, a ROCK inhibitor and / or a WNT pathway activator causes the mesodermal cells with secondary HE potential to differentiate into secondary HE cells, which are also expanded during differentiation.

[0208] The methods provided herein for obtaining cells of the hematopoietic lineage are superior to EB-mediated pluripotent stem cell differentiation because EB formation results in moderate to minimal cell expansion, monolayer culture, which is important for many applications requiring homogeneous expansion, does not allow for homogeneous differentiation of cells within the population, is difficult, and is less efficient.

[0209] The provided monolayer differentiation platform facilitates differentiation into secondary hemogenic endothelium, resulting in the derivation of hematopoietic stem cells and differentiated progeny such as T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion, enabling the delivery of therapeutically relevant numbers of pluripotent stem cell-derived hematopoietic cells for a variety of therapeutic applications. Furthermore, monolayer culture using the methods provided herein yields functional hematopoietic lineage cells capable of a full range of in vitro differentiation, ex vivo conditioning, and in vivo long-term hematopoietic self-renewal, reconstitution, and engraftment. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, secondary hemogenic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem cells and progenitors, T cell progenitors, NK cell progenitors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0210] A method for directing differentiation of pluripotent stem cells into cells of a secondary hematopoietic lineage, the method comprising: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate the differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor (optionally not including a TGFβ receptor / ALK inhibitor) to initiate the differentiation and expansion of mesodermal cells having secondary HE potential from the mesodermal cells; and (iii) contacting the mesodermal cells having secondary HE potential with a composition comprising one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor; bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator (optionally not including a TGFβ receptor / ALK inhibitor) to initiate the differentiation and expansion of secondary hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having secondary hemogenic endothelium potential.

[0211] In some embodiments, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (the composition does not comprise a TGFβ receptor / ALK inhibitor) to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs comprising one or more genetic imprints, wherein the one or more genetic imprints comprised in the iPSC are retained in hematopoietic cells differentiated therefrom. In some embodiments of the method for directing differentiation of pluripotent stem cells into cells of a hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of a hematopoietic lineage lacks the generation of embryoid bodies and is in a monolayer culture form.

[0212] In some embodiments of the above methods, the obtained pluripotent stem cell-derived secondary hemogenic endothelial cells are CD34+. In some embodiments, the obtained secondary hemogenic endothelial cells are CD34+CD43-. In some embodiments, the secondary hemogenic endothelial cells are CD34+CD43-CXCR4-CD73-. In some embodiments, the secondary hemogenic endothelial cells are CD34+CXCR4-CD73-. In some embodiments, the secondary hemogenic endothelial cells are CD34+CD43-CD93-. In some embodiments, the secondary hemogenic endothelial cells are CD34+CD93-.

[0213] In some embodiments of the above method, the method further includes (i) contacting secondary hemogenic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator to initiate differentiation of the secondary hemogenic endothelium into pre-T cell precursors, and optionally (ii) contacting pre-T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor to initiate differentiation of the pre-T cell precursors into T cell precursors or T cells. In some embodiments of the method, the T cell precursors derived from pluripotent stem cells are CD34+CD45+CD7+. In some embodiments of the method, the T cell precursors derived from pluripotent stem cells are CD45+CD7+.

[0214] In some further embodiments of the above-described methods for inducing differentiation of pluripotent stem cells into cells of a hematopoietic lineage, the method further comprises: (i) contacting secondary hemogenic endothelium derived from the pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor; VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, to initiate differentiation of the secondary hemogenic endothelium into pre-NK cell precursors; and, optionally, (ii) contacting pre-NK cell precursors derived from the pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 (the medium does not contain one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor), to initiate differentiation of the pre-NK cell precursors into NK cell precursors or NK cells. In some embodiments, the NK cell precursors derived from the pluripotent stem cells are CD3-CD45+CD56+CD7+. In some embodiments, pluripotent stem cell-derived NK cells are CD3-CD45+CD56+, and optionally further defined by NKp46+, CD57+, and CD16+.

[0215] Therefore, using the above differentiation method, 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 hemogenic endothelial (iHE) cells using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; and (iii) iMPP-A, iTC-A2, iTC-B2, iNK-A2. (iv) multipotent progenitor cells (iMPP) using iMPP-A, (v) T cell progenitor cells (ipro-T) using one or more culture media selected from iTC-A2 and iTC-B2, (vi) T cells (iTC) using iTC-B2, (vii) NK cell progenitor cells (ipro-NK) using one or more culture media selected from iNK-A2 and iNK-B2, and / or (viii) NK cells (iNK), and iNK-B2. In some embodiments, the culture medium is: a. iCD34-C comprises one or more growth factors and cytokines selected from the group consisting of a ROCK inhibitor, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator, but does not comprise 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 comprises a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7, and optionally a BMP activator; d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; e. iNK-A2 comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15; and f. iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0216] In some embodiments, the genomically engineered iPSC-derived cells resulting from the above methods comprise one or more inducible suicide genes integrated at one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In some other embodiments, the genomically engineered iPSC-derived cells comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote stem and / or progenitor cell trafficking, homing, viability, self-renewal, persistence, and / or survival. In some embodiments, the genomically engineered iPSC-derived cells comprising one or more suicide genes further comprise one or more in / dels in one or more endogenous genes associated with regulating and mediating immune responses, including, but not limited to, checkpoint genes, endogenous T cell receptor genes, and MHC class I inhibitory genes. In one embodiment, the genomically engineered iPSC-derived cells comprising one or more suicide genes further comprise an in / del in the B2M gene, wherein B2M is knocked out.

[0217] In addition, dedifferentiation methods and compositions applicable for obtaining second-fate genomically engineered hematopoietic cells from first-fate genomically engineered hematopoietic cells include, for example, those set forth in International Publication No. WO 2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided therein partially reprogram starting non-pluripotent cells into non-pluripotent intermediate cells by limiting expression of the endogenous Nanog gene during reprogramming, and allow the non-pluripotent intermediate cells to be subjected to conditions for differentiating the intermediate cells into a desired cell type. In some embodiments, the genomically modified iPSCs and their derivatives obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0218] V. Therapeutic Applications of Derived Immune Cells with Exogenous Functional Modalities Differentiated from Genetically Engineered iPSCs In some embodiments, the present invention provides compositions comprising 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 iPSCs contain one or more targeted gene edits that can be retained in the iPSC-derived immune cells, and the engineered iPSCs and their derivative cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises iPSC-derived CD34 cells. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises iPSC-derived proT or T cells. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises iPSC-derived proNK or NK cells. In one embodiment, the isolated population or subpopulation of engineered immune cells comprises iPSC-derived immune regulatory cells or myeloid-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived engineered immune cells are further conditioned ex vivo for improved therapeutic potential. In one embodiment, the isolated population or subpopulation of iPSC-derived engineered immune cells comprises an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of iPSC-derived engineered immune cells comprises an increased number or proportion of type I NKT cells. In another embodiment, the isolated population or subpopulation of iPSC-derived engineered immune cells comprises an increased number or proportion of adaptive NK cells. In some embodiments, the isolated population or subpopulation of iPSC-derived engineered CD34 cells, HSC cells, T cells, NK cells, or myeloid-derived suppressor cells is allogeneic. In some other embodiments, the isolated population or subpopulation of iPSC-derived engineered CD34 cells, HSC cells, T cells, NK cells, NKT cells, or MDSC is autologous.

[0219] In some embodiments, iPSCs for differentiation contain genetic imprints selected to convey desired therapeutic attributes in effector cells, provided that cell developmental biology is not disrupted during differentiation and that the genetic imprints are retained and functional in differentiated hematopoietic cells derived from the iPSCs.

[0220] In some embodiments, the genetic imprints of the pluripotent stem cells comprise (i) one or more genetically modified modalities obtained by genomic insertion, deletion, or substitution in the genome of the pluripotent cells during or after reprogramming of 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 therapeutic response-specific, wherein the pluripotent cells are reprogrammed from source-specific immune cells and the iPSCs retain the therapeutic attributes of the source that are also contained in the iPSC-derived hematopoietic lineage cells.

[0221] In some embodiments, the genetic modification modalities comprise one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, and / or survival of iPSCs or their derived cells. In some embodiments, the genetically modified iPSCs and their derived effector cells comprise a genotype listed in Table 1. In some other embodiments, the genetically modified iPSCs and derived effector cells thereof comprising the genotypes listed in Table 1 further comprise additional genetic modification modalities including: (1) deletion or reduced expression of one or more of TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any gene in the chromosome 6p21 region; and (2) introduced or increased expression of HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, antigen-specific TCR, Fc receptor, checkpoint inhibitor, antibody or functional fragment or variant thereof, or surface triggering receptor for coupling to a bispecific or multispecific or universal engager.

[0222] In some other embodiments, the hematopoietic lineage cells comprise therapeutic attributes of source-specific immune cells related to a combination of at least two of the following: (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) tolerance to the tumor microenvironment, (iv) recruitment and immunomodulation of bystander immune cells, (iv) improved target specificity with reduced extratumoral effects, and (v) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0223] In some embodiments, the iPSC-derived hematopoietic cells comprise a genotype listed in Table 1, and the cells express at least one cytokine and / or its receptor, including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, or any modified protein thereof, and express at least a CAR. In some embodiments, the engineered expression of the cytokine and CAR is NK cell-specific. In some other embodiments, the engineered expression of the cytokine and CAR is T cell-specific. In one embodiment, the CAR comprises a BCMA-binding domain. In some embodiments, the iPSC-derived hematopoietic effector cells are antigen-specific. In some embodiments, the antigen-specific derived effector cells target liquid tumors. In some embodiments, the antigen-specific derived effector cells target solid tumors. In some embodiments, the antigen-specific iPSC-derived hematopoietic effector cells are capable of rescuing tumor antigen escape.

[0224] By introducing the immune cells of the present invention into a subject suitable for adoptive cell therapy, various diseases can be ameliorated. In some embodiments, the iPSC-derived hematopoietic cells provided are for allogeneic adoptive cell therapy. Additionally, the present invention provides, in some embodiments, therapeutic uses of the above-described therapeutic compositions by introducing the compositions into a subject suitable for adoptive cell therapy, the subject having an autoimmune disorder, a hematological malignancy, a solid tumor, or an infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes. Examples of solid cancers include, but are not limited to, cancer of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testicle, bladder, kidney, 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), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple myeloma, and esophageal cancer. Examples of viral infections include, but are not limited to, rheumatoid arthritis, pemphigoid / bulbous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV (human immunodeficiency virus), HSV (herpes simplex virus), KSHV (Kaposi's sarcoma-associated herpesvirus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), VZV (varicella-zoster virus), adenovirus, lentivirus, and BK polyomavirus-associated disorders.

[0225] Treatment using the derived hematopoietic lineage cells of the embodiments disclosed herein can be performed in response to symptoms or to prevent recurrence. The terms "treating," "treatment," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in that a disease is completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects resulting from the disease are partially or completely cured. As used herein, "treatment" encompasses any intervention in a subject, including: preventing a disease from occurring in a subject who is susceptible to the disease but has not yet been diagnosed with it; inhibiting a disease, i.e., arresting its development; or alleviating a disease, i.e., reversing a disease. Therapeutic agents or compositions can be administered before, during, or after the onset of a disease or injury. Treatment of ongoing diseases, in which treatment stabilizes or reduces undesirable clinical symptoms in the patient, is also of particular interest. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury in which at least one associated symptom can be suppressed, ameliorated, and / or ameliorated by cell therapy. Certain embodiments contemplate that subjects in need of cell therapy include, but are not limited to, bone marrow or stem cell transplant candidates, subjects who have undergone chemotherapy or radiation therapy, subjects having or at risk of developing a hyperproliferative disorder or cancer, e.g., a hyperproliferative disorder or cancer of the hematopoietic system, subjects having or at risk of developing a tumor, e.g., a solid tumor, subjects having or at risk of developing a viral infection or a disease associated with a viral infection.

[0226] When assessing responsiveness to a treatment comprising the derived hematopoietic lineage cells of the embodiments disclosed herein, response may be measured by criteria including at least one of clinical benefit rate, survival to death, pathological complete response, semi-quantitative measurement of pathological response, clinical complete remission, clinical partial remission, clinical stable disease, relapse-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Response Evaluation Criteria in Solid Tumors) criteria.

[0227] Therapeutic compositions containing the disclosed derived hematopoietic lineage cells can be administered to a subject before, during, and / or after other treatments. Thus, combination therapy methods can involve the administration or preparation of iPSC-derived immune cells before, during, and / or after the use of additional therapeutic agents. As provided above, the one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, vectors containing one or more polynucleic acids of interest, antibodies or functional variants or fragments thereof, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). Administration of iPSC-derived immune cells can be separated from administration of the additional therapeutic agent by hours, days, or even weeks. Additionally or alternatively, administration can be combined with other biologically active agents or modalities, such as, but not limited to, anti-tumor agents, non-drug therapies such as surgery, etc.

[0228] In some embodiments of the combined cell therapy, the therapeutic combination comprises the iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent that is an antibody or antibody fragment. In some embodiments, the additional therapeutic agent comprises multiple antibodies targeting one or more antigens. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody can be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor- or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells, enhancing their killing capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to the administered iPSC-derived hematopoietic lineage cells include CD20 antibodies (e.g., rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), HER2 antibodies (e.g., trastuzumab, pertuzumab), CD52 antibodies (e.g., alemtuzumab), EGFR antibodies (e.g., certuximab), GD2 antibodies (e.g., diamantezomib), and the like. nutuximab), 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 humanized or Fc-modified variants or fragments thereof, or functional equivalents and biosimilars thereof.

[0229] In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. Checkpoints are cellular molecules, often cell surface molecules, that, if uninhibited, can suppress or downregulate an immune response. Checkpoint inhibitors are antagonists that can reduce checkpoint gene expression or gene products or decrease the activity of checkpoint molecules. Suitable checkpoint inhibitors for combination therapy with derived effector cells, including 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, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2) antagonists.

[0230] 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, three, or more inhibitors such that two, three, or more checkpoint molecules are targeted. In some embodiments, the derived effector cells for the combination therapy are functionally enhanced as provided herein. In some embodiments, two, three, or more checkpoint inhibitors can be administered in combination therapy with, before, or after administration of the derived effector cells. In some embodiments, two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

[0231] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of whole antibodies and may be more cost-effective to produce, easier to use, or more sensitive than whole antibodies. In some embodiments, the one, or two, or three or more checkpoint inhibitors comprise at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0232] Combination therapy including derived effector cells and one or more check inhibitors is indicated for the treatment of cutaneous T-cell lymphoma, non-Hodgkin's lymphoma (NHL), mycosis fungoides, Paget's reticulosis, Sézary syndrome, granulomatous lax skin, lymphomatoid papulosis, chronic pityriasis lichenoides, acute pityriasis lichenoides, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennart's lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, and cutaneous T-cell lymphoma. It is applied to the treatment of liquid and solid tumors, including but not limited to, Hodgkin's lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyosarcoma, Lewis lung carcinoma (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostate small cell neuroendocrine carcinoma (SCNC), liver cancer, glioblastoma, liver cancer, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid cancer, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastases, and nasopharyngeal carcinoma.

[0233] In some embodiments, in addition to the derived effector cells provided herein, the combination for therapeutic use includes one or more additional therapeutic agents, including chemotherapeutic agents or radioactive moieties. Chemotherapeutic agents refer to cytotoxic anti-tumor agents, i.e., chemical agents that are found to preferentially kill tumor cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the growth of neoplastic cells. Chemotherapeutic agents, sometimes referred to as anti-tumor or cytotoxic drugs or agents, are well known in the art.

[0234] In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamine, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing agents, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, mechlorethamine, mephalin, chlorambucil, hememethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), animavorites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisantrene, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine.Additional agents include 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, calcitonin, capecitabine, celecoxib, and ceftazidime. Cimab, cladribine, cloflavine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, ibuprofen Darubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegadromase, pemetrexed Suitable agents include guaspargase, pentostatin, pipobroman, plicamycin, porifeprosan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those approved as chemotherapeutic or radiotherapeutic agents and known in the art.Such agents can be found in any of several standard physician and oncologist reference works (e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, NY, 1995) or through the National Cancer Institute's website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated from time to time.

[0235] 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.

[0236] 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., a pharmaceutically acceptable medium, e.g., cell culture medium), or other pharmaceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents will be determined, in part, by the particular composition being administered, as well as by the particular method used to administer the therapeutic composition. Accordingly, there are a wide variety of suitable formulations of the therapeutic compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17 th ed. 1985, the disclosure of which is incorporated herein by reference in its entirety).

[0237] In one embodiment, a therapeutic composition comprises T cells derived from pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, a therapeutic composition comprises NK cells derived from pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, a therapeutic composition comprises CD34+ HE cells derived from pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, a therapeutic composition comprises HSCs derived from pluripotent cells generated by the methods and compositions disclosed herein. In one embodiment, a therapeutic composition comprises MDSCs derived from pluripotent cells generated by the methods and compositions disclosed herein. Therapeutic compositions comprising populations of iPSC-derived hematopoietic lineage cells disclosed herein can be administered intravenously, intraperitoneally, enterally, or intratracheally, either separately or in combination with other appropriate compounds, to affect a desired therapeutic goal.

[0238] These pharmaceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Thus, the buffering agent can be as much as about 5% on a weight-to-weight basis of the total composition. Electrolytes, such as, but not limited to, sodium chloride and potassium chloride, can also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition 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 comprises a buffer having a pH within one of the above pH ranges. In 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.

[0239] The present invention also provides, in part, the use of pharmaceutically acceptable cell culture media in certain compositions and / or cultures of the invention. Such compositions are suitable for administration to human subjects. Generally speaking, any medium that supports the maintenance, growth, and / or health of iPSC-derived immune cells according to embodiments of the invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharmaceutically acceptable cell culture medium is serum-free and / or feeder-free. In various embodiments, the serum-free medium is animal-free and optionally protein-free. Optionally, the medium may contain recombinant proteins acceptable for biologicals. Animal-free medium refers to a medium whose components are derived from sources other than animals. Recombinant proteins replace natural animal proteins in animal-free media, and nutrients are derived from synthetic, plant, or microbial sources. In contrast, protein-free medium is defined as being 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 known to those of skill in the art.

[0240] Isolated pluripotent stem cell-derived hematopoietic lineage cells may comprise at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+ HE cells, HSCs, B cells, myeloid-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, isolated pluripotent stem cell-derived hematopoietic lineage cells comprise about 95% to about 100% T cells, NK cells, proT cells, proNK cells, CD34+ HE cells, or myeloid-derived suppressor cells (MDSCs). In some embodiments, the present invention provides therapeutic compositions comprising purified T cells or NK cells, such as compositions comprising an isolated population of about 95% T cells, NK cells, proT cells, proNK cells, CD34+ HE cells, or myeloid-derived suppressor cells (MDSCs), for treating a subject in need of cell therapy. In some other embodiments, the isolated pluripotent stem cell-derived hematopoietic lineage cells may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% effector cells with functional or structural characteristics that are absent or atypical of T, NK, or NKT cells, or any other immune cells of natural origin.

[0241] In one embodiment, the combination cell therapy comprises a therapeutic protein or peptide and a population of effector cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, wherein the derived effector cells comprise a BCMA-CAR. In another embodiment, the combination cell therapy comprises a CD38-specific therapeutic protein or peptide and a population of effector cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, wherein the derived effector cells comprise a BCMA-CAR and CD38 null. In some embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202 and a population of effector cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, wherein the derived NK cells or T cells comprise a BCMA-CAR, CD38 null, and exogenous CD16. In still other embodiments, the combination cell therapy comprises daratumumab and a population of effector cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, wherein the derived effector cells comprise a BCMA-CAR, CD38 null, hnCD16, and a second CAR targeting at least one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1. In yet some additional embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202 and a population of effector cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, wherein the derived effector cells comprise a BCMA-CAR, CD38 null, hnCD16, exogenous CD16, and a CAR and one or more protein complexes comprising full-length or partial-length cytokines, cytokine receptors, or combinations thereof.In yet another embodiment, the combination cell therapy comprises a therapeutic protein or peptide and a population of NK lineage cells derived from genomically engineered iPSCs comprising the genotypes set forth in Table 1, wherein the derived NK cells comprise B2M- / -CIITA- / - comprising BCMA-CAR, CD38 null, hnCD16, CAR, one or more exogenous cytokines, and at least one of HLA-G overexpression or CD58 knockout and CD54 knockout.

[0242] As those skilled in the art will understand, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs based on the methods and compositions herein can be used for the above-mentioned cell therapy.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 HLA-mismatched with the subject, and the derived hematopoietic lineage cells are effector cells with HLA I and HLA II null.

[0243] In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is at least 0.1 x 10 per dose. 5 cells, at least 1 x 10 5 Cells, at least 5 x 10 5 cells, at least 1 x 10 6 Cells, at least 5x10 6 Cells, at least 1x10 7 Cells, at least 5x10 7 Cells, at least 1x10 8 Cells, at least 5x10 8 Cells, at least 1x10 9 cells, or at least 5x10 9 In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1 x 10 per dose. 5 cells ~ approx. 1x10 6 cells, approximately 0.5x10 per dose 6 cells ~ approx. 1x10 7 cells, approximately 0.5x10 per dose 7 cells ~ approx. 1x10 8 cells, approximately 0.5x10 per dose 8cells ~ approx. 1x10 9 cells, approximately 1x10 per dose 9 Cells ~ approx. 5x10 9 cells, approximately 0.5x10 per dose 9 cells ~ approx. 8x10 9 cells, approximately 3x10 per dose 9 cells ~ approx. 3x10 10 cells, or any range in between. Generally, for a 60 kg patient, 1 x 10 8 Cells / dose: 1.67 x 10 6 Converted to cells / kg.

[0244] In one embodiment, the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a portion of blood or a single umbilical cord, or at least 0.1 x 10 5 At least 0.5 × 10 cells / kg body weight 5 At least 1 x 10 cells / kg body weight 5 At least 5 x 10 cells / kg body weight 5 At least 10 x 10 cells / kg body weight 5 At least 0.75 x 10 cells / kg body weight 6 At least 1.25 x 10 cells / kg body weight 6 At least 1.5 x 10 cells / kg body weight 6 At least 1.75 x 10 cells / kg body weight 6 At least 2 x 10 cells / kg body weight 6 At least 2.5 x 10 cells / kg body weight 6 At least 3 x 10 cells / kg body weight 6 At least 4 x 10 cells / kg body weight 6 At least 5 x 10 cells / kg body weight 6 At least 10 x 10 cells / kg body weight 6 At least 15 x 10 cells / kg body weight 6 At least 20 x 10 cells / kg body weight 6 At least 25 x 10 cells / kg body weight 6 At least 30 x 10 cells / kg body weight 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 cells / kg body weight, or 1 x 109 cells / kg body weight.

[0245] In one embodiment, a 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 2×10 6 cells / kg, at least 3 × 10 6 cells / kg, at least 4 × 10 6 cells / kg, at least 5 × 10 6 cells / kg, at least 6 × 10 6 cells / kg, at least 7 × 10 6 cells / kg, at least 8 × 10 6 cells / kg, at least 9 × 10 6 cells / kg, or at least 10 × 10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0246] In another exemplary embodiment, the effective amount of effector cells provided to a subject is about 2×10 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 cells / kg, or more cells / kg, including all intervening cell doses.

[0247] In another exemplary embodiment, the effective amount of effector cells provided to a subject is about 2×10 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 6 cells / kg, approximately 5×10 6 cells / kg ~ approx. 10×10 6cells / kg, 2×10 6 cells / kg~about 6×10 6 cells / kg, 2×10 6 cells / kg ~ approx. 7×10 6 cells / kg, 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 3×10 6 cells / kg~about 6×10 6 cells / kg, 3×10 6 cells / kg ~ approx. 7×10 6 cells / kg, 3×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 4×10 6 cells / kg~about 6×10 6 cells / kg, 4×10 6 cells / kg ~ approx. 7×10 6 cells / kg, 4×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 5×10 6 cells / kg~about 6×10 6 cells / kg, 5×10 6 cells / kg ~ approx. 7×10 6 cells / kg, 5×10 6 cells / kg ~ approx. 8×10 6 cells / kg, or 6 × 10 6 cells / kg ~ approx. 8×10 6 cells / kg and includes all intervening cell doses.

[0248] In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a single-dose treatment. In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a multiple-dose treatment (also called multi-cycle or fractionated treatment). In some embodiments, the multiple-dose treatment is once every day, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days, every 45 days, or every 50 days, or any number of days in between.

[0249] Compositions comprising a population of derived hematopoietic lineage cells of the present invention can be sterile, suitable for administration to a human patient, and ready for administration (i.e., can be administered without further processing). A cell-based composition that is ready for administration means that the composition does not require any further processing or manipulation before transplantation or administration to a subject. In other embodiments, the present invention provides isolated populations of derived hematopoietic lineage cells that are expanded and / or conditioned before administration of one or more agents. In the case of derived hematopoietic lineage cells that have been genetically engineered to express a recombinant TCR or CAR, the cells can be activated and expanded using, for example, the methods described in U.S. Patent No. 6,352,694.

[0250] In certain embodiments, the primary stimulatory signal and the costimulatory signal for the derived hematopoietic lineage cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. If bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal can be bound to the cell surface, and the agent providing the primary activation signal can be in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then crosslinked to a surface, such as an antibody or other binding agent that binds to cells expressing Fc receptors, or agents disclosed in U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810, for artificial antigen-presenting cells (aAPCs) contemplated for use in activating and expanding T lymphocytes in embodiments of the present invention.

[0251] Some variation in dosage, frequency, and protocol will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the dosage, frequency, and protocol appropriate for the individual subject. [Example]

[0252] The following examples are offered by way of illustration and not by way of limitation.

[0253] Example 1 - Materials and Methods To effectively select and test suicide lines under the control of various promoters and in combination with different safe harbor locus integration strategies, we use the Applicant's proprietary hiPSC platform, which allows single cell passaging and high-throughput 96-well plate-based flow cytometry sorting, and enables the derivation of clonal hiPSCs with single or multiple gene regulation.

[0254] Maintenance of hiPSCs in small-cell culture: Once the culture reached 75%–90% confluency, hiPSCs were routinely passaged as single cells. For single-cell dissociation, hiPSCs were washed once with PBS (Mediatech) and treated with Accutase (Millipore) for 3–5 minutes at 37°C, followed by pipetting to ensure single-cell dissociation. The single-cell suspension was then mixed with equal volumes of conventional medium, centrifuged at 225 x g for 4 minutes, resuspended in FMM, and plated on a Matrigel-coated surface. Passages were typically performed at a ratio of 1:6–1:8 for 2–4 hours at 37°C, transferred to pre-coated Matrigel tissue culture plates, and fed with FMM every 2–3 days. Cell cultures were maintained in a humidified incubator set at 37°C and 5% CO2.

[0255] Human iPSC manipulation with ZFNs and CRISPR for targeted editing of the desired modality: Using ROSA26-targeted insertion as an example, for ZFN-mediated genome editing, a mixture of 2.5 μg of ZFN-L (FTV893), 2.5 μg of ZFN-R (FTV894), and 5 μg of donor construct was transfected into 2 million iPSCs for AAVS1-targeted insertion. For CRISPR-mediated genome editing, a mixture of 5 μg of ROSA26-gRNA / Cas9 (FTV922) and 5 μg of donor construct was transfected into 2 million iPSCs for ROSA26-targeted insertion. Transfection was performed using a Neon transfection system (Life Technologies) with parameters of 1500 V, 10 ms, and 3 pulses. On the second or third day after transfection, transfection efficiency was measured using flow cytometry if the plasmid contained an artificial promoter-driver GFP and / or RFP expression cassette. On day 4 after transfection, targeted cells were selected by adding puromycin to the medium at a concentration of 0.1 μg / ml for the first 7 days and 0.2 μg / ml after 7 days. During puromycin selection, cells were passaged onto new Matrigel-coated wells on day 10. From day 16 of puromycin selection onwards, surviving cells were analyzed by flow cytometry for the percentage of GFP+ iPS cells.

[0256] Bulk and clonal sorting of genome-edited iPSCs: iPSCs with genome-targeted edits using ZFN or CRISPR-Cas9 were bulk- and clonal-sorted for GFP+SSEA4+TRA181+ iPSCs after 20 days of puromycin selection. Single-cell dissociated targeted iPSC pools were resuspended in freshly prepared, chilled staining buffer containing Hank's balanced 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 7 μL per million cells in 100 μL of staining buffer. The solution was washed once with staining buffer, spun down at 225 x g for 4 minutes, resuspended in staining buffer containing 10 µM thiazolinone, and kept on ice for flow cytometry sorting. Flow cytometry sorting was performed using a FACS Aria II (BD Biosciences). For bulk sorting, GFP+SSEA4+TRA181+ cells were gated and sorted into standard 15 ml tubes filled with 7 ml of FMM. For clonal sorting, sorted cells were ejected directly into a 96-well plate using a 100 µM nozzle at a concentration of three events per well. Each well was preloaded with 200 µL of FMM supplemented with 5 µg / mL fibronectin and 1x penicillin / streptomycin (Mediatech) and pre-coated with 5x Matrigel overnight. 5x Matrigel pre-coating involves adding one aliquot of Matrigel to 5 mL of DMEM / F12, followed by overnight incubation at 4°C to allow for proper resuspension, and finally adding 50 μL per well to a 96-well plate, followed by overnight incubation at 37°C. The 5x Matrigel is aspirated immediately before adding medium to each well. Once sorting is complete, the 96-well plate is centrifuged at 225 g for 1-2 minutes before incubation. The plate is left undisturbed 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, the wells were refed with an additional 100 μL of FMM. Colony formation was detected as early as day 2, with most colonies expanding 7–10 days after sorting. For the first passage, wells were washed with PBS and dissociated with 30 μL of Accutase at 37°C for approximately 10 minutes. The need for extended Accutase treatment reflects the compactness of colonies that had been left idling in culture for extended periods. After cells were observed to be dissociated, 200 μL of FMM was added to each well and disrupted by pipetting several times. Dissociated colonies were transferred to separate wells of a 96-well plate pre-coated with 5x Matrigel and then centrifuged at 225 g for 2 minutes before incubation. This 1:1 passage was performed to expand the initial colonies before expansion. Subsequent passages were routinely performed with 3-5 min of Accutase treatment and 1:4-1:8 expansion at 75-90% confluency into larger wells pre-coated with 1x Matrigel in FMM. Each clonal cell line was analyzed for GFP fluorescence and TRA1-81 expression levels. Clonal lines approaching 100% GFP+ and TRA1-81+ were selected for further PCR screening and analysis. Flow cytometry analysis was performed on a Guava EasyCyte 8 HT (Millipore) and analyzed using Flowjo (FlowJo, LLC).

[0257] Example 2 - iPSCs and effector cells differentiated therefrom, including BCMA-CAR To confirm that the BCMA-CAR construct exhibited surface expression, 293T cells were transfected with a plasmid containing CAR-2A-IL15 / IL15Rα (truncated) (IL15Δ) with and without a plasmid expressing DAP10-Thy1.1. Cells were harvested and examined for surface expression on day 3. Cells were stained with antibodies against Thy1.1 and IL15Ra, biotinylated BCMA protein, and then streptavidin-FITC. Cell surface expression of BCMA-CAR, IL15Δ, and Thy1.1 was assessed by flow cytometry. As shown in Figure 5, exogenous DAP10 appeared to promote surface expression of both BCMA-CAR and IL15Δ in 293T cells.

[0258] To determine whether the BCMA-CAR construct also exhibits surface expression in iPS cells, we transduced the CAR-2A-IL15Δ lentiviral vector into engineered iPSCs containing CD38 knockout and hnCD16, with or without DAP10-Thy1.1. The CD38 knockout and hnCD16 iPSCs were sequentially or simultaneously engineered to obtain CD38 knockout and high-affinity, uncleavable CD16 expression. After each engineering step, iPSCs were selected for the desired phenotype before the next engineering step, e.g., loss of HLA-I by knocking out the B2M gene, loss of HLA-II by knocking out CIITA, or the introduction of BCMA-CAR and IL15Δ. The engineered iPSCs could then be maintained in vitro or for derivative cell generation.

[0259] CD38 - / -Surface expression of Thy1.1, IL15Δ, and BCMA-CAR proteins after transduction of hnCD16 iPSCs was examined by flow cytometry on day 3. In iPSCs, exogenous DAP10 does not appear to be necessary to promote surface expression of as-assembled BCMA-CAR; however, IL15Δ is detectable only in the presence of exogenous DAP10 (Figure 6). To determine the BCMA-CAR expression pattern in iNK cells, IL15Δ-positive iPSCs were sorted from iPSCs transduced with or without DAP10 and then differentiated into iNK cells using the methods and compositions provided herein. iNK cells differentiated from iPSCs containing BCMA-CAR and IL15Δ, or from iPSCs containing BCMA-CAR and IL15Δ plus DAP10, were surface stained to assess transgene expression as previously described. As shown in Figures 7A-C, expression of BCMA-CAR and IL15Δ is independent of the introduction and expression of DAP10.

[0260] Telomere shortening occurs with cellular aging and is associated with stem cell dysfunction and cellular senescence. Generated derivative cells, including T cells and NK cells, demonstrated that these genetically engineered modalities in iPSCs maintain the directed development of cells toward their desired cell fate in vitro during hematopoietic differentiation without disrupting their development. Mature derivative cells were shown here to maintain longer telomeres compared to their adult peripheral blood counterparts. For example, telomere length was measured using the 1301 T cell leukemia line as a control (100%) and G 0 / 1 The DNA index of cells was corrected and determined by flow cytometry for iPSCs, adult peripheral blood NK lineage cells, and iPSC-derived NK cells. As shown in Figure 8, iPSC-derived NK cells maintained significantly longer telomere lengths compared to adult peripheral blood NK cells (p=0.105, ANOVA), indicating a higher likelihood of proliferation, survival, and persistence in iPSC-derived NK cells. Similar observations with iPSC-derived T cells are known in the art.

[0261] Example 3 - Functional profiling of derived effector cells expressing BCMA-CAR To detect antigen-specific activity directed by the BCMA-CAR, a cytotoxicity assay targeting BCMA-expressing Nalm6 cells (Nalm-BCMA) and parental Nalm6 cells (Nalm-P, no BCMA expression) was performed. Nalm6-BCMA and Nalm6-P target cells were labeled with the cell proliferation dyes eFlour-450 and eFlour670, respectively. IL15Δ / CD38null / hnCD16 iNK effector cells with or without the BCMA-CAR were titrated and added to target cells at effector:target ratios (E:T) ranging from 30:1 to 0.03:1. Target cells without effectors were used as a control for spontaneous cell death. Samples were incubated for 4 hours at 37°C, and CellEvent® Caspase-3 / 7 Green Detection Reagent (1x400 dilution) was added during the last 30 minutes of incubation. As shown in Figure 9, iNK cells expressing BCMA-CAR demonstrate superior antigen specificity and induce significant killing of the BCMA-expressing Nalm6 line compared to the parental Nalm6.

[0262] To analyze BCMA-CAR function in T cells, bead-stimulated T cells were transduced with lentivirus carrying BCMA-CAR. Six days after transduction, T cells were tested for their ability to target BCMA-expressing tumor targets. Nalm6-BCMA and parental Nalm6 lines were prepared and incubated with the transduced T cells as described above. As shown in Figure 10, BCMA-CAR-transduced T cells demonstrated clear antigen degradation and induced greater cytotoxicity in the Nalm6-BCMA tumor group compared to non-transduced T cells.

[0263] To test the ADCC-mediated dual-targeting ability of BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK cells, MM.1R myeloma cells were used as targets in a restimulation cytotoxicity assay using BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK cells and CD19-CAR / hnCD16 / IL15Δ / iNK cells as effectors. Target and effector cells were incubated with or without daratumumab at a 3:1 effector-to-target ratio for 48 hours. After 48 hours, all effector cells were collected and transferred to new wells containing new MM.1R targets. After another 48-hour restimulation period, the number of remaining MM.1R target cells was determined. As shown in Figure 11, to effectively eliminate multiple myeloma cancer cells, daratumumab synergizes with BCMA-CAR via hnCD16-mediated ADCC, allowing BCMA-CAR / CD38null / hnCD16 / IL15Δ effector cells to enhance antitumor activity by targeting multiple antigens on cancer cells.

[0264] The in vivo function of BCMA-CAR was evaluated using mouse melanoma cells expressing human BCMA as tumor cell targets or human cell lines expressing endogenous BCMA. For in vivo evaluation, mouse or human T cells were transduced with BCMA-CAR along with other constructs, IL15Δ, CD38null, and hnCD16, and used as effectors in addition to iPSC-derived NK cells containing BCMA-CAR / CD38null / hnCD16 / IL15Δ.

[0265] The efficacy of BCMA-CAR / CD38null / hnCD16 was evaluated in a mouse melanoma model. The mouse melanoma cell line B16F10 was transduced with human BCMA (B16F10-BCMA), and these cells were implanted intravenously (IV) or subcutaneously (SC) into immunocompetent C57BL / 6 or immunocompromised NSG mice. Intravenous injection of B16F10-BCMA tumor cells resulted in lung metastases in C57BL / 6 mice and lung and liver metastases in NSG mice, while SC implantation resulted in single solid tumors in both mouse strains. Lung tumor nodules (metastases) were counted in C57BL / 6 mice after IV implantation of B16F10-BCMA cells. Adoptive transfer of BCMA-CAR / CD38null / hnCD16- T or NK cells was performed after tumor implantation to assess the ability of these cells to reduce the number of tumor nodules that developed in these animals. Tumor nodules were further evaluated by gross morphology and microscopic examination of tissue sections. In the subcutaneous B16-F10-BCMA model, tumor progression was monitored by caliper measurement of tumor size. A reduction in the number and / or size of lung tumor nodules compared to mice treated with mock-transduced T or NK cells reflected the efficacy of treating C57BL / 6 mice intravenously implanted with B16F10-BCMA cells using BCMA-CAR / CD38null / hnCD16-T or NK cells. Similarly, in the subcutaneous model of B16-F10-MICA tumor growth, the efficacy of BCMA-CAR / CD38null / hnCD16-T or NK cell treatment was demonstrated by delaying tumor progression, prolonging survival, inducing tumor regression, or a combination of the above.

[0266] In NSG mice, both lung and liver tumor nodules were counted, and mice treated with mock-transduced T cells were compared to BCMA-CAR / CD38null / hnCD16 T cells for their ability to reduce the number of nodules in each organ. Both murine and human BCMA-CAR / CD38null / hnCD16 T cells were evaluated for their ability to control tumor growth in NSG mice. A reduction in the number and size of tumor nodules in the lungs and liver of NSG mice IV-transferred with BCMA-CAR / CD38null / hnCD16 T cells from either human or murine sources indicates efficacy of treatment and is associated with extended survival of the mice.

[0267] Similar results are expected for treatment of B16-F10-BCMA tumor-bearing NSG mice with BCMA-CAR / CD38null / hnCD16 iNK cells. Figures 12A-B show the in vivo efficacy of BCMA-CAR / CD38null / hnCD16 iNK cells in combination with daratumumab in a disseminated xenograft MM.1S multiple myeloma model using representative bioluminescence images (Figure 12A) or cumulative data from n=10 mice per study group (Figure 12B), demonstrating complete tumor clearance with no signs of recurrence for at least 30 days compared to treatment with cells or daratumumab alone.

[0268] The function of BCMA-CAR against various human tumor cell lines was also evaluated. BCMA-expressing human cell lines, including CI-H929, RPMI-8226, U266, and KMS11, were implanted into immunocompromised NSG mice. Treatment of NSG mice bearing any of these tumor types using either BCMA-CAR / CD38null / hnCD16 T cells or BCMA-CAR / CD38null / hnCD16 iNK cells was evaluated for delaying tumor progression, inducing tumor regression, and extending survival. Figure 13 shows that BCMA-CAR / CD38null / hnCD16 iNK cells effectively target BCMA+ multiple myeloma tumor cell lines via CAR- and ADCC-mediated mechanisms.

[0269] Further cytotoxicity characterization of BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK cells was performed using a serial restimulation assay. The assay was performed against MM.1S cells using BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK as effector cells at a 1:1 E:T ratio in the presence or absence of daratumumab. The effector BCMA-CAR-negative parental cells (CD38null / hnCD16 / IL15Δ) were used as a control. Cells were co-cultured for 48 hours (R1), and then all non-adherent cells were collected and transferred to freshly plated MM.1S target cells for an additional round of stimulation (R2). This procedure was repeated once more (R3), for a total of three rounds of stimulation. As shown in Figure 14, BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK cells demonstrated excellent dual-target killing capabilities against tumor cells. BCMA-CAR / CD38null / hnCD16 / IL15Δ effector cells also exhibited consistent and effective cytotoxicity over three consecutive challenges with MM.1S cells. This cytotoxicity was evident both in the presence and absence of daratumumab, demonstrating significantly improved cytotoxicity compared to the CAR-negative parental control. Notably, BCMA-CAR / CD38null / hnCD16 / IL15Δ effector cells demonstrated improved cytotoxicity over each stimulation round in both the monotherapy and combination arms, suggesting evasion of antigen-mediated expansion and exhaustion.

[0270] In cytokine production assays, BCMA-CAR iNK cells were used as effector cells and stimulated with the indicated tumor cell lines for 4 hours in the presence of daratumumab. Supernatants were collected, and TNFα and IFNγ levels were assessed using a MesoScale Diagnostics (MSD) electrochemiluminescence platform. As shown in Figure 15, increased cytokine production could be promoted by either CAR targeting or ADCC-mediated targeting, depending on the tumor cell, reflecting the advantages of the dual-targeting strategy in BCMA-CAR / CD16 effector cells, which utilize both CAR and ADCC for enhanced cytotoxicity.

[0271] Furthermore, we observed that BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK effector cells maintained their in vivo efficacy in the absence of exogenous cytokine support (see Figure 16). NSG mice were implanted with MM.1S-Luc cells and treated with BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK effector cells on days 2, 9, and 16, or with BCMA-CAR T cells on day 2. Two batches of iNK effector cells were used, and all three treatment groups were split and either left without additional cytokine support or supplemented with twice-weekly injections of IL-15 and IL-2. BLI images for each cell group in the presence or absence of in vivo exogenous cytokine support are shown in Figure 16. Treatment of MM.1S-implanted mice with iNK effector cells resulted in tumor regression and delayed tumor growth in all three batches tested, with no significant differences observed between cytokine support and no cytokine support. In contrast, primary CAR-T cells were effective in the presence of exogenous IL-2 and IL-15 support, but the efficacy of these cells was significantly lower without cytokine administration. Thus, BCMA-CAR / CD38null / hnCD16 / IL15Δ iNK effector cells exhibited autonomous and durable properties in vivo without the need for cytokine support while maintaining antitumor potential.

[0272] MM.1S cells were transplanted into mice at a dose of 2.5E5 on day 0. Mice were treated with three doses of CAR / CD38null / hnCD16 / IL15Δ iNK cells at a dose of 5E6 on days 3, 10, and 17. As shown...

Claims

1. A cell or population thereof, (i) the cell is an induced pluripotent cell (iPSC) or a derivative cell obtained from iPSC differentiation; (ii) A cell or a population thereof, wherein the cell comprises a polynucleotide encoding at least BCMA-CAR (chimeric antigen receptor).

2. The derivative cells obtained from the iPSC differentiation are hematopoietic cells and contain longer telomeres compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue, or the BCMA-CAR has the following characteristics: (i) being T cell specific; (ii) NK cell-specific; (iii) binding to surface BCMA; (iv) an insertion at one of the following loci: AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1; or 2. The cell or population thereof of claim 1, wherein the cell or population thereof has at least one of the following loci: (v) an insertion at one of the loci B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, wherein the insertion optionally knocks out expression of the gene at the locus.

3. The cells (i) CD38 knockout, (ii) B2M null or low, and optionally CIITA null or low, compared to the native counterpart; (iii) introduction of expression of HLA-G or uncleavable HLA-G, or knockout of one or both of CD58 and CD54; (iv) exogenous CD16 or a variant thereof; (v) chimeric antigen receptors (CARs) with target specificity other than BCMA; (vi) a cell surface expressed protein complex comprising a partial or full length cytokine, cytokine receptor, or any combination thereof; (vii) at least one of the genotypes listed in Table 1; (viii) deleted or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT compared to a native corresponding cell; and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A, compared to the native counterpart. ２Ａ 2. The cell or population thereof of claim 1, further comprising one or more of: an R, an antigen-specific TCR, an Fc receptor, a checkpoint inhibitor, an antibody or functional fragment and variant thereof, an engager, and introduced or increased expression in at least one of a surface triggering receptor for binding with a bi- or multispecific or universal engager.

4. The BCMA-CAR comprises at least (a) a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37; (b) a light chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38; or (c) a scFV represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39-44.

5. The cells are derived NK or T cells and have the following characteristics compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue: (i) improved durability and / or survival; (ii) increased tolerance to natural immune cells; (iii) increased cytotoxicity; (iv) improving tumor invasion; (v) enhancing or obtaining ADCC; (vi) an enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site; (vii) enhanced ability to reduce tumor immunosuppression; (viii) improved ability to rescue tumor antigen escape; (ix) the ability to stabilize tumor antigens, and (x) the ability to avoid fratricide; The cell or population thereof according to claim 1, having at least one of the following characteristics:

6. The cell or population thereof of claim 3, wherein the exogenous CD16 or variant thereof comprises high-affinity non-cleavable CD16 (hnCD16).

7. the exogenous CD16 or variant thereof (a) F176V and S197P in the ectodomain of CD16; (b) a complete or partial ectodomain derived from CD64; (c) a non-native (or non-CD16) transmembrane domain; (d) a non-native (or non-CD16) intracellular domain; (e) a non-native (or non-CD16) signaling domain; (f) a non-naturally occurring stimulatory domain; and (g) a cell or population thereof according to claim 3, comprising at least one of transmembrane, signal transduction, and stimulatory domains not derived from CD16 but derived from the same or a different polypeptide;

8. (a) the non-native transmembrane domain is derived from a 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, or T cell receptor (TCR) polypeptide; (b) the non-native stimulatory domain is derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide; (c) the non-native signaling domain is derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide; or (d) the non-native transmembrane domain is derived from NKG2D, the non-native stimulatory domain is derived from 2B4, and the non-native signaling domain is derived from CD3ζ, or a population thereof according to claim 7.

9. The cells further comprise a CAR having a target specificity other than BCMA, wherein the CAR (i) T cell-specific or NK cell-specific; (ii) is a bispecific antigen-binding CAR; (iii) is a switchable CAR; (iv) is a dimerized CAR; (v) is a split CAR; (vi) is a multi-chain CAR; (vii) is an inducible CAR; (viii) co-expressed, optionally in a separate or bicistronic construct, with a cell surface expressed protein complex comprising partial or full length of a cytokine, cytokine receptor, or any combination thereof; (xi) co-expressed with an antibody or functional fragment or variant thereof, or a checkpoint inhibitor, optionally in a separate construct or a bicistronic construct; (xii) specific for at least one of CD19, MICA / B, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, 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, cytomegalovirus (CMV)-infected cell antigen, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase ( hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG 4. The cell or population thereof of claim 3, which is specific for any one of 2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms' tumor protein (WT-1), and a pathogen antigen.

10. 4. The cell or population thereof of claim 3, wherein the cell comprises at least a CAR inserted into the TRAC locus and / or is driven by the endogenous promoter of the TCR and / or the TCR is knocked out by the CAR insertion.

11. the cell surface-expressed protein complex (a) comprising at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or variants thereof, and their receptors or variants thereof; (b) (i) Co-expression of IL15 and IL15Rα by using a self-cleaving peptide; (ii) a fusion protein of IL15 and IL15Rα; (iii) an IL15 / IL15Rα fusion protein having a truncated intracellular domain of IL15Rα; (iv) a fusion protein of IL15 and the membrane-binding Sushi domain of IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and common receptor γC, wherein the common receptor γC is native or modified; and (vii) a homodimer of IL15Rβ; (Any one of (i) to (vii) may be co-expressed with the CAR in a separate or bicistronic construct); and optionally, (c) A cell or population thereof according to claim 3, which is transiently expressed.

12. 4. The cell or population thereof of claim 3, wherein the cell is a derived NK cell or a derived T cell, the derived NK cell is capable of recruiting and / or migrating T cells to tumor sites, and the derived NK cell or the derived T cell is capable of reducing tumor immune suppression in the presence of one or more checkpoint inhibitors.

13. 13. The cell or population thereof of claim 3 or 12, wherein the checkpoint inhibitor is an antagonist of 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, or inhibitory KIR.

14. the checkpoint inhibitor (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (b) the cell or population thereof of claim 13, comprising at least one of atezolizumab, nivolumab, and pembrolizumab.

15. The cell or population thereof of claim 1, wherein the derivative cells comprise derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T cells, derived NKT cells, derived NK cells, or derived B cells.

16. The cells (i) one or more exogenous polynucleotides integrated into one safe harbor locus or selected locus; or (ii) more than two exogenous polynucleotides integrated into different safe harbor loci or two or more selected loci; or (iii) a polynucleotide encoding IL15Δ comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 17, 19, or 21;

17. 17. The cell or population thereof of claim 16, wherein the safe harbor loci include at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, and the selected locus is one of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and wherein the integration of the exogenous polynucleotide optionally knocks out expression of the gene at the locus.

18. The cell or population thereof described in claim 17, wherein the TCR locus is the constant region of TCR alpha or TCR beta.

19. A cell or population thereof comprising a BCMA-CAR and one or more of (i) a CD38 knockout, (ii) exogenous CD16 or a variant thereof, and (iii) a cell surface-expressed protein complex comprising a partial or full-length of a cytokine, a cytokine receptor, or any combination thereof, wherein the cell or population thereof is an immune effector cell.

20. The BCMA-CAR comprises at least (a) a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37; (b) a light chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38; or (c) a scFV represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39-44.

21. The following characteristics compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue: (i) improved durability and / or survival; (ii) increased tolerance to natural immune cells; (iii) increased cytotoxicity; (iv) improving tumor invasion; (v) enhancing or obtaining ADCC; (vi) an enhanced ability to migrate and / or activate or recruit bystander immune cells to the tumor site; (vii) enhanced ability to reduce tumor immunosuppression; (viii) improved ability to rescue tumor antigen escape; (ix) the ability to stabilize tumor antigens, and (x) the ability to avoid fratricide; 20. The cell or population thereof according to claim 19, comprising at least one of:

22. 20. The cell or population thereof of claim 19, wherein the immune effector cell is a T lineage or NK lineage cell.

23. A composition comprising a cell or a population thereof according to any one of claims 1 to 22.

24. A composition for therapeutic use comprising the cells of any one of claims 1 to 22 and one or more therapeutic agents.

25. 25. The composition of claim 24, wherein the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector comprising one or more polynucleic acids of interest, an antibody or functional variant or fragment thereof, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

26. (1) The checkpoint inhibitor is (a) one or more antagonists of checkpoint molecules, including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab; or (2) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide; or (3) The composition of claim 25, wherein the therapeutic agent comprises a gamma secretase inhibitor (GSI).

27. The antibody (a) anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies; (b) one or more of rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, certuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, and functional equivalents and biosimilars thereof; or 26. The composition of claim 25, comprising (c) daratumumab.

28. 28. Therapeutic use of the composition of any one of claims 24 to 27 by introducing said composition into a subject suitable for adoptive cell therapy, wherein the subject has: (i) an autoimmune disorder, a hematological malignancy, a solid tumor, a cancer, or a viral infection; (ii) an inflammatory autoimmune disease, a cancer of plasma cells, or a cancer of B lymphocytes; (iii) an autoimmune disease associated with autoreactive plasma cells and / or autoreactive memory B cells; (iv) systemic lupus erythematosus (SLE), or rheumatoid arthritis, multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, plasma cell leukemia, or Hodgkin's disease; or (v) multiple myeloma.

29. differentiating iPSCs, wherein the iPSCs are differentiated with a polynucleotide encoding a BCMA-CAR and optionally (i) CD38 knockout, (ii) B2M null or low, and optionally CIITA null or low, compared to the native counterpart; (iii) introduction of expression of HLA-G or uncleavable HLA-G, or knockout of one or both of CD58 and CD54; (iv) high-affinity non-cleavable CD16 (hnCD16) or a variant thereof; (v) chimeric antigen receptors (CARs) with target specificity other than BCMA; (vi) a cell surface expressed protein complex comprising a partial or full length cytokine, cytokine receptor, or any combination thereof; (vii) at least one of the genotypes listed in Table 1; (viii) deleted or reduced expression of at least one of TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT compared to a native corresponding cell; and (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A, compared to the native counterpart. ２Ａ and one or more of: an R, an antigen-specific TCR, an Fc receptor, a checkpoint inhibitor, an antibody or functional fragment or variant thereof, an engager, and introduced or increased expression of at least one surface triggering receptor for binding with a bi- or multispecific or universal engager.

30. The clonal iPSCs are genomically engineered to knock in a polynucleotide encoding BCMA-CAR, and optionally, (i) knocking out CD38, or 30. The method of producing a derivative cell of claim 29, further comprising (ii) knocking out B2M and CIITA, knocking out one or both of CD58 and CD54, or introducing expression of a cell surface expressed protein complex comprising partial or full length of HLA-G or non-cleavable HLA-G, exogenous CD16 or a variant thereof, a second CAR, and / or a cytokine, cytokine receptor, or any combination thereof.

31. 31. The method of producing a derivative cell of claim 30, wherein said genome engineering comprises targeted editing.

32. 32. The method of producing a derivative cell of claim 31 , wherein the targeted edit comprises a deletion, insertion, or in / del, and the targeted edit is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation of these methods.

33. 1. CRISPR-mediated editing of clonal iPSCs, wherein the editing comprises knock-in of a polynucleotide encoding a BCMA-CAR, and wherein the edited clonal iPSCs comprise at least one of the genotypes listed in Table 1.

34. The editing further comprises knocking out CD38, or the BCMA-CAR has the following characteristics: (i) being T cell specific; (ii) NK cell-specific; (iii) binding to surface BCMA; (iv) comprising a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37; (vi) comprising a light chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38; (vii) comprising an scFv represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39, 40, 41, 42, 43, or 44; and (viii) the CRISPR-mediated editing of Claim 33 is performed at least one of the following loci: B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, wherein the insertion knocks out expression of the gene at the locus.

35. 34. The CRISPR-mediated editing of Claim 33, wherein the editing further comprises insertion of the BCMA-CAR or a second CAR at the constant region of a TCR locus, and / or wherein the CAR is driven by the endogenous promoter of the TCR, and / or wherein the TCR is knocked out by insertion of the CAR.

36. 1. A method for improving the treatment of multiple myeloma, comprising administering to a subject undergoing said treatment effector cells comprising a BCMA-CAR, a CD38 knockout, and a high-affinity uncleavable CD16 or a variant thereof, wherein the BCMA-CAR has the following characteristics: (i) being T cell specific; (ii) NK cell-specific; (iii) binding to and stabilizing cell surface BCMA; (iv) comprising a heavy chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 33, 35, or 37; (vi) comprising a light chain variable region represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 34, 36, or 38; (vii) comprising an scFv represented by an amino acid sequence that is at least about 99%, 98%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 39, 40, 41, 42, 43, or 44; and (viii) an insertion in one of the gene loci B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, wherein the insertion knocks out expression of the gene at the locus.

37. the effector cells comprise derivative hematopoietic cells, including derivative NK cells or derivative T cells, and the derivative NK cells or derivative T cells are (i) B2M and CIITA knockout; (ii) introduction of expression of HLA-G or uncleavable HLA-G, or knockout of one or both of CD58 and CD54; (iii) introduction of a second CAR, and / or expression of a cell surface expressed protein complex comprising partial or full length of a cytokine, cytokine receptor, or any combination thereof; and / or 37. The method of claim 36, further comprising one or more of: (iii) at least one of the genotypes listed in Table 1.

38. 37. The method of claim 36, further comprising administering an anti-CD38 antibody and / or a GSI.

39. 37. The method of claim 36, wherein the effector cells comprising a BCMA-CAR, a CD38 knockout, and a high-affinity non-cleavable CD16 or variant thereof have been or are being contacted with a GSI.

40. 37. The method of claim 36, wherein the multiple myeloma is a relapsed or refractory form of multiple myeloma.

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