Enhancement of iPSC-derived effector immune cells using small compounds

Abstract

Methods and compositions are provided for obtaining functionally enhanced derivative effector cells obtained from directed differentiation of genomically engineered iPSCs. The derivative cells provided herein contain stable, functional genome edits that result in improved or enhanced therapeutic efficacy. Therapeutic compositions and uses thereof comprising the functionally enhanced derivative effector cells, alone or in combination with antibodies or checkpoint inhibitors, are also provided.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 62 / 945,040, filed on 6 December 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This disclosure broadly relates to the field of ready-made immune cell products. More specifically, this disclosure relates to strategies for developing multifunctional effector cells that can deliver therapeutically relevant properties in vivo. Cell products developed under this disclosure address significant limitations of patient-derived cell therapies. [Background technology]

[0003] The field of adoptive cell therapy currently focuses on the use of patient-derived and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapy and deliver treatment to all patients who could potentially benefit. To promote favorable patient outcomes, there is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes. Lymphocytes such as T cells and natural killer (NK) cells are potent antitumor effectors that play a crucial role in innate and adaptive immunity. However, the use of these immune cells for adoptive cell therapy remains challenging, and there is an unmet need for improvement. Therefore, there remains a significant opportunity to fully utilize the potential of T cells, NK cells, or other lymphocytes in adoptive immunotherapy. [Overview of the project]

[0004] Functionally improved effector cells are needed to address issues related to the effectiveness against solid tumors, i.e., the tumor microenvironment and associated immunosuppression, recruitment, transport, and invasion, from response rate, cell depletion, loss of transfused cells (survival and / or persistence), tumor escape due to target loss or lineage change, accuracy of tumor targeting, extra-target toxicity, and extratumor effects. While characterizing the in vitro behavior of therapeutic cell populations is important, it is often even more important to evaluate in vivo function and performance (i.e., potency, efficacy, and safety profiles) using animal models and / or early clinical trials. Furthermore, the use of effector cells for cell therapy will preferably utilize manufacturing processes that not only enable scale-up but also maintain and / or enhance cell potency, cell efficacy, and patient safety. Among the many important aspects of the manufacturing process for cell therapies, this application identifies cell proliferation and cryopreservation as key areas of interest, as these significantly impact cell viability and functionality during the freeze-thaw cycle, and the in vivo efficacy and persistence of effector cells derived from iPSC differentiation are complexly affected by the effector cell proliferation stage after iPSC differentiation. This application provides for the fine-tuning of effector cells by treating iPSC-derived effector cells with one or more selected compounds during cell proliferation, resulting in sustainable therapeutic effector cells through a cryogenic freeze-thaw manufacturing process while enhancing in vivo potency and efficacy, including but not limited to persistence, tumor invasion, tumor killing, and tumor clearance, compared to iPSC-derived effector cells without compound treatment.

[0005] The object of the present invention is to provide a method and composition for generating derived non-pluripotent cells differentiated from a single-cell-derived iPSC (induced pluripotent stem cell) clone line, wherein the iPSC line contains one or more gene modifications in its genome. These one or more gene modifications include DNA insertions, deletions, and substitutions, and these modifications are retained and continue to function in subsequent derived cells after differentiation, expansion, passage, and / or transplantation.

[0006] The iPSC-derived non-pluripotent cells of this application include, but are not limited to, CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NKT cells, NK cells, and B cells. The iPSC-derived non-pluripotent cells of this application have one or more gene modifications in their genome through differentiation from iPSCs that contain the same gene modifications. In the engineered clonal iPSC differentiation strategy for obtaining genetically engineered derived cells, it is also necessary that the possibility of iPSC development in the directed differentiation is not adversely affected by the engineered modality of the iPSC, and that the engineered modality functions as intended in the derived cells. Furthermore, this strategy overcomes the current barriers to manipulating primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely the difficulty in manipulating such cells, which often result in cells that lack reproducibility and uniformity, exhibit insufficient cell persistence with high cell death and low cell proliferation. Furthermore, this strategy avoids the generation of heterogeneous effector cell populations obtained by other methods, by initially using a heterogeneous primary cell source.

[0007] Several aspects of the present invention provide genome-engineered iPSCs obtained using genome editing strategies after, concurrent with, and prior to a reprogramming process. In one embodiment of the methods described above, at least one targeted genome edit at one or more selected sites includes 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, transport, homing, viability, self-renewal, persistence, and / or survival of genome-engineered iPSCs or their derived cells. In one embodiment, the resulting genome-engineered iPSCs containing at least one targeted genome edit are functional, differentiateable, and can differentiate into non-pluripotent cells containing the same functional genome edit.

[0008] Accordingly, in one embodiment, the present invention also provides a method for producing immune cells or populations thereof, wherein immune cells are treated with a small compound comprising at least one of dexamethasone, lenalidomide, AQX-1125, or derivatives or analogs thereof, thereby obtaining immune cells with enhanced cytotoxicity after thawing compared to corresponding immune cells without the same small compound treatment. In some embodiments, the immune cells are derived effector immune cells differentiated from induced pluripotent stem cells (iPSCs), and the effector immune cells include derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional features not present in the corresponding primary T, NK, NKT, and / or B cells.In some embodiments, the iPSC is edited as follows: (i) a first chimeric antigen receptor (CAR) having a first target specificity; (ii) CD38 knockout; (iii) HLA-I deficiency and / or HLA-II deficiency compared to the corresponding native cell; (iv) introduction of HLA-G or non-cleavable HLA-G expression, or knockout of one or both CD58 and CD54; (v) CD16 or a variant thereof; (vi) a second CAR having a second target specificity; (vii) a signaling complex comprising a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor; (viii) at least one of the genotypes listed in Table 2; (ix) B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RF compared to the corresponding native cell. Effector immune cells differentiated from iPSCs include deletion or reduced expression of at least one of the following: XAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or introduced or increased expression of at least one of the following: (x)HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for binding to bispecific or multispecific or universal engagers, wherein effector immune cells differentiated from iPSCs include the same one or more edits as the iPSCs.

[0009] In some embodiments, the small compound treatment (i) comprises dexamethasone or its derivatives or analogs; (ii) does not contain or essentially does not contain cytokine IL7, and optionally the immune cells during treatment are T cells; (iii) does not contain or essentially does not contain cytokine IL2 and / or cytokine IL15, and optionally the immune cells during treatment are NK cells; (iv) contains dexamethasone but does not contain cytokine IL7; (v) does not contain or essentially does not contain cytokines; (vi) is during cell culture and / or before or after cell storage; (vii) is during immune cell proliferation after differentiation of cells from iPSCs, and / or (viii) lasts for about 1 to about 12 days, or about 3 to about 6 days, before cryopreservation. In some embodiments, dexamethasone is present in a concentration range of about 10 nM to about 20 μM.

[0010] In these embodiments, the iPSC comprises a first chimeric antigen receptor (CAR) having a first target specificity, the first CAR comprising: (i) an ectodomain comprising at least one antigen recognition region, a transmembrane domain, and at least one signaling domain, wherein the at least one signaling domain comprises an ectodomain comprising an endodomain derived from the cytoplasmic domain of a signaling transduction protein specific for T and / or NK cell activation or functionalization; (ii) an antigen recognition domain that specifically binds to antigens associated with disease, pathogens, humoral tumors, or solid tumors; or (iii) any one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1; or ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD2 2, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV) infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFR-VIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, E GFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19).9) may include Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1). In some embodiments, the first CAR is contained in a bisistronic construct co-expressing the following: (1) Partial or full-length peptides of the cell surface expressing exogenous cytokines or their receptors, (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors; and (b) (i) co-expression of IL15 and IL15Rα using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is cleaved or excluded, and (iv) IL15 and (1) A partial or full-length peptide of an exogenous cytokine or its receptor, comprising at least one of the following: (v) a fusion protein of the membrane-bound Sushi domain of IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and its common receptor γC, wherein the common receptor γC is native or modified; and (vii) a homodimer of IL15Rβ; (2) an antibody or fragment thereof; or (3) an enforcer; or (4) a checkpoint inhibitor.

[0011] In some embodiments, the treatment of immune cells with small compounds occurs before or after cryopreservation of the immune cells. In some embodiments, the method further includes cryopreserving the immune cells that have been treated with small compounds. In certain embodiments, cryopreservation does not involve or substantially does not involve one or more of the small compounds of the treatment.

[0012] In some embodiments, enhanced post-thaw cytotoxicity includes enhanced in vivo efficacy of immune cells thawed after cryopreservation, where post-thaw immune cells having small compound treatment include at least one of the following features: (i) enhanced ability in tumor control, tumor clearance, and / or reduction of tumor recurrence; (ii) improved tumor penetration; or (iii) enhanced ability in migration to the bone marrow and / or tumor site compared to corresponding post-thaw immune cells without the same small compound treatment.

[0013] In another embodiment, the present invention provides cells or populations thereof, (i) the cells are immune cells treated with a small compound comprising at least one of dexamethasone, lenalidomide, AQX-1125, and their derivatives or analogs, and (ii) the immune cells have enhanced cytotoxicity after thawing compared to corresponding immune cells without the same small compound treatment. In some embodiments, of the cells or population thereof, (iii) the immune cells are derived effector immune cells differentiated from induced pluripotent stem cells (iPSCs), and (iv) the effector immune cells include derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional features not present in the corresponding primary T, NK, NKT, and / or B cells.In some embodiments, the iPSC is edited as follows: (i) a first chimeric antigen receptor (CAR) having a first target specificity; (ii) CD38 knockout; (iii) HLA-I deficiency and / or HLA-II deficiency compared to the corresponding native cell; (iv) introduction of HLA-G or non-cleavable HLA-G expression, or knockout of one or both CD58 and CD54; (v) CD16 or a variant thereof; (vi) a second CAR having a second target specificity; (vii) a signaling complex comprising a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor; (viii) at least one of the genotypes listed in Table 2; (ix) B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RF compared to the corresponding native cell. Effector immune cells differentiated from iPSCs include deletion or reduced expression of at least one of the following: XAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or introduced or increased expression of at least one of the following: (x)HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for binding to bispecific or multispecific or universal engagers, wherein effector immune cells differentiated from iPSCs include the same one or more edits as the iPSCs.

[0014] In some embodiments of cells or populations thereof, the small compound treatment is (i) comprising dexamethasone; (ii) not comprising or essentially not comprising cytokine IL7, and optionally the immune cells during treatment are T cells; (iii) not comprising or essentially not comprising cytokine IL2 and / or cytokine IL15, and optionally the immune cells during treatment are NK cells; (iv) comprising dexamethasone but not comprising cytokine IL7; (v) not comprising or essentially not comprising cytokines; (vi) during cell culture and / or before or after cell storage; (vii) during immune cell proliferation after differentiation of cells from iPSCs, and / or (viii) lasting about 1 to about 12 days, or about 3 to about 6 days, before cryopreservation. In some embodiments, dexamethasone is present in a concentration range of about 10 nM to about 20 μM.

[0015] In some embodiments of cells or populations thereof, immune cells are contained in a culture medium which (i) contains dexamethasone; (ii) contains lenalidomide; (iii) contains AQX-1125; (iv) contains dexamethasone and lenalidomide; (v) contains dexamethasone but does not contain cytokine IL7, and optionally the immune cells are T cells; (vi) contains dexamethasone but does not contain cytokine IL2 or cytokine IL15, and optionally the immune cells are NK cells; (vii) contains dexamethasone but does not contain cytokines, or does not contain cytokines in any meaningful way.

[0016] In these embodiments, the iPSC comprises a first chimeric antigen receptor (CAR) having a first target specificity, the first CAR comprising: (i) an ectodomain comprising at least one antigen recognition region, a transmembrane domain, and at least one signaling domain, wherein the at least one signaling domain comprises an ectodomain comprising an endodomain derived from the cytoplasmic domain of a signaling transduction protein specific for T and / or NK cell activation or functionalization; (ii) an antigen recognition domain that specifically binds to antigens associated with disease, pathogens, humoral tumors, or solid tumors; or (iii) any one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1; or ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD2 2, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV) infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFR-VIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, E GFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19).9) May contain Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), and Wilms' tumor protein (WT-1).

[0017] In some embodiments, the first CAR comprises a bicistronic construct co-expressing: (1) a partial or full-length cell surface peptide expressing an exogenous cytokine or its receptor, (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors; and (b) (i) co-expression of IL15 and IL15Rα using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, and (iii) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is cleaved or excluded, and (i (v) a fusion protein of the membrane-bound Sushi domain of IL15 and IL15Rα; (v) a fusion protein of IL15 and IL15Rβ; (vi) a fusion protein of IL15 and common receptor γC in which the common receptor γC is native or modified; and (vii) a homodimer of IL15Rβ; (2) a partial or full-length peptide of an exogenous cytokine or its receptor on the cell surface; (3) an antibody or fragment thereof; or (4) a checkpoint inhibitor.

[0018] In some embodiments of cells or populations thereof, small compound treatment of immune cells is performed before cryopreservation of the immune cells. In some embodiments, immune cells treated with small compounds are (i) in pre-cryopreservation medium, (ii) in cryopreservation medium, (iii) during cryopreservation, or (iv) after thawing from cryopreservation. In some embodiments, cryopreservation does not include or substantially does not include one or more small compounds of treatment. In some embodiments, enhanced post-thaw cytotoxicity includes enhanced in vivo efficacy of immune cells thawed after cryopreservation, where post-thaw immune cells having pre-cryopreservation small compound treatment include at least one of the following features: (i) enhanced ability in tumor control, tumor clearance, and / or reduction of tumor recurrence, (ii) improved tumor penetration, or (iii) enhanced ability in migration to bone marrow and / or tumor sites compared to corresponding post-thaw immune cells without the same small compound treatment.

[0019] In some embodiments, immune cells include one or more differentially expressed genes, including (i) upregulation of SPOCK2, PTGDS, IL7R, LCNL1, RASGRP2, SMAP2, IL6ST, IL-7R, and IL2RA, or (ii) downregulation of JCHAIN, KLF3, KLRB1, IGFBP4, NUCB2, CSF2RB, and CXCR6, compared to corresponding immune cells without the same small compound treatment.

[0020] In yet another aspect, the present invention provides a method for producing immune cells or populations thereof, the method comprising (a) differentiating genetically engineered iPSCs to obtain immune cells, the iPSCs having the following characteristics: (i) a first chimeric antigen receptor (CAR) having first target specificity; (ii) CD38 knockout; (iii) HLA-I deficiency and / or HLA-II deficiency compared to the corresponding natural cells; (iv) introduction of HLA-G or non-cleavable HLA-G expression, or one or both of CD58 and CD54 knockout. (v) CD16 or a variant thereof; (vi) a second CAR with second target specificity; (vii) a signaling complex comprising a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor; (viii) at least one of the genotypes listed in Table 2; (ix) B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG Deletion or reduced expression of at least one of 2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; or (x)HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and bispecific or multispecific or universal - comprising at least one of introduced or increased expression in at least one of surface trigger receptors for binding to a sulfenager, the immune cells differentiated from the iPSC comprising the same one or more edits as the iPSC, (b) the immune cells undergo treatment with a small compound comprising at least one of dexamethasone, lenalidomide, AQX-1125, or derivatives or analogs thereof, thereby obtaining immune cells having enhanced post-thaw cytotoxicity compared to the corresponding immune cells without the same small compound treatment. In some embodiments, the method further comprises (c) cryopreserving the treated immune cells from step (b).

[0021] In some embodiments, the method further comprises genomically manipulating a cloned iPSC to knock in a polynucleotide encoding a first CAR, optionally (ii) knocking out B2M and CIITA, (iii) knocking out one or both of CD58 and CD54, and / or (iv) introducing the expression of HLA-G or an uncleavable HLA-G, CD16 or a variant thereof, a second CAR, and / or an exogenous cytokine-expressing cell surface partial or complete peptide or its receptor. In some embodiments, the genomic manipulation includes targeted deletions, insertions, or in / dels, and the genomic manipulation is carried out by CRISPR, ZFN, TALEN, homing nucleases, homologous recombination, or any other functional mutation of these methods. In some embodiments, immune cells differentiated from induced pluripotent stem cells (iPSCs) include derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional features not present in the corresponding primary T, NK, NKT, and / or B cells. In some embodiments, the method further includes (d) thawing cryopreserved immune cells from step (c).

[0022] In yet another embodiment, the present invention provides compositions for therapeutic use comprising immune cells as described herein and one or more therapeutic agents. In some embodiments, one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory agents (IMiDs). In some embodiments, the therapeutic agent is a checkpoint inhibitor, and the checkpoint inhibitors are (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 (b) one or more antagonists to checkpoint molecules including 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 their derivatives or functional equivalents; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab. In some embodiments, the therapeutic agent may include one or more of venetoclax, azacitidine, and pomalidomide.In these embodiments where the therapeutic agent is an antibody, the antibody is (a) anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibody; (b) rituximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cerutuximab, dinutuximab, avelumab, (c) daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and one or more of their humanized or Fc-modified variants or fragments and their functional equivalents and biosimilars; or (c) daratumumab, inducing hematopoietic cells including CD38 knockout in induced NK cells or induced T cells, and optionally expressing CD16 or its variants. Thus, in another embodiment, the present invention provides therapeutic use of the compositions provided herein by introducing the compositions into subjects suitable for adoptive cell therapy, the subjects having autoimmune disorders, hematological malignancies, solid tumors, cancer, or viral infections.

[0023] In yet another embodiment, the present invention provides a method for treating a disease or condition comprising (i) thawing one or more units of cryopreserved immune cells produced according to a method disclosed herein, wherein the cryopreserved immune cells are treated with a small compound treatment described herein prior to cryopreservation, and (ii) administering a composition comprising the immune cells after thawing in step (i) to a subject. In some embodiments, the immune cells are iPSC-derived NK cells, iPSC-derived T cells, or iPSC-derived effector cells having one or more functional features not present in the corresponding primary T, NK, NKT, and / or B cells.

[0024] Various purposes and advantages of the compositions and methods provided herein will become apparent from the following description, together with the accompanying drawings illustrating certain embodiments of the invention, as examples and illustrations. [Brief explanation of the drawing]

[0025] [Figure 1A]This figure shows that dexamethasone treatment reduces granzyme B protein levels in iNK cells. [Figure 1B] This figure shows that dexamethasone treatment reduces granzyme B protein levels in primary NK cells. Granzyme B levels were determined by flow cytometry staining, and the geometric mean fluorescence intensity (GMFI) is shown. [Figure 2A] Figure 2A shows that treatment of CAR-expressing iNK cells with small compounds improves antigen-specific recognition by thawed iNK cells. [Figure 2B] This figure shows that treatment of CAR-expressing iNK cells with small compounds improves antigen-specific recognition of iNK cells after thawing and resting overnight. [Figure 3A] This figure shows a long-term killing assay using treated, thawed CD19-CAR-expressing iNK cells that target CD19+ lymphoma target cells. [Figure 3B] This figure shows a long-term killing assay using treated, thawed CD19-CAR-expressing iNK cells targeting CD19+ lymphoma target cells. The increased cytotoxicity of treated, thawed iNK cells is indicated using (Figure 3A) the normalized number of target cells remaining at each time point (target alone = 100) and (Figure 3B) the area on the curve (AOC). [Figure 4A] This figure shows the in vivo efficacy of previously treated small compound thawed CAR-expressing iNK cells compared to untreated counterpart cells, using bioluminescence imaging of NSG mice transplanted with 1E5 Nalm6-luciferase cells. [Figure 4B] This figure shows the in vivo efficacy of thawed CAR / hnCD16-expressing iNK cells that had previously been treated with small compounds in combination therapy with rituximab. [Figure 4C] This figure shows the in vivo efficacy of thawed CAR-expressing iNK cells previously treated with small compounds compared to untreated corresponding cells in a mouse model of solid tumor metastasis. [Figure 4D]This figure shows the in vivo persistence of CAR-expressing iNK cells after thawing, previously treated with a small compound, compared to untreated corresponding cells in the spleen of a mouse model. [Figure 4E] This figure shows the in vivo persistence of CAR-expressing iNK cells after thawing, which were previously treated with a small compound, compared to untreated corresponding cells in the peripheral blood of mice in the absence of tumors. [Figure 5] This figure shows differential gene expression analysis of iNK cells treated with small compounds using RNA-seq. [Figure 6] This figure shows the differentially expressed genes in iT cells treated with dexamethasone. [Figure 7A] This figure shows that the removal of IL7 during dexamethasone treatment of iT cells does not affect cell proliferation. [Figure 7B] This figure shows that IL7 removal during dexamethasone treatment of iT cells does not affect the cellular phenotype. [Figure 7C] This figure shows that IL7 removal during dexamethasone treatment of iT cells does not affect the cellular phenotype. [Figure 8A] This figure shows the in vivo efficacy of CAR-i T cells without dexamethasone treatment. [Figure 8B] This figure shows the in vivo efficacy of CAR-iT cells treated with dexamethasone. Treatment with small compounds improves the in vivo function of CAR-iT cells. [Figure 9A] This figure shows that CAR-iT cells treated with dexamethasone control tumor growth in a systemic xenography model of lymphocytic leukemia compared to primary CAR-T cells. [Figure 9B] This figure shows that CAR-iT cells treated with dexamethasone control tumor growth in a systemic xenographic model of lymphocytic leukemia compared to primary CAR-T cells. In Figure 9B, the clusters of lines from the highest to the lowest represent tumors only, primary CAR-T, CAR-iT+Dex, and IVIS. [Figure 10A]This figure shows that CAR-i T cells treated with dexamethasone survive in mouse bone marrow tissue in a systemic xenography model of lymphocytic leukemia. [Figure 10B] This figure shows that CAR-i T cells treated with dexamethasone survive in mouse bone marrow tissue in a systemic xenography model of lymphocytic leukemia. [Figure 11A] This figure shows the T cell phenotypic expression profiles of CAR-i T cells proliferated by dexamethasone treatment alone and IL-7 supplementation. [Figure 11B] This figure shows the T cell phenotypic expression profiles of CAR-i T cells proliferated by dexamethasone treatment alone and IL-7 supplementation. [Figure 11C] This figure shows that dexamethasone treatment supplemented with IL7 resulted in improved CAR-iT cell proliferation compared to dexamethasone treatment in the absence of cytokines. [Figure 11D] This figure shows that CAR-i T cells treated with dexamethasone alone and those treated with dexamethasone plus IL7 exhibit improved efficacy compared to untreated cells. [Modes for carrying out the invention]

[0026] definition

[0027] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular forms shall include plural forms and plural forms shall include singular forms.

[0028] The present invention is not limited to, and therefore may differ from, the specific methodologies, protocols, and reagents described herein. The terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

[0029] As used herein, the articles “a,” “an,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “element” means one or more elements.

[0030] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination thereof of the alternatives.

[0031] The term "and / or" should be understood to mean either one or both of the alternatives.

[0032] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “about” or “approximately” refer to a range of a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0033] As used herein, the terms “substantially” or “essentially” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “essentially the same” or “substantially the same” refer to a range of quantities, levels, values, numbers, frequencies, percentages, dimensions, size, volume, weight, or length that is approximately the same as the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0034] As used herein, the terms “substantially absent” and “essentially absent” are interchangeable and, when used to describe a composition such as a cell population or culture medium, refer to a composition that does not contain a particular substance or its source, for example, a composition that is 95%, 96%, 97%, 98%, 99% absent of a particular substance or its source, or undetectable when measured by conventional means. The terms “absent” or “essentially absent” of a particular component or substance in a composition also mean that such component or substance is (1) not present in the composition at any concentration, or (2) present in the composition at a low concentration, but functionally inactive. A similar meaning can be applied to the term “absent,” which refers to the absence of a particular substance or its source in a composition.

[0035] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” are understood to mean including the steps or elements or groups of steps or elements described, but not to mean excluding any other steps or elements or groups of steps or elements. In certain embodiments, the terms “include,” “have,” “contain,” and “comprise” are used as synonyms.

[0036] The phrase "consists of" means that what follows the phrase is included and limited to it. Therefore, the phrase "consists of" indicates that the listed elements are necessary or essential, and that other elements cannot exist.

[0037] "Essentially consisting of" means including any elements listed after the phrase, but limited to other elements that do not interfere with or contribute to the activity or action identified in the disclosure of the listed elements. Thus, the phrase "essentially consisting of" indicates that the listed elements are necessary or essential, but the other elements are not optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0038] Throughout this specification, references to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “a particular embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, mean that any specific features, structures, or characteristics described in relation to an embodiment are included in at least one embodiment of the present invention. Therefore, occurrences of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in any preferred manner in one or more embodiments.

[0039] The term “ex vivo” generally refers to activities performed outside of a living organism, such as experiments or measurements, conducted in or on living tissue in an artificial environment outside of a living organism, preferably with minimal alteration of natural conditions. In certain embodiments, “ex vivo” procedures involve living cells or tissues taken from a living organism and cultured in an experimental apparatus, usually under sterile conditions, for typically several hours or up to about 24 hours (but depending on the circumstances, up to 48 or 72 hours or more). In certain embodiments, such tissues or cells may be collected and frozen and later thawed for ex vivo treatment. Tissue culture experiments or procedures using living cells or tissues that last longer than several days are typically considered “in vitro,” but in certain embodiments, this term may be used interchangeably with “ex vivo.”

[0040] The term "in vivo" generally refers to activities performed within a living organism.

[0041] As used herein, the terms “drug,” “compound,” and “minor compound” are interchangeable herein and refer to compounds or molecules that can fine-tune the gene expression profile or biological properties of cells, including immune cells derived from the differentiation of pluripotent stem cells or progenitor cells. A drug may be a single compound or molecule, or a combination of multiple compounds or molecules.

[0042] Where used herein, in relation to the manufacture or production of immune cells, the terms “contact,” “treat,” or “treat” are interchangeable herein to mean culturing, incubating, or exposing immune cells with one or more of the agents disclosed herein so that the gene expression profile or one or more biological properties of the cells are regulated, fine-tuned, or modified.

[0043] As used herein, “uncontacted” or “untreated” cells are cells that have not been treated, for example, cultured, in contact with, or incubated with a drug other than the control drug. Cells that have been in contact with a control drug such as DMSO, or cells that have been in contact with another vehicle, are examples of uncontacted cells.

[0044] As used herein, the terms “reprogramming,” “dedifferentiation,” “increased differentiation potential,” and “increased developmental potential” refer to methods of increasing a cell’s differentiation potential or dedifferentiating a cell to a less differentiated state. For example, a cell with increased differentiation potential has greater developmental plasticity (i.e., can differentiate into more cell types) than the same cell that has not been reprogrammed. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell that has not been reprogrammed.

[0045] As used herein, the term “differentiation” is the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or differentiated cells are cells that have taken on a more specialized ("committed") position within a cell lineage. When applied to the process of differentiation, the term “committed” refers to a cell that, under normal circumstances, has progressed along the differentiation pathway to the point where it continues to differentiate into a particular cell type or subset of cell types, and under normal circumstances cannot differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term “pluripotency” refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo itself). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, ranging from incomplete or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce complete organisms to more primitive and pluripotent cells that can produce complete organisms (e.g., embryonic stem cells).

[0046] As used herein, the terms “induced pluripotent stem cells” or “iPSCs” mean stem cells produced from induced or modified, differentiated adult, neonatal, or fetal cells, i.e., cells that have been reprogrammed to differentiate into all tissues of all three germ layers or dermis: mesoderm, endoderm, and ectoderm. Produced iPSCs do not refer to cells found in nature.

[0047] As used herein, the term “embryonic stem cells” refers to the naturally occurring pluripotent stem cells in the inner cell mass of a blastocyst. Embryonic stem cells are pluripotent and give rise to all three major germ layer derivatives during development: the ectoderm, endoderm, and mesoderm. They do not contribute to the extraembryonic membrane or placenta; that is, they are not totipotent.

[0048] As used herein, the term “multipotent stem cell” refers to a cell that has the potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not all three. Thus, multipotent cells are also called “partially differentiated cells.” Multipotent cells are well known in the art, and examples of multipotent cells include, for example, adult stem cells such as hematopoietic stem cells and neural stem cells. “Multipotency” indicates that a cell may form many cell types within a given lineage, but not cells within other lineages. For example, a multipotent hematopoietic cell may form many different blood cell types (red, white, platelet, etc.), but not neurons. Thus, the term “multipotency” refers to a state of cell development that has a lower degree of developmental potential than totipotency and pluripotency.

[0049] Pluripotency can be determined in part by evaluating the pluripotent characteristics of cells. These characteristics include, but are not limited to, (i) the morphology of pluripotent stem cells, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) the formation of teratomas consisting of the three somatic cell lineages, and (vi) the formation of embryoid bodies consisting of cells from the three somatic cell lineages.

[0050] Two types of pluripotency have been previously described: a pluripotent "priming" or "metastable" state similar to that of late blastocyst epiblast stem cells (EpiSCs), and a pluripotent "naive" or "grounded" state similar to that of early / preimplantation blastocyst cell masses. Both pluripotent states exhibit the characteristics described above, but the naive or grounded state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single-cell culture, (iii) overall decreased DNA methylation, (iv) decreased deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) decreased expression of differentiation markers compared to pluripotent cells in the priming state. Standard methodologies of cell reprogramming in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or removed from the resulting pluripotent cells are generally considered to have the characteristics of the priming state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in a priming state and exhibit ground state characteristics unless the expression of exogenous transgenes that exhibit ground state characteristics is maintained.

[0051] As used herein, the term “pluripotent stem cell morphology” refers to the classic morphological features of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a high nucleus-to-cytoplasm ratio, prominent nucleoli, typical intercellular spacing, and a round, small shape.

[0052] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0053] A “pluripotency factor” or “reprogramming factor” refers to a drug that can increase the developmental potential of cells, either alone or in combination with other drugs. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of cells. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0054] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in each case, the singular "medium"), "supplementary components," and "medium supplementary components" refer to the nutritional composition used to culture cells.

[0055] "Culturing" or "maintaining" refers to maintaining, proliferating (growing), and / or differentiating cells outside of tissue or in vitro, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" or "maintaining" may utilize the culture medium as a source of nutrients, hormones, and / or other factors that help proliferate and / or maintain the cells.

[0056] As used herein, the term “mesoderm” refers to one of the three germ layers that appear during early embryonic development and give rise to various specialized cell types, including circulatory blood cells, muscle, heart, dermis, skeleton, and other supporting and connective tissues.

[0057] As used herein, the terms “secondary hematopoietic endothelial cells” (HE) or “pluripotent stem cell-derived secondary hematopoietic endothelial cells” (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem cells and progenitor cells in a process called endothelial hematopoietic transition. In embryos, hematopoietic cell development progresses sequentially from the lateral plate mesoderm through angioblasts to secondary hematopoietic endothelial cells and hematopoietic progenitor cells.

[0058] The terms “hematopoietic stem cells and progenitor cells,” “hematopoietic stem cells,” “hematopoietic progenitor cells,” or “hematopoietic progenitor cells” refer to cells that are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation, including pluripotent hematopoietic stem cells (hemocytoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including bone marrow (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid cells (T cells, B cells, NK cells). As used herein, the term “secondary hematopoietic stem cells” refers to CD34+ hematopoietic cells that can give rise to both mature bone marrow and lymphoid cell types, including T lineage cells, NK lineage cells, and B lineage cells. Hematopoietic cells include various subsets of primitive hematopoietic cells that give rise to primitive red blood cells, megakaryocytes, and macrophages.

[0059] As used herein, the terms “T lymphocyte” and “T cell” are interchangeable and refer to the major type of leukocyte that completes maturation in the thymus and plays various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells in an MHC class I-restrictive manner. T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from mammals, pluripotent stem cells, or T cells obtained from direct hematopoietic differentiation of progenitor cells. T cells are CD3 +A T cell can be any type of T cell, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), etc., and can be at any developmental stage. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). A T cell can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also differentiate from stem cells or progenitor cells. T cell-like derived effector cells may have T cell lineage in some respects, but at the same time possess one or more functional features that are not present in primary T cells.

[0060] As used herein, “CD4+ T cells” refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a post-stimulation secretion profile, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. “CD4” is a 55kD glycoprotein initially defined as a differentiation antigen on T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as an associated recognition element in the MHC (major histocompatibility complex) class II restriction immune response. In T lymphocytes, they define a helper / trigger subset.

[0061] As used herein, “CD8+ T cells” refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The “CD8” molecule is a differentiation antigen found in thymocytes, as well as cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a relevant recognition element in major histocompatibility complex class I restriction interactions.

[0062] As used herein, the terms “NK cells” or “natural killer cells” refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms “adaptive NK cells” and “memory NK cells” are interchangeable and phenotypically CD3 - and CD56 + This refers to a subset of NK cells that express at least one of NKG2C and CD57, and optionally CD16, but lack the expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, CD56 + The isolated subpopulation of NK cells includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and / or DNAM-1. + This can be dim or bright expression. NK cells, or NK cell-like effector cells, can be differentiated from stem cells or progenitor cells. NK cell-like derived effector cells may have some NK cell lineage but also possess one or more functional features not present in primary NK cells.

[0063] As used herein, the terms “NKT cells” or “natural killer T cells” refer to CD1d-restricted T cells that express a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire, i.e., standard α chains (Vα24-Jα18 in humans) associated with a limited spectral β chain (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, exhibits a more heterogeneous use of TCRαβ. Type I NKT cells are considered suitable for immunotherapy. Adapted or invariant (type I) NKT cells can be identified by the expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0064] As used herein, the term “isolated” means cells or populations of cells separated from their original environment; that is, the environment of isolated cells substantially does not contain at least one component found in the environment in which “unisolated” reference cells exist. This term includes cells isolated from some or all components found in a natural environment, for example, cells isolated from tissue or biopsy specimens. This term also includes cells isolated from at least one, some or all components, such as cells isolated from a cell culture or cell suspension, because the cells are found in an environment in which they do not naturally exist, for example. Thus, isolated cells, when found in nature or when grown, preserved, or surviving in an environment in which they do not naturally exist, are partially or completely separated from at least one component, including other substances, cells, or populations of cells. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in media that do not naturally exist. Isolated cells can be obtained by separating desired cells or populations of cells from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0065] As used herein, terms such as "purify" refer to increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0066] As used herein, the term “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, which has either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties derived therefrom, and which serve as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, may be said to code for the protein or other product of that gene or cDNA.

[0067] "Construction" refers to a macromolecule or molecular complex containing polynucleotides that is delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct that can be directed to deliver or transport foreign genetic material to a target cell and can be replicated and / or expressed in the target cell. As used herein, the term "vector" includes the construct to be delivered. A vector may be a linear or cyclic molecule. A vector may be embedded or unembedded. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, and Sendai virus vectors.

[0068] "Integration" means that one or more nucleotides of a construct are stably inserted into the cellular genome, i.e., covalently bonded to a nucleic acid sequence within the cell's chromosomal DNA. "Targeted integration" means that the nucleotides of a construct are inserted into the cell's chromosome or mitochondrial DNA at a pre-selected site or "integration site." As used herein, the term "integration" further refers to the process of inserting one or more exogenous sequences or nucleotides of a construct, with or without deletion of endogenous sequences or nucleotides at the integration site. If there is a deletion at the insertion site, "integration" may further include replacing the deleted endogenous sequence or nucleotide with one or more inserted nucleotides.

[0069] As used herein, the term “exogenous” is intended to mean that the referenced molecule or activity is introduced into the host cell or is not native to the host cell. The molecule may be introduced, for example, by introducing the coding nucleic acid into the host's genetic material, for example, by incorporating it into the host's chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used in relation to the expression of coding nucleic acid, the term refers to the introduction of the coding nucleic acid into the cell in an expressible form. The term “endogenous” refers to the referenced molecule or activity that is present in the host cell. Similarly, when used in relation to the expression of coding nucleic acid, the term refers to the expression of coding nucleic acid that is present in the cell and not introduced exogenously.

[0070] As used herein, “Gene of Interest” or “Polynucleotide Sequence of Interest” is a DNA sequence that, under the control of an appropriate regulatory sequence, is transcribed into RNA and, possibly translated in vivo, into a polypeptide. Genes of Interest or polynucleotides may include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from DNA of eukaryotes (e.g., mammals), and synthetic DNA sequences. For example, a gene of Interest may encode miRNA, shRNA, natural polypeptides (i.e., polypeptides found in nature) or fragments thereof; variant polypeptides (i.e., variants of natural polypeptides having less than 100% sequence identity with the natural polypeptide) or fragments thereof; engineered polypeptides or peptide fragments, therapeutic peptides or polypeptides, imaging markers, selectable markers, etc.

[0071] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, which is either a deoxyribonucleotide or a ribonucleotide or an analog thereof. The sequence of a polynucleotide consists of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) for thymine. Polynucleotides may include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to both double-stranded and single-stranded molecules.

[0072] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, also commonly referred to in the art as polypeptides or proteins. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0073] "Operatively linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is influenced by the other. For example, a promoter is operationally linked to a coding sequence or functional RNA if it can influence the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operationally linked to a regulatory sequence in sense or antisense orientation.

[0074] As used herein, the term “genetic imprint” refers to genetic or epigenetic information that contributes to preferred therapeutic attributes in source cells or iPSCs and is retainable in source cell-derived iPSCs and / or iPSC-derived hematopoietic cells. As used herein, “source cell” is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, and source cell-derived iPSCs can be further differentiated into specific cell types, including any hematopoietic cell lineage. Source cell-derived iPSCs, and cells differentiated therefrom, are sometimes collectively referred to as “derived” or “derived” cells, depending on the context. For example, derived effector cells, derived NK lineage cells, or derived T lineage cells as used throughout this specification are cells differentiated from iPSCs compared to primary corresponding cells obtained from natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, gene imprints conferring preferred therapeutic attributes are incorporated into iPSCs either by reprogramming donor-specific, disease-specific, or treatment-response-specific selected source cells, or by introducing a genetically modified modality into iPSCs using genome editing. In the case of source cells obtained from a specifically selected donor, disease, or treatment situation, the gene imprints contributing to preferred therapeutic attributes may include situation-specific genetic or epigenetic traits that represent a retainable phenotype, i.e., preferred therapeutic attribute, which are passed on to derived cells of the selected source cells, regardless of whether the underlying molecular event has been identified. Donor-specific, disease-specific, or treatment-response-specific source cells may include gene imprints that can be retained in iPSCs and derived hematopoietic lineage cells, which may include, but are not limited to, pre-configured single-specific TCRs from virus-specific T cells or invariant natural killer T (iNKT) cells; traceable and desirable gene polymorphisms, such as homozygosity for point mutations encoding high-affinity CD16 receptors in selected donors; and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting an increased population haplotype.When used herein, preferred therapeutic attributes include improved engraftment, transport, homing, viability, self-renewal, persistence, regulation of immune responses, survival, and cytotoxicity of derived cells. Preferred therapeutic attributes may also relate to antigen-targeting receptor expression, HLA presentation or absence, resistance to the tumor microenvironment, induction and immune regulation of bystander immune cells, improved target specificity through reduced extratumor effects, and / or resistance to treatments such as chemotherapy.

[0075] As used herein, the term “enhanced therapeutic properties” refers to the therapeutic properties of a cell that are enhanced compared to a typical immune cell of the same common cell type. For example, NK cells with “enhanced therapeutic properties” have enhanced, improved, and / or increased therapeutic properties compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, transport, homing, viability, self-renewal, persistence, regulation of immune responses, survival, and cytotoxicity. Therapeutic properties of immune cells can also be indicated by the expression of antigen-targeting receptors, HLA presentation or absence, resistance to the tumor microenvironment, induction and immune regulation of bystander immune cells, improved target specificity by reducing extratumor effects, and / or resistance to treatments such as chemotherapy.

[0076] As used herein, the term “engager” refers to a molecule, such as a fusion polypeptide, that can form links between immune cells, such as T cells, NK cells, NKT cells, B cells, macrophages, neutrophils, and tumor cells, and activate immune cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), triplicate killer cell engagers, multispecific killer cell engagers, or universal engagers compatible with multiple immune cell types.

[0077] As used herein, the term “surface trigger receptor” refers to a receptor that can induce or initiate an immune response, such as a cytotoxic response. Surface trigger receptors can be manipulated and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, surface trigger receptors facilitate the binding of bispecific or multispecific antibodies between effector cells and specific target cells, such as tumor cells, regardless of the effector cell's native receptor and cell type. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and then differentiated into populations of various effector cell types expressing the universal surface trigger receptor. “Universal” means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind to or ligate to an engager having the same epitope recognizable by the surface trigger receptor, regardless of the engager’s tumor-binding specificity. In some embodiments, an engager having the same tumor-targeting specificity is used to bind to the universal surface trigger receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to universal surface trigger receptors. Therefore, one or more effector cell types may be used to kill one specific type of tumor cell, or two or more types of tumor cells. Surface trigger receptors generally include a co-stimulatory domain for effector cell activation and an epitope-binding region specific to the engager's epitope. A bispecific engager is specific to the epitope-binding region of the surface trigger receptor at one end and specific to the tumor antigen at the other end.

[0078] As used herein, the term “safety switch protein” refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of cell therapy. In some cases, the expression of a safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells, in which the gene encoding the safety switch protein is permanently incorporated into its genome. This conditional control may be variable and may include control by post-translational activation via small molecules and control by tissue-specific and / or transient transcriptional regulation. Safety switches may mediate the induction of apoptosis, inhibition of protein synthesis, arrest of DNA replication and growth, transcription and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some cases, safety switch proteins are activated by exogenous molecules, such as prodrugs, which, upon activation, induce apoptosis and / or cell death in therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered in the event of an adverse event is activated by a suicide gene product, killing the transduced cells.

[0079] As used herein, the term “pharmaceutically active protein or peptide” refers to a protein or peptide capable of achieving biological and / or pharmaceutical effects on an organism. A pharmaceutically active protein may have curative, radical, or palliative properties to a disease and may be administered to restore, alleviate, reduce, reverse, or mitigate the severity of the disease. A pharmaceutically active protein may also have prophylactic properties and may be used to prevent the onset of a disease or, when it appears, to mitigate the severity of such a disease or pathological condition. A pharmaceutically active protein may include whole proteins or peptides, or pharmaceutically active fragments thereof. It may also include pharmaceutically active analogues of proteins or peptides, or analogues of protein or peptide fragments. The term pharmaceutically active protein may also refer to multiple proteins or peptides that act synergistically or cooperatively to provide therapeutic benefits. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0080] As used herein, the term “signaling molecule” refers to any molecule that affects, is involved in, inhibits, activates, reduces, or increases cellular signaling. “Signaling” refers to the transmission of molecular signals in the form of chemical modifications by the recruitment of protein complexes along pathways that ultimately lead to intracellular biochemical events. Signaling pathways are well known in the art and include, but are not limited to, G protein-bound receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Tollgate signaling, ligand-dependent ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0081] As used herein, the term “targeting modality” refers to molecules, e.g., polypeptides, that are genetically incorporated into cells and promote antigen and / or epitope specificity, including but not limited to: i) antigen specificity when related to a unique chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when related to a monoclonal antibody or bispecific engager; iii) targeting of transformed cells; iv) targeting of cancer stem cells; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0082] As used herein, the terms “specific” or “singularity” may be used to refer to the ability of a molecule, such as a receptor or engager, to selectively bind to a target molecule, as opposed to nonspecific or nonselective binding.

[0083] When used herein, the term “adoptive cell therapy” refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, which are identified as T cells or B cells, whether genetically modified or not, that have been expanded in vivo before infusion.

[0084] As used herein, “therapeutably sufficient amount” means, within its meaning, a sufficient and / or effective amount of the particular therapeutic and / or pharmaceutical composition it refers to, that is nontoxic but provides the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient’s general health, the patient’s age, and the stage and severity of the condition. In a particular embodiment, a therapeutically sufficient amount is sufficient and / or effective to restore, reduce, and / or improve at least one symptom associated with the disease or condition being treated.

[0085] Differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium or changes in the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a general and important intermediate to initiate lineage-specific differentiation. Embryoid bodies are three-dimensional clusters that have been shown to mimic embryogenesis by giving rise to numerous lineages within a three-dimensional region. Through a differentiation process that typically lasts from a few hours to several days, simple EBs (e.g., aggregated pluripotent stem cells that induce differentiation) continue to mature and develop into cystic EBs, at which point (typically several days to several weeks) they are further treated and continue to differentiate. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other within a three-dimensional multilayer cell cluster, which is typically achieved by one of several methods, including settling the pluripotent cells into droplets, settling the cells into a "U" bottom well plate, or by mechanical agitation. Aggregates maintained in pluripotent culture maintenance medium do not form proper EBs, so pluripotent stem cell aggregates require further differentiation cues to promote EB development. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cues for induction toward the selected lineage. EB-based culture of pluripotent stem cells typically results in the generation of differentiated cell populations (ectoderm, mesoderm, and endoderm) with moderate proliferation within EB cell clusters. While proven to promote cell differentiation, EBs produce heterogeneous cells in different differentiation states because the three-dimensional cells are not consistently exposed to differentiation cues from the environment. In addition, EBs are difficult to produce and maintain. Furthermore, EB-mediated cell differentiation involves moderate cell expansion, which also contributes to reduced differentiation efficiency.

[0086] In contrast, "aggregate formation," distinct from "EB formation," can be used to expand populations of pluripotent stem cell-derived cells. For example, during aggregate-based expansion of pluripotent stem cells, culture media are selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of aggregates, which form larger aggregates, and these aggregates can routinely dissociate into smaller aggregates mechanically or enzymatically to maintain cell proliferation in culture and increase cell number. Unlike EB culture, cells cultured in aggregates under maintenance culture retain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0087] As used herein, “monolayer differentiation” refers to a differentiation method distinct from differentiation by three-dimensional multilayer clustering of cells, i.e., “EB formation.” Among the other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Because monolayer culture does not mimic embryonic development such as EB formation, differentiation into a particular lineage is considered minimal compared to the differentiation of all three germ layers in EB.

[0088] As used herein, “dissociated” cells refer to cells that have been substantially separated or purified from other cells or from a surface (e.g., the surface of a culture plate). For example, cells may be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro may be dissociated from each other enzymatically or mechanically, such as by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture plate or other surface. Thus, dissociation may involve disruption of cell interactions with the extracellular matrix (ECM) and substrate (e.g., the culture surface), or disruption of the ECM between cells.

[0089] As used herein, “feeder cells” or “feeder” is a term describing one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, expand, or differentiate, by providing stimuli, growth factors, and nutrients to support the second cell type. Feeder cells are, by choice, from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with mitotic antagonists such as mitomycin to prevent them from growing more than the cells they support when co-cultured with other cells. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type may be a human feeder, such as human dermal fibroblasts. Another feeder cell type may be a mouse embryonic fibroblast (MEF). In general, various feeder cells can be used partially to maintain pluripotency, direct differentiation into a particular lineage, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells.

[0090] As used herein, “feeder-free” (FF) environment refers to an environment such as culture conditions, cell cultures, or culture media that essentially do not contain feeder cells or stromal cells and / or are not pre-conditioned by feeder cell culture. “Pre-conditioned” medium refers to a medium that has been collected after feeder cells have been cultured in the medium for a period of at least one day. Pre-conditioned 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 pre-conditioned by feeder cell culture.

[0091] When used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, “functional” means (1) good knock-in, knock-out, knock-down gene expression, transgenic or controlled gene expression at the gene level achieved by direct genome editing or modification, or by “transmission” through differentiation or reprogramming from the initial genomically engineered initiating cell, e.g., inducible or transient expression at a desired cell developmental stage, or (2)(i) gene expression obtained in the cell by direct genome editing. (ii) modification of gene expression maintained in the cell by “transmission” through differentiation or reprogramming from the initial genomically engineered initiating cell, (iii) downstream gene regulation in the cell as a result of gene expression modification that appears only in the early developmental stages of the cell, or only in the initiating cell that gives rise to the cell through differentiation or reprogramming, or (iv) removal, addition, or alteration of favorable cellular functions / characteristics at the cellular level by enhanced or newly acquired cellular functions or attributes presented in the mature cell product, which originate from genome editing or modification performed initially on an iPSC, progenitor cell, or dedifferentiated cell origin.

[0092] "HLA deficiency," including HLA class I deficiency, HLA class II deficiency, or both, refers to any cell in which the surface expression of the complete MHC complex, comprising the HLA class I protein heterodimer and / or the HLA class II heterodimer, is absent, no longer maintained, or has a reduced level such that the reduction or decrease is lower than that naturally detectable by other cells or synthetic methods.

[0093] As used herein, “modified HLA-deficient iPSC” refers to an HLA-deficient iPSC that is further modified by introducing genes expressing proteins associated with, but not limited to, improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, co-stimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 41BB, DAP10, DAP12, CD24, CD3ζ, 41BBL, CD47, CD113, and PDL1. “Modified HLA-deficient” cells also include cells other than iPSCs.

[0094] Fc receptors, abbreviated as "FcR," are classified based on the type of antibody they recognize. For example, those that bind to IgG, the most common class of antibody, are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). The classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells, and B cells) and the signaling characteristics of each receptor. Fc-gamma receptors (FcγR) include several members such as FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), and FcγRIIIB(CD16b), which have different antibody affinities due to their different molecular structures.

[0095] The term "chimeric Fc receptor," abbreviated as "CFcR," is used to describe engineered Fc receptors in which the native transmembrane and / or intracellular signaling domains are modified or replaced with non-native transmembrane and / or intracellular signaling domains. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane and / or signaling domains being non-native, one or more stimulating domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance cell activation, expansion, and function through receptor induction. Unlike chimeric antigen receptors (CARs) that contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to Fc fragments, or the Fc region of an antibody, or the Fc region contained in an engager or binding molecule, activating cells regardless of whether the targeted cell is in proximity. For example, the Fcγ receptor may be engineered to include a selected transmembrane domain, a stimulating domain, and / or signaling domains within the intracellular region that respond to the binding of IgG at the extracellular domain, thereby generating a CFcR. In one example, CFcRs are generated by manipulating the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domains. To further improve the binding affinity of CD16-based CFcRs, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcRs with a high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is eliminated, or the extracellular domain of the receptor is replaced in a way that makes it uncleavable, i.e., unaffected by shedding, thereby yielding an hnCD16-based CFcR.

[0096] The FcγR receptor CD16 has been identified to have two isoforms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and facilitating antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high affinity CD16," "uncleavable CD16," or "high affinity uncleavable CD16 (hnCD16)" refer to native or non-native variants of CD16, respectively. Wild-type CD16 has low affinity and undergoes ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes, upon activation by NK cells. F176V and F158V (without signal peptide) are exemplary native CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane proximal region (positions 189-212) is modified or eliminated are not subject to shedding. The cleavage site and membrane proximal region are described in detail in International Publication 2015 / 148926, the full disclosure of which is incorporated herein by reference. The CD16 S197P variant is an engineered non-cleavable version of CD16. CD16 variants containing both F158V and S197P are highly affinity and non-cleavable. Another exemplary highly affinity and non-cleavable CD16 (hnCD16) variant is engineered CD16 containing an ectodomain derived from one or more of the three exons of the CD64 ectodomain. I. Agents for the production of effector cells and improvement of in vivo efficacy in adoptive immunotherapy

[0097] Cryopreservation is a process known to have a significant impact on cell viability, function, and stability. In some embodiments, the present disclosure provides compositions comprising one or more agents in sufficient quantities to improve the production of effector cells suitable for adoptive cell-based therapies and their in vivo efficacy, particularly when effector cells need to be cryopreserved and thawed before use.

[0098] In various embodiments, immune cells suitable for adoptive cell-based therapy are contacted or treated with one or more drugs, including but not limited to dexamethasone, lenalidomide, AQX-1125, and derivatives, analogues, or pharmaceutically acceptable salts of the drugs(s), esters, ethers, solvates, hydrates, stereoisomers, and prodrugs. Treatment with the selected drugs(s) can enhance the biological properties of cells or cell subpopulations, including regulating cell enlargement, maintenance, survival, proliferation, cytotoxicity, persistence, and / or cellular memory, and thus enhance the therapeutic potential of the cells. Dexamethasone is a glucocorticoid that binds to cytoplasmic glucocorticoid receptors to form ligand receptor complexes, which then translocate to the cell nucleus, where the complex binds to glucocorticoid response elements in its promoter region, resulting in transcriptional activation of target genes associated with anti-inflammatory and immunosuppressive effects. Dexamethasone, for example, a glucocorticoid receptor agonist, is a synthetic glucocorticoid used in this application, known for its potent anti-inflammatory and immunosuppressive properties, to unexpectedly modulate differentiated effector cells and achieve enhanced cryopreservation durability and in vivo efficacy.

[0099] Additional exemplary glucocorticoids suitable for use in the methods of this disclosure include medrizone, alclomethasone, alclomethasone dipropionate, amcinonide, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone benzoate, betamethasone valerate, budesonide, ciclesonide, clobetasol, clobetasol butyrate, clobetasol propionate, clobetazone, crocortol, cloprednol, cortisol, cortisone, cortibazo Flumethasone, deflazacort, desonide, deoxymethasone, deoxycorton, deoxymethasone, diflorason, diflorason diacetate, diflucortone, diflucortone valerate, difluorocortone, difluprednate, fluchlorolone, fluchlorolone acetonide, fludroxycortide, flumethasone (flumetasone, flumethasone), flumethasone pivalate, flunisolid, flunisolid hemihydrate, fluocinolone, fluosinolone Nolon acetonide, fluocinonide, fluocortin, fluocortin butyl, fluocortolone, fluorocortisone, fluorometholone, fluperolone, flupredniden, flupredniden acetate, fluprednisolone, fluticasone, fluticasone propionate, formocortal, halcinonide, halomethasone, hydrocortisone, hydrocortisone acetate, hydrocortisone aceponate, hydrocortisone buteplat, hydrocortisone butyrate This includes, but is not limited to, roteprednol, meprednisone, 6a-methylprednisolone, methylprednisolone acetate, methylprednisolone aceponate, mometasone, mometasone florate, mometasone florate monohydrate, parametasone, prednicarbart, prednisolone, prednisone, prednylidene, rimexolone, thixocortol, triamcinolone, triamcinolone acetonide, and urobetasol, as well as combinations thereof. In certain embodiments, the glucocorticoid includes medrisone, hydrocortisone, triamcinolone, alclometasone, or dexamethasone. In more specific embodiments, the glucocorticoid is dexamethasone or its derivatives, analogs, or pharmaceutically acceptable salts thereof.

[0100] In some embodiments, a composition for improving the therapeutic potential of immune cells suitable for adoptive cell-based therapy comprises at least one of dexamethasone, lenalidomide, AQX-1125, or derivatives or analogs thereof. In one embodiment, a composition for improving the therapeutic potential of immune cells comprises dexamethasone and lenalidomide, and / or combinations of derivatives and analogs thereof.

[0101] In one embodiment, a composition comprising at least one of dexamethasone, lenalidomide, AQX-1125, or derivatives or analogs thereof further comprises an organic solvent. In certain embodiments, the organic solvent is substantially free of methyl acetate. In certain embodiments, the organic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, and combinations thereof. In some embodiments, the organic solvent is DMSO. In some embodiments, the organic solvent is ethanol. In some other embodiments, the organic solvent is a mixture of DMSO and ethanol.

[0102] In some embodiments, a composition comprising one or more of dexamethasone, lenalidomide, AQX-1125, or their derivatives or analogues further comprises one or more additional additives selected from the group consisting of peptides, cytokines, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or substitution factors, vectors comprising one or more polynucleic acids of interest, antibodies, and antibody fragments thereof. In some embodiments, the additional additives comprise antibodies or antibody fragments. In some of these 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.

[0103] In some embodiments, cytokines and growth factors include one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia suppressor factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF) transferrin, various interleukins (IL-1 to IL-18, etc.), various colony-stimulating factors (granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (IFN-γ, etc.), stem cell factor (SCF), and erythropoietin (Epo). In some embodiments, the cytokines include at least interleukin-2 (IL-2 or IL2), interleukin-7 (IL-7 or IL7), interleukin-12 (IL-12 or IL12), interleukin-15 (IL-15 or IL15), interleukin-18 (IL-18 or IL18), interleukin-21 (IL-21 or IL21), or any combination thereof. In some embodiments, the growth factors of the composition include fibroblast growth factors. These cytokines can be commercially available, for example, from R&D Systems (Minneapolis, Minn.), and may be either natural or recombinant. In certain embodiments, the growth factors and cytokines may be added at concentrations intended herein. In certain embodiments, the growth factors and cytokines may be added at concentrations determined empirically or as guided by established cytokine techniques. In some other embodiments, compositions comprising dexamethasone, lenalidomide, AQX-1125, or one of their derivatives or analogues, do not contain IL7 for T-cell therapy. In certain embodiments, compositions comprising dexamethasone, lenalidomide, AQX-1125, or one of their derivatives or analogues, do not contain cytokines for T-cell therapy.In some other embodiments, a composition comprising dexamethasone, lenalidomide, AQX-1125, or one of their derivatives or analogues, does not contain IL2 and / or IL15 for NK cell therapy. In some other embodiments, a composition comprising dexamethasone, lenalidomide, AQX-1125, or one of their derivatives or analogues, does not contain IL7 for NK cell therapy. In certain embodiments, a composition comprising dexamethasone, lenalidomide, AQX-1125, or one of their derivatives or analogues, does not contain any cytokines for NK cell therapy.

[0104] Cells suitable for therapy using a composition comprising dexamethasone, lenalidomide, AQX-1125, or one of their derivatives or analogues include, but are not limited to, naturally occurring cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue, such as T, NK, NKT, B cells or any of their subtypes; or derived cells obtained from differentiated induced pluripotent stem cells (iPSCs). Derived cells may be any one of derived CD34 cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, or derived B cells. In some embodiments, the population of immune cells for therapy may be differentiated in vitro from stem cells, hematopoietic stem or progenitor cells, or progenitor cells, or trans-differentiated from hematopoietic or non-hematopoietic lineage non-pluripotent cells. In some embodiments, the stem cells, hematopoietic stem cells, or progenitor cells, progenitor cells, or non-pluripotent cells that induce immune cells for regulation are genomically engineered, including insertions, deletions, and / or nucleic acid substitutions, and the immune cells induced for therapeutic purposes contain the same gene modality as that introduced into the source cells by genomic engineering.

[0105] In one embodiment, a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy comprises contacting immune cells with a composition comprising at least one small compound provided herein, wherein the contacted immune cells have enhanced post-thaw cytotoxicity and comprise enhanced in vivo efficacy characterized by enhanced ability in tumor control, tumor clearance, and / or reduction of tumor recurrence, compared with the corresponding post-thaw immune cells without treatment with the same small compound, resulting in improved tumor penetration and / or enhanced ability to migrate to the bone marrow and / or tumor site.

[0106] In some embodiments, a method for treating a population or subpopulation of immune cells suitable for adoptive cell-based therapy involves contacting the immune cells with a composition comprising at least one small compound provided herein in an amount sufficient to enhance cellular efficacy. In one embodiment, the small compound for immunotherapy is present at a concentration of about 10 nM to about 20 μM. In one embodiment, the compound for immunotherapy is present at a concentration of about 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 3 μM, 5 μM, 10 μM, 15 μM, or 20 μM, or any concentration in between. In one embodiment, the concentration of the compound for immunotherapy is approximately 10 nM to 100 nM, approximately 50 nM to 250 nM, approximately 100 nM to 500 nM, approximately 250 nM to 1 μM, approximately 500 nM to 5 μM, approximately 1 μM to 5 μM, approximately 3 μM to 10 μM, approximately 5 μM to 15 μM, approximately 8 μM to 12 μM, or approximately 15 μM to 20 μM.

[0107] In some embodiments, a method for modulating a population or subpopulation of immune cells suitable for adoptive cell-based therapy involves contacting immune cells with a composition comprising at least one of the compounds provided herein for a period of time sufficient to enhance cellular efficacy. In one embodiment, immune cells are contacted with one or more of the provided compounds for at least 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, or any period in between. In one embodiment, immune cells are contacted with one or more of the provided compounds for about 18 hours to about 2 days, about 1 day to about 3 days, about 2 days to about 5 days, about 3 days to about 6 days, about 5 days to about 8 days, about 7 days to about 10 days, about 8 days to about 12 days, about 11 days to about 14 days. In some embodiments, immune cells are exposed to one or more compounds provided herein for a period of at least 2 days, 1 day, 18 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or any period in between. Thus, the length of such sufficient time is, for example, 48, 24, 15, 13, 11, 9, 7, 5, 3, or 1 hour or more. In some other embodiments of this method, the length of such sufficient time is at least 5 days, 4 days, 3 days, 2 days, or any period in between. Thus, the length of such sufficient time is, for example, 5, 4, 3, or 2 days or more.

[0108] Cells treated with a composition containing dexamethasone, lenalidomide, AQX-1125, or one of their derivatives or analogs may be in static / maintenance culture or culture for cell proliferation. In some embodiments, treatment of derived cells obtained from iPSC differentiation with the small compound composition may occur during the post-differentiation proliferation stage. In some embodiments, cells are treated with the small compound composition before cryopreservation. The treatment lasts for a sufficient period, and may extend for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days or more. In some embodiments, the treatment lasts for 2 to 7 days. In some embodiments, the treatment lasts for 5 days. In some embodiments, if the treated cells are cryopreserved, the culture medium does not contain, or essentially does not contain, the compound(s) used in the treatment prior to cryopreservation. II. Adoptive cells suitable for functional regulation

[0109] Suitable adoptive cells for functional modulation provided herein include derived effector cells obtained from differentiating genome-engineered iPSCs, both of which include CAR, CD38 knockout; CD16; exogenous cytokines and / or their signaling components; HLA-I and / or HLA-II deficiency; HLA-G overexpression and knockout of one or both CD58 and CD54; TCR null; CD3-presenting surface; antigen-specific TCR; NKG2C; DAP10 / 12; NKG2C-IL15-CD33 ("2C1533"), one or more of the additional edits described in further detail in this specification or known in the art. 1. Chimeric antigen receptor (CAR)

[0110] Applicable to genetically engineered iPSCs and their derived effector cells may be any chimeric antigen receptor (CAR) design known in the art. A CAR is a fusion protein generally comprising an ectodomain, which includes an antigen recognition region, a transmembrane domain, and an endodomain. In some embodiments, the ectodomain may further comprise a signal peptide or leader sequence and / or a spacer. In some embodiments, the endodomain may further comprise a signaling peptide that activates effector cells expressing the CAR. In some embodiments, the CARs described herein are designed to be expressed and function in induced pluripotent stem cells (iPSCs) and derived effector cells differentiated from iPSCs engineered to constitute a CAR. In some embodiments, the CARs described herein are designed not to interfere with iPSC differentiation and / or to promote the differentiation of iPSCs directed toward a desired effector cell type. In some embodiments, CARs enhance the proliferation, persistence, survival, cytotoxicity, resistance to allorejection, tumor penetration, migration, ability to activate and / or recruit bystander immune cells, and / or ability to overcome tumor suppression of effector cells. In embodiments, the CARs provided herein may also be directly expressed in cell lines and cells from primary sources, i.e., natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissues.

[0111] In some embodiments, the CAR is suitable for activating either T cells or NK cell lineage cells that express the CAR. In some embodiments, the CAR containing an NK-specific signaling component is NK cell-specific. In certain embodiments, the T cells are derived from a CAR-expressing iPSC, and the derived T cells may include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or a combination thereof. In certain embodiments, the NK cells are derived from a CAR-expressing iPSC. In some embodiments, the CAR containing an NK cell-specific signaling component is NK cell-specific. In some embodiments, the CAR containing an NK cell-specific signaling component is also suitable for T cells or other cell types. In some embodiments, the CAR containing a T cell-specific signaling component is T cell-specific. In some embodiments, the CAR containing a T cell-specific signaling component is also suitable for NK cells or other cell types. In some embodiments, the CAR containing an NKT cell-specific signaling component is NKT cell-specific. In some embodiments, the CAR containing an NKT cell-specific signaling component is also suitable for NK cells or T cells, or other cell types.

[0112] In some embodiments, the CARs described herein include at least an ectodomain, a transmembrane domain, and an endodomain. The endodomain of the CAR includes at least one signaling domain that influences the proliferation and function of cells expressing the CAR and activates effector cells expressing the CAR upon antigen binding. In some embodiments of the CAR endodomain, one or more co-stimulatory domains (often also called additional signaling domains) are further included to influence cell lifespan, memory differentiation, and metabolic properties. Here, signaling proteins specific to T cells and / or NK cells are used to provide the building blocks of a CAR fusion protein, e.g., the transmembrane domain and one or more signaling domains contained in the endodomain of the CAR. Exemplary signaling proteins suitable for CAR design include, but are not limited to, 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, and CD8. A description of exemplary signaling proteins, including their transmembrane and cytoplasmic sequences, is provided below.

[0113] In some embodiments of the CAR, the endodomain of the CAR includes at least a first signaling domain having an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the cytoplasmic domain or portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8. In some embodiments, the signaling domain of the CAR disclosed herein comprises only a portion of the cytoplasmic domain of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8. In some embodiments, the portion of the cytoplasmic domain selected for the CAR signaling domain is an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with ITAM (immune receptor tyrosine-based activation motif), YxxM motif, TxYxxV / I motif, FcRγ, hemi-ITAM, and / or ITT-like motif.

[0114] In some embodiments of the CAR, the endodomain of the CAR comprises a first signaling domain further comprising a second signaling domain having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the cytoplasmic domain or portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ / 1XX (i.e., CD3ζ or CD3ζ1XX), CS1, or CD8, wherein the second signaling domain is distinct from the first signaling domain.

[0115] In some embodiments of CAR, the endodomain of CAR comprises first and second signaling domains further comprising a third signaling domain having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the cytoplasmic domain or portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ / 1XX (i.e., CD3ζ or CD3ζ1XX), CS1, or CD8, wherein the third signaling domain is distinct from the first and second signaling domains.

[0116] In some exemplary embodiments of a CAR having an end domain consisting of only one signaling domain, the end domain comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with a cytoplasmic domain or portion thereof of a protein including, but not limited to, DNAM1, CD28H, KIR2DS2, DAP12, or DAP10.

[0117] In some exemplary embodiments of a CAR having an end domain composed of two different signaling domains, the end domain is 2B4-CD3ζ / 1XX, 2B4-DNAM1, 2B4-FcERIγ, 2B4-DAP10, CD16-DNAM1, CD16-DAP10, CD16-DAP12, CD2-CD3ζ / 1XX, CD2-DNAM1, CD2-FcERIγ, CD2-DAP10, CD28-DNAM1, CD28-F It contains a fused cytoplasmic domain or a portion thereof, including but not limited to cERIγ, CD28-DAP10, CD28-DAP12, CD28H-CD3ζ / 1XX, DAP10-CD3ζ / 1XX, or DAP10-DAP12, DAP12-CD3ζ / 1XX, DAP12-DAP10, DNAM1-CD3ζ / 1XX, KIR2DS2-CD3ζ / 1XX, KIR2DS2-DAP10, KIR2DS2-2B4, or NKp46-2B4.

[0118] In some exemplary embodiments of a CAR having an end domain composed of three different signaling domains, the end domain includes, but is not limited to, a fusion cytoplasmic domain or a portion thereof in a form that includes 2B4-DAP10-CD3ζ / 1XX, 2B4-IL21R-DAP10, 2B4-IL2RB-DAP10, 2B4-IL2RB-CD3ζ / 1XX, 2B4-41BB-DAP10, CD16-2B4-DAP10, or KIR2DS2-2B4-CD3ζ / 1XX.

[0119] In some embodiments, the transmembrane domain of CAR contains an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the full-length or partial transmembrane domain of CD2, CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or T cell receptor polypeptide. In some other embodiments, the transmembrane domain of CAR contains an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the full-length or partial transmembrane domain of 2B4, CD2, CD16, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8. In some embodiments of CAR, the transmembrane domain and its immediately linked signaling domain originate from the same protein. In some other embodiments of CAR, the transmembrane domain and its immediately linked signaling domain originate from different proteins.

[0120] Generally, a CAR construct comprises a transmembrane domain and an end domain containing one or more signaling domains derived from the cytoplasmic region of one or more signaling proteins. In some embodiments, the one or more signaling domains contained in the CAR end domain are derived from the same or different proteins from those from which the TM is derived. As provided herein, the portion representing the transmembrane domain (TM) of a CAR is underlined, and the domains contained in the end domain are enclosed in parentheses "()", and each of the TM and signaling domains is indicated by the name of the signaling protein from which the domain sequence is derived. In some embodiments, the amino acid sequence of each TM or signaling domain may be approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% identical to the full-length or partial transmembrane or cytoplasmic region of the corresponding signaling protein. Exemplary CAR constructs containing transmembrane and end domains provided herein include NKG2D-(2B4-IL2RB-CD3ζ), CD8-(41BB-CD3 ζ1XX), CD28-(CD28-2B4-CD3ζ), CD28H-(CD28H-CD3ζ), DNAM1-(DNAM1-CD3ζ), DAP10-(DAP10-CD3ζ), KIR2DS2-(KIR2DS2-CD3ζ), KIR2DS2-(KIR2DS2-DAP10), KIR2DS2-(KIR2DS2-2B4), CD16-(CD16-2B4-D This includes, but is not limited to, AP10, CD16-(CD16-DNAM1), NKp46-(NKp46-2B4), NKp46-(NKp46-2B4-CD3ζ), NKp46-(NKp46-CD2-Dap10), CD2-(CD2-CD3ζ), 2B4-(2B4-CD3ζ), 2B4-(2B4-FcERIg), and CS1-(CS1-CD3ζ).

[0121] A CAR containing any of the above TM-(endodomains) can be constructed to specifically target at least one antigen determined by the antigen-binding domain contained in the ectodomain of the CAR. In some embodiments, the CAR can specifically target antigens associated with a disease or pathogen. In some embodiments, the CAR can specifically target tumor antigens, which may be humoral or solid tumors. The ectodomain of the CAR contains one or more antigen-recognition domains for antigen-specific binding. In some embodiments, the ectodomain may further include a signal peptide or reader sequence and / or a spacer.

[0122] In certain embodiments, the ectodomain of the provided CAR includes an antigen recognition region comprising a mouse antibody, a human antibody, a humanized antibody, camel Ig, a shark heavy chain-only antibody (VNAR), an IgNAR, 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 fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody.Non-exclusive examples of antigens that may be targeted by CARs (cancellable antigens) contained in genetically modified iPSCs and derived effector cells include ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, and CD13. 8, CD269 (BCMA), CDS, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 ( GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), mucin 1 (Muc-1), whip Examples include Muc-16, mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0123] In some embodiments, the ectodomain of the provided CAR further comprises a signal peptide. The signal peptide guides the CAR polypeptide to the endoplasmic reticulum (ER) for proper glycosylation and fixation to the plasma membrane. In general, any eukaryotic signal sequence that targets secreted proteins to the ER pathway can be used. Suitable example signal peptides include, but are not limited to, IL-2 signal sequences, kappa reader sequences, CD8α reader sequences, albumin signal sequences, prolactin signal sequences, and IgG signal peptides, as well as GM-CSF signal peptides.

[0124] In some embodiments, the ectodomain of the provided CAR may optionally include a hinge (also called a spacer) region to provide flexibility between the antigen recognition domain and the transmembrane domain of the CAR. In some exemplary and non-limiting embodiments, the hinge of the CAR includes an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the hinge region of a known polypeptide such as the immunoglobulin, CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8α, CD8β, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, IgG1, or CH2 / CH3 domain, or a combination thereof. In some embodiments, the hinge region of the CAR contains amino acids having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with respect to the CH2 / CH3 domain of the immunoglobulin.

[0125] In some embodiments, effector cells containing one or more CARs as provided herein can be used to treat autoimmune disorders, hematological malignancies, solid tumors, or infections associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemia (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML)), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes. Examples of solid tumors include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), several forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, several forms of myocarditis, multiple sclerosis, bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, several forms of thyroiditis, several 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 (herpes zoster virus associated with Kaposi's sarcoma), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), VZV (varicella-zoster virus), adenovirus, lentivirus, and BK polyomavirus-associated disorders.

[0126] One aspect of the present invention provides iPSCs and derived effector cells differentiated therefrom, comprising a polynucleotide encoding a CAR containing one of the endodomains provided herein. In one embodiment of the CAR, the CAR is CD19-specific. In another embodiment, the CAR is MICA / B-specific. In yet another embodiment, the CAR is BCMA-specific. In yet another embodiment, the CAR is CD38-specific. In yet another embodiment, the CAR is HER2-specific. In yet another embodiment, the CAR is MSLN-specific. In yet another embodiment, the CAR is PSMA-specific. In yet another embodiment, the CAR is VEGF-R2-specific.

[0127] In another aspect of the present invention, iPSCs and derived effector cells differentiated therefrom, comprising a polynucleotide encoding a first CAR including one of the provided endodomains, may further comprise a second CAR having different antigen specificity. The endodomain of the second CAR may be the same as or different from that of the first CAR. In some embodiments, the second CAR comprises an endodomain different from that of the first CAR, which is one of the endodomains provided herein. In some other embodiments, the second CAR comprises an endodomain different from that of the first CAR, which is not one of the endodomains provided herein.

[0128] Non-restrictive CAR strategies include conditionally activated heterodimerized CARs via dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9,587,020); CAR splits where CARs are generated by homologous recombination of antigen-binding, hinge, and endo-domains (see, e.g., U.S. Patent No. 2017 / 0183407); multi-chain CARs that enable non-covalent bonding between two transmembrane domains connected to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Patent No. 2014 / 0134142); CARs with bispecific antigen-binding domains (see, e.g., U.S. Patent No. 9,447,194); or CARs 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). This includes Invest.2016;126(8):3036-3052); inductive CARs (see, for example, U.S. Patents 2016 / 0046700, 2016 / 0058857, and 2017 / 0166877); switchable CARs (see, for example, U.S. Patent 2014 / 0219975); and any other designs known in the art.

[0129] Suitable genomic loci for CAR insertion provided herein include loci that meet the criteria for a genome-safe harbor provided herein, and / or loci where knockdown or knockout of a gene at a selected locus as a result of insertion is desired. In some embodiments, suitable genomic loci for CAR insertion include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0130] In one embodiment, the iPSC and its derived cells contain a CAR inserted into the TCR constant region (TRAC or TRBC), resulting in TCR knockout and optionally placing CAR expression under the control of an endogenous TCR promoter. In a particular embodiment of the iPSC derived cell containing a TCR null and a CAR, the derived cell is a T cell. In another embodiment, the iPSC and its derived cell containing a CAR has a CAR inserted into the NKG2A or NKG2D locus, resulting in NKG2A or NKG2D knockout and optionally placing CAR expression under the control of an endogenous NKG2A or NKG2D promoter. In a particular embodiment of the iPSC derived cell containing an NKG2A or NKG2D null and a CAR, the derived cell is an NK cell. In a further embodiment, the iPSC and its derived cell containing a CAR has a CAR inserted into the CD38 coding region, resulting in CD38 knockout and optionally placing CAR expression under the control of an endogenous CD38 promoter. In one embodiment of a cell comprising a CD38 null and a CAR comprising one of the provided endodomains, the CAR is specific to CD38. In one embodiment, an iPSC and its derived cells comprising a CAR comprising one of the provided endodomains have a CAR inserted into a CD58 coding region, resulting in CD58 knockout. In one embodiment, an iPSC and its derived cells comprising a CAR comprising one of the provided endodomains have a CAR inserted into a CD54 coding region, resulting in CD54 knockout. In one embodiment, an iPSC and its derived cells comprising a CAR comprising one of the endodomains have a CAR inserted into a CIS (cytokine-induced SH2-containing protein) coding region, resulting in CIS knockout. In one embodiment, an iPSC and its derived cells comprising a CAR comprising one of the provided endodomains have a CAR inserted into a CBL-B (E3 ubiquitin protein ligase CBL-B) coding region, resulting in CBL-B knockout. In one embodiment, an iPSC and its derived cells comprising a CAR as provided have a CAR inserted into a SOCS2 coding region, resulting in SOCS2 knockout.In one embodiment, an iPSC and its derivative cells containing a CAR as provided have a CAR inserted into the CD56 (NCAM1) coding region. In another embodiment, an iPSC and its derivative cells containing a CAR as provided have a CAR inserted into one of the coding regions of PD1, CTLA4, LAG3, and TIM3, resulting in checkpoint receptor knockout or knockdown at the insertion site. In a further embodiment, an iPSC and its derivative cells containing a CAR as provided have a CAR inserted into the TIGIT coding region, resulting in TIGIT knockout.

[0131] Further embodiments provided include derived effector cells obtained from differentiating genome-engineered iPSCs, where both the iPSCs and derived cells contain the CARs described herein, and the iPSCs and derived cells further include, but are not limited to, one or more additional modification modalities including, CD38 knockout; CD38-CAR; CD16 or its variants; cell surface-expressed exogenous cytokines and / or their receptors, partial or complete peptides; HLA-I and / or HLA-II deficiency; HLA-G overexpression and knockout of one or both CD58 and CD54; TCR null; surface-presented CD3; antigen-specific TCR; NKG2C; DAP10 / 12; NKG2C-IL15-CD33 ("2C1533"), which are further described in detail herein. 2. CD38 Knockout

[0132] The cell surface molecule CD38 is highly upregulated in several hematological malignancies originating from both lymphoid and myeloid lines, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapies aimed at depleting cancer cells. Antibody-mediated depletion of cancer cells typically results from a combination of direct induction of apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cytotoxicity). In addition to ADCC, immune effector mechanisms that work in conjunction with therapeutic antibodies may also include phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0133] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells and NK cells, as well as activated T cells and B cells. During hematopoiesis, CD38 is expressed during the final stage of maturation that continues to the plasma cell stage in CD34 + stem cells, as well as progenitor cells committed to the lymphoid, erythroid, and myeloid lineages. As a type II transmembrane glycoprotein, CD38 performs cellular functions both as a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 catalyzes the synthesis and hydrolysis of the reaction from NAD + to ADP-ribose, thereby producing the second messengers CADPR and NAADP that stimulate the release of calcium from the endoplasmic reticulum and lysosomes, which is important for the process of cell adhesion (this process is calcium-dependent). As a receptor, CD38 recognizes CD31 and controls cytokine release and cytotoxicity in activated NK cells. CD38 has also been reported to associate with cell surface proteins in lipid rafts, control cytoplasmic Ca 2+ flux, and mediate signal transduction in lymphoid and myeloid cells.

[0134] In the treatment of malignant tumors, when T cells transduced with the CD38 antigen-binding receptor are used systemically, CD34 + hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells express CD38 +It has been shown that fraction lysis leads to recipient immune effector cell dysfunction, resulting in an incomplete therapeutic response and reduced or eliminated efficacy. In addition, in multiple myeloma patients treated with daratumumab, a CD38-specific antibody, a reduction in NK cells was observed in both bone marrow and peripheral blood, while other immune cell types such as T cells and B cells were unaffected despite CD38 expression (Casneuf et al., Blood Advances. 2017;1(23):2105-2114). Without being limited by theory, this application provides a strategy to maximize the potential of CD38-targeted cancer therapy by overcoming the depletion or reduction of CD38-specific antibodies and / or CD38 antigen-binding domain-induced effector cells by fructoliside. In addition, CD38 can be used to eliminate activated lymphocytes or suppress the activation of these lymphocytes in recipients of allogeneic effector cells. By suppressing the activation of these recipient lymphocytes using CD38-specific antibodies such as daratumumab, CD38 is upregulated by activated lymphocytes such as T cells or B cells, thereby reducing and / or preventing allo-rejection of these effector cells, and thereby increasing the survival and persistence of effector cells.

[0135] Therefore, this application also provides strategies to enhance the persistence and / or survival of effector cells by using CD38-specific antibodies, secreted CD38-specific enforcers, or CD38CARs (chimeric antigen receptors) against the activation of recipient T and B cells, and / or to eliminate activated recipient T and B cells, i.e., by reducing or preventing allo-rejection by lymphocyte depletion of activated T and B cells, often prior to adoptive cell transplantation. Specifically, the strategies provided herein, in some embodiments, involve generating a master cell bank containing CD38 knockout iPSC lines, single-selected and expanded clonal CD38-negative iPSCs, and CD38-negative (CD38) iPSCs through targeted differentiation of the manipulated iPSC lines. neg) Obtaining derived effector cells, and including the use of a CD38-targeted therapeutic portion in conjunction with effector cells, the derived effector cells are protected from fructoliside and allo-rejection, among other advantages. In addition, anti-CD38 monoclonal antibody therapy significantly depletes the patient's activated immune system without adversely affecting the patient's hematopoietic stem cell compartment. CD38-negative derived cells have the ability to resist CD38 antibody-mediated depletion and can be effectively administered in combination with anti-CD38 or CD38-CAR without the use of toxic conditioning agents, thus reducing and / or replacing chemotherapy-based lymphocyte depletion.

[0136] In one embodiment provided herein, CD38 knockout in an iPSC strain is a biallelic knockout. As disclosed herein, the provided CD38 null iPSC strain can differentiate as directed to produce functional derived hematopoiesis, including but not limited to mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional features not present in primary NK, T and / or NKT cells. In some embodiments, when an anti-CD38 antibody is used to induce ADCC or an anti-CD38 CAR is used for targeted cell killing, CD38 - / -iPSCs and / or their derived effector cells are not eliminated by anti-CD38 antibodies, anti-CD38CARs, or recipient-activated T or B cells, thereby increasing the persistence and / or survival of iPSCs and their effector cells in the presence of and / or after exposure to such therapeutic agents. In some embodiments, effector cells exhibit increased in vivo persistence and / or survival in the presence of and / or after exposure to such therapeutic agents. In some embodiments, CD38 null effector cells are NK cells derived from iPSCs. In some embodiments, CD38 null effector cells are T cells derived from iPSCs. In some embodiments, CD38 null iPSCs and derived cells include, but are not limited to, one or more additional genome editing methods described herein, including CD16 or its variants, CAR expression, signaling complexes comprising cell surface-expressed exogenous cytokines and / or partial or complete peptides of their receptors, HLAI and / or HLAII knockout, and additional modalities provided herein.

[0137] In another embodiment, inserting one or more transgenes provided herein at a selected location on CD38 while simultaneously knocking out CD38 can be achieved, for example, by a CD38-targeted knock-in / knockout (CD38-KI / KO) construct. In some embodiments of the construct, the construct includes a pair of CD38-targeting homology arms for site-selective insertion within the CD38 locus. In some embodiments, the pre-selected targeting site is located 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 the exogenous promoter contained in the construct. When two or more transgenes are inserted at a selected location on the CD38 locus, a linker sequence, e.g., a 2A linker or IRES, is positioned between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-I, and TaV (also referred to as "F2A," "E2A," "P2A," and "T2A," respectively), enabling the production of distinct proteins from a single translation. In some embodiments, the insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter included in the CD38-KI / KO construct may be CAG, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, including but not limited to CMV, EF1α, PGK, and UBC. 3. CD16 knock-in

[0138] CD16 has been identified as two isoforms: the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and facilitating antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, “high affinity CD16,” “uncleavable CD16,” “high affinity uncleavable CD16,” or “hnCD16” refers to various CD16 variants. Wild-type CD16 has low affinity and undergoes ectodomain shedding, a proteolytic cleavage process that controls the cell surface density of various cell surface molecules on leukocytes, upon activation of NK cells. F176V (also referred to as F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is a genetically engineered exemplary non-cleavable version of CD16. Engineered CD16 variants, including both F176V and S197P, are high affinity and non-cleavable, as described in detail in International Publication 2015 / 148926, the full disclosure of which is incorporated herein by reference. In addition, chimeric CD16 receptors 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 substituted ectodomain of the chimeric CD16 includes one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoform or polymorphic variant). Therefore, various embodiments of exogenous CD16 introduced into cells include functional CD16 variants and their chimeric receptors. In some embodiments, the functional CD16 variant is a high-affinity, non-cleavable CD16 receptor (hnCD16). In some embodiments, hnCD16 includes both F176V and S197P, and in some embodiments, it includes F176V with the cleavage region eliminated.

[0139] Accordingly, among other edits intended and described herein, genetically engineered cloned iPSCs containing a high-affinity, non-cleavable CD16 receptor (hnCD16) are provided herein, and the genetically engineered iPSCs can differentiate into effector cells containing the hnCD16 introduced into the iPSCs. In some embodiments, the derived effector cells containing hnCD16 are NK cells. In some embodiments, the derived effector cells containing hnCD16 are T cells. Exogenous hnCD16 or its functional variant contained in iPSCs or derived cells exhibit high affinity not only for ADCC antibodies or fragments thereof, but also for binding to bispecific, tripspecific, or multispecific engagers or binders that recognize the extracellular binding domains of CD16 or CD64 of the hnCD16. Bispecific, tripspecific, or multispecific engagers or binders are further described below in this application. Thus, this application provides derived effector cells or a population of such cells that are pre-loaded with one or more pre-selected ADCC antibodies via exogenous CD16 expressed on derived effector cells in amounts sufficient for therapeutic use in the treatment of conditions, diseases, or infections as further detailed in the following sections, wherein the hnCD16 comprises the extracellular binding domain of CD64, or of CD16 having F176V and S197P.

[0140] In some other embodiments, exogenous CD16 includes CD16 or a variant thereof based on CFcR. By modifying or replacing the native CD16 transmembrane and / or intracellular domains, chimeric Fc receptors (CFcRs) are produced to include a non-native transmembrane domain, a non-native stimulating domain, and / or a non-native signaling domain. As used herein, the term “non-native” means that the transmembrane domain, stimulating domain, or signaling domain originates from a different receptor other than the receptor that provides the extracellular domain. In the figures herein, CFcRs based on CD16 or a variant thereof do not have a transmembrane domain, stimulating domain, or signaling domain derived from CD16. In some embodiments, the exogenous hnCD16-based CFcR includes a non-innate transmembrane domain derived from CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide. In some embodiments, the exogenous hnCD16-based CFcR includes a non-innate stimulative / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD1, LAG3, 2B4, BTLA, DAP10, DAP12, CTLA4, or NKG2D polypeptide. In some embodiments, the exogenous hnCD16-based CFcR includes a non-native signaling domain derived from the CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of the CD16-based CFcR, the provided chimeric Fc receptor includes a transmembrane domain and a signaling domain, both derived from one of the IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, and NKG2D polypeptides.One particular embodiment of the CD16-based chimeric Fc receptor comprises a transmembrane domain of NKG2D, a stimulating domain of 2B4, and a signaling domain of CD3ζ, where the extracellular domain of CFcR is derived from the extracellular domain of CD64 or CD16 in full length or partial sequence, and the extracellular domain of CD16 comprises F176V and S197P. Another embodiment of the CD16-based chimeric Fc receptor comprises a transmembrane domain and a signaling domain of CD3ζ, where the extracellular domain of CFcR is derived from the extracellular domain of CD64 or CD16 in full length or partial sequence, and the extracellular domain of CD16 comprises F176V and S197P.

[0141] The various embodiments of the CD16-based chimeric Fc receptor described above can bind with high affinity to the Fc region of antibodies or fragments thereof, or to bispecific, triplicate, or multispecific engagementrs or binders. Upon binding, the stimulating and / or signaling domains of the chimeric receptor enable activation of effector cells and cytokine secretion, as well as the killing of tumor cells targeted by the antibody or tumor antigen-binding component and the bispecific, triplicate, or multispecific engagementr or binder having the Fc region. Without being limited by theory, CFcRs contribute to the effector cell killing ability and increase the proliferation and / or potential of effector cells through the non-native transmembrane, stimulating, and / or signaling domains of the CD16-based chimeric Fc receptor, or through the binding of an engagementr to the ectodomain. Antibodies and engagementrs can bring tumor cells expressing the antigen and effector cells expressing CFcR into close proximity, which also contributes to the enhancement of tumor cell killing. Exemplary tumor antigens that are bispecific, tripspecific, or multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, tripspecific, or multispecific engagers or binders suitable for binding effector cells expressing CD16-based CFcRs to attack tumor cells include CD16(or CD64)-CD30, CD16(or CD64)-BCMA, CD16(or CD64)-IL15-EPCAM, and CD16(or CD64)-IL15-CD33.

[0142] Unlike endogenous CD16 expressed by primary NK cells, which is cleaved from the cell surface after NK cell activation, various non-cleavable versions of CD16 in derived NK cells avoid CD16 shedding and maintain constant expression. In derived NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, which indicate improved cellular function. Non-cleavable CD16 also enhances antibody-dependent cytotoxicity (ADCC) and the binding of bispecific, tripspecific, or multispecific engagers. ADCC is an NK cell-mediated lysis mechanism via the binding of CD16 to antibody-coated target cells. The additional high-affinity characteristics of introduced hnCD16 in derived NK cells also allow for in vitro loading of ADCC antibodies into hnCD16-mediated NK cells before administering the cells to subjects requiring cell therapy. As provided herein, hnCD16 may comprise F176V and S197P in some embodiments, or further comprise at least one of non-natural transmembrane domains, stimulatory domains, and signaling domains. As disclosed, the application also provides derived NK cells or populations thereof pre-filled with one or more pre-selected ADCC antibodies in quantities sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed below.

[0143] Unlike primary NK cells, mature T cells from primary sources (i.e., natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing expressed exogenous, uncleavable CD16 could differentiate into functionally derived T cells without impairing the developmental biology of T cells. Unlike primary NK cells, mature T cells from primary sources (i.e., natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing expressed exogenous, uncleavable CD16 could differentiate into functionally derived T cell lineages that not only express exogenous CD16 but can also perform functions via acquired ADCC mechanisms, without impairing the developmental biology of T cells. This acquired ADCC in derived T cell lineages can be further used as an approach for dual targeting and / or as an approach to rescue antigen escape, which often occurs in CAR-T cell therapy, where tumors relapse with reduced or lost expression of CAR-T targeted antigens, or with mutant antigens that evade recognition by CARs. If the derived T cell lineages contain acquired ADCC via exogenous CD16 expression, including functional variants and CD16-based CFcRs, and if 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 regrowth of targeted tumors commonly seen in CAR-T therapy. Such strategies for reducing and / or preventing antigen escape while achieving dual targeting are equally applicable to NK cells expressing one or more CARs. A variety of CARs that can be used in this antigen escape reduction and prevention strategy are described in more detail below.

[0144] Accordingly, embodiments of the present invention provide derived T cell lines containing exogenous CD16 in addition to signaling complexes and CARs as provided herein. In some embodiments, the CD16 contained in the derived T cell lines is hnCD16 containing CD16 ectodomains including F176V and S197P. In some other embodiments, the hnCD16 contained in the derived T cells may contain a complete or partial ectodomain derived from CD64, or further contain at least one of a non-natural transmembrane domain, a stimulating domain, and a signaling domain. As described, such derived T cells have an acquisition mechanism to target tumors with monoclonal antibodies meditated by ADCC to enhance the therapeutic effect of the antibodies. As disclosed, the application also provides derived T cells or cell populations pre-filled with one or more pre-selected ADCC antibodies in amounts sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed below.

[0145] In this application, a master cell bank is further provided, comprising single-cell sorted, enlarged, clonalized iPSCs having at least one phenotype provided herein, including but not limited to exogenous CD16 or its variants, the cell bank providing a platform for additional iPSC manipulations and a renewable source for manufacturing ready-made, manipulated, homogeneous cell therapy products, including but not limited to derived NK cells and T cells, that are well-defined, homogeneous in composition, and can be mass-produced on a large scale in a cost-effective manner. 4. Exogenously introduced cytokines and / or cytokine signaling

[0146] By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicity associated with such practices is reduced, while cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional soluble cytokine administration, signaling complexes containing one or more partial-length or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors are introduced into cells to enable cytokine signaling, with or without expression of the cytokines themselves, 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 (signaling complexes) for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or ephemeral.

[0147] Various construct designs for introducing protein complexes into cells for cytokine signaling, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, are provided herein. The following exemplary example is provided when the signaling complex is for IL15.

[0148] Design 1: IL15 and IL15Rα are co-expressed without excluding cis-presentation of IL15, using a self-cleaving peptide that mimics the trans-presentation of IL15.

[0149] Design 2: IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans presentation without excluding cis presentation of IL15, and ensuring membrane binding of IL15.

[0150] Design 3: IL15Rα with a cleaved intracellular domain is fused to IL15 at its C-terminus via a linker, mimicking the trans presentation of IL15, maintaining the membrane binding of IL15, and excluding cis presentation and / or any other potential signaling pathways mediated by normal IL15R via its intracellular domain. The intracellular domain of IL15Rα is thought to be crucial for the receptor to be expressed in IL15-responsive cells, which then expand and function. Design 4 is a construct that provides another practical alternative to Design 3, in which the Sushi domain is fused with IL15 at one end and the transmembrane domain at the other (mb-Sushi), and essentially the entire IL15Rα is removed, except that, optionally, it has a linker between the Sushi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of any membrane-bound protein. In constructs such as Design 4, unwanted signaling via IL15Rα, including cis presentation, is eliminated, while only the desired trans presentation of IL15 is retained.

[0151] Design 5: Natural or modified IL15Rβ is fused to IL15 at its C-terminus via a linker, enabling constitutive signaling and maintaining IL15 membrane binding and trans presentation.

[0152] Design 6: The native or modified common receptor γC is fused to IL15 at its C-terminus via a linker for constitutive signaling and membrane-bound trans presentation of cytokines. The common receptor γC is also known as the common gamma chain or CD132, and is also known as the IL2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to many interleukin receptor complexes, including but not limited to the IL2, IL4, IL7, IL9, IL15, and IL21 receptors.

[0153] Design 7: Manipulated IL15Rβ, which forms homodimers in the absence of IL15, is useful for generating constitutive signaling of cytokines.

[0154] In some embodiments, one or more cytokines IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, and / or their receptors, can be introduced into iPSCs and their derived cells during iPSC differentiation using one or more of the designs provided. In some embodiments, IL2 or IL15 cell surface expression and signaling are mediated through a construct illustrated in any one of designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling are mediated through a construct illustrated in design 5, 6, or 7, by using either a common receptor or a cytokine-specific receptor. In some embodiments, IL7 surface expression and signaling are mediated through a construct illustrated in design 5, 6, or 7, by using either a common receptor or a cytokine-specific receptor (e.g., IL4 receptor). The transmembrane (TM) domain of any of the above designs may be naturally occurring for the corresponding cytokine receptor or may be modified or substituted with the transmembrane domain of any other membrane-bound protein.

[0155] In iPSCs and cells derived therefrom that contain both CARs and exogenous cytokines and / or cytokine receptor signaling (signaling complexes, or "ILs"), CARs and ILs may be expressed in separate constructs or co-expressed in a bisistronic construct containing both CARs and ILs. In some further embodiments, the signaling complex may be ligated to either the 5' or 3' end of a CAR expression construct via an autocleaved 2A coding sequence, exemplified by, for example, CAR-2A-IL15 or IL15-2A-CAR. Thus, IL15 and CAR reside in a single open reading frame (ORF). The CAR-2A-IL15 or IL15-2A-CAR bisistonic design enables the coordinated expression of the CAR and IL15 signaling complex in terms of both timing and quantity, and under the same regulatory mechanisms, which may be selected to incorporate, for example, an inducible promoter for the expression of a single ORF. Self-cleaving peptides are found in foot-and-mouth disease viruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tesiovirus-1 (PTV-I) (Donnelly, ML, et al., J. Gen. Virol, 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732 (2001)), as well as in cardioviruses such as tyrovirus (e.g., Tyler mouse encephalomyelitis virus) and encephalomyocarditis virus. 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively.

[0156] Embodiments of the bicistronic CAR-2A-IL15 or IL15-2A-CAR disclosed herein for IL15 also intend to express any other cytokines or cytokine signaling complexes provided herein, such as IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, IL2 cell surface expression and signaling are via constructs illustrated in any of designs 1-7. In some embodiments, IL4, IL7, IL9, or IL21 cell surface expression and signaling are via constructs illustrated in designs 5, 6, or 7, using either a common receptor and / or a cytokine-specific receptor. 5. HLA-I- and HLA-II- deficiency

[0157] Often, to avoid the problem of allogeneic rejection, it is necessary to match multiple HLA class I and class II proteins for histocompatibility in allogeneic recipients. iPSC cell lines and their derivative cells differentiated therefrom in which the expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced are provided herein. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or by deletion or reduced expression levels of HLA class I-related genes, including but not limited to the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for the cell surface expression of all HLA class I heterodimers. B2M null cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion or reduction of HLA-II-related genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through the activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA null cells are HLA-II deficient. For example, iPSC lines and their derived cells that have both HLA-I and HLA-II deficiencies to lack both B2M and CIITA expression are provided herein, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoid recognition and killing by host (allogeneic) T cells.

[0158] However, in some cell types, the absence of class I expression leads to lysis by NK cells. To overcome this "self-loss" response, HLA-G can be optionally 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 include HLA-G knock-in. Alternatively, in some embodiments, the provided HLA-I-deficient iPSCs and their derived cells further include one or both of CD58 knockout and CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and facilitate cell migration, including immune cells. It was previously unclear whether, and how, disruption of CD58 and / or CD54 in iPSCs affects pluripotency and developmental biology in iPSC differentiation into functional immune effector cells, including T cells and NK cells. Furthermore, it was previously unclear whether CD58 and / or CD54 knockout could effectively and / or sufficiently reduce the sensitivity of effector cells derived from HLA-I-deficient iPSCs to allogeneic NK cell killing. Here, we showed that CD58 knockout was more efficient than CD54 knockout in reducing allogeneic NK cell activation, while double knockout of both CD58 and CD54 most effectively enhanced the reduction in NK cell activation. In some observations, CD58 and CD54 double knockout is even more effective than HLA-G overexpression in HLA-I-deficient cells in overcoming the "self-loss" effect.

[0159] As provided above, in some embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells have an exogenous polynucleotide encoding HLA-G. In some embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD58 null. In some other embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD54 null. In some yet other embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD58 null and CD54 null.

[0160] In some embodiments, the procedure for HLA-I and / or HLA-II deficiency may be bypassed or kept intact by expressing an inactivating CAR that targets upregulated surface proteins in activated recipient immune cells to avoid allo-rejection. In some embodiments, the upregulated surface proteins in activated recipient immune cells include, but are not limited to, CD38, CD25, CD69, or CD44. When cells express such an inactivating CAR, it is preferable that the cells do not express or knock out the same surface proteins targeted by the CAR. 6. Genetically modified iPSC lines and derived cells provided herein

[0161] In light of the foregoing, this application provides iPSCs, iPS cell lines, or populations thereof, and derived functional cells obtained from differentiating said iPSCs, each cell comprising at least one CAR having the endodomain described herein. In some embodiments, derived effector cells include, but are not limited to, mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, myeloid common progenitor cells, lymphoid common progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and derived immune effector cells having one or more functional features not present in primary NK, T and / or NKT cells.

[0162] Therefore, this application provides iPSCs and their functionally derived hematopoietic cells, comprising any one of the following genotypes in Table 2. The "CAR" provided in Table 2 of this application (2nd)"IL" represents a CAR with a different target specificity than the first CAR, and non-limiting examples include CARs that target at least one of CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1. As provided in Table 2, "IL" represents one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which particular cytokine / receptor expression is selected. Furthermore, "IL" also encompasses the IL15Δ embodiment, which is detailed above as a cleaved fusion protein of IL15 and IL15Rα, but does not include the intracellular domain. Furthermore, if the iPSC and its functionally derived hematopoietic cells have a genotype containing both CAR (first CAR or second CAR) and IL, in one embodiment of the cells, CAR and IL are contained in a bicistrolytic expression cassette containing a 2A sequence. For comparison, in some other embodiments, CAR and IL are in separate expression cassettes contained in the iPSC and its functionally derived hematopoietic cells. In a particular embodiment, the iPSC and its functionally derived effector cells expressing both CAR and IL contain IL15 as described in design 3 or 4, and the IL15 construct is contained in the expression cassette together with or separately from CAR. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] 7. Additional modifications

[0163] In some embodiments, iPSCs and their derived effector cells containing any one of the genotypes in Table 2 may further include deletion or reduced expression in at least one of the following genes: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The present invention may further include introduced or increased expression of at least one of R, antigen-specific TCRs, Fc receptors, engagers, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers.

[0164] A bispecific or multispecific engager is a fusion protein comprising two or more single-chain variable fragments (scFv) of different antibodies, where at least one scFv binds to an effector cell surface molecule and at least one other binds to a tumor cell via a tumor-specific surface molecule. Exemplary effector cell surface molecules or surface trigger receptors that can be used for bispecific or multispecific engager recognition or 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 includes the extracellular domains of CD16 (including F176V and optionally S197P) or CD64, as described in Section I.2, as well as native or non-native transmembrane domains, stimulating domains, and / or signaling domains. In some embodiments, the CD16 expressed on the surface of effector cells for engager recognition is an hnCD16-based chimeric Fc receptor (CFcR). In some embodiments, the hnCD16-based CFcR comprises a transmembrane domain of NKG2D, a stimulating domain of 2B4, and a signaling domain of CD3ζ, the extracellular domain of hnCD16 is derived from the extracellular domain of CD64 or a full-length or partial sequence of CD16, and the extracellular domain of CD16 comprises F176V and optionally S197P. Exemplary tumor cell surface molecules that recognize bispecific or multispecific engagers include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. In one embodiment, the bispecific antibody is CD3-CD19.In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In yet another embodiment, the bispecific antibody is CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody is CD3-CD33. In yet another embodiment, the bispecific antibody further includes a linker between the effector cell and the tumor cell antigen-binding domain, for example, a modified IL15 (referred to in some publications as TriKE, or triplicate killer enhancer) as a linker for effector NK cells that promotes 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").

[0165] In some embodiments, the surface trigger receptor for a bispecific or multispecific engager may be endogenous to the effector cell, sometimes depending on the cell type. In some other embodiments, the methods and compositions provided herein can be used to further manipulate iPSCs containing the genotypes listed in Table 2, directing the differentiation of the iPSCs into T cells, NK cells, or any other effector cells containing the same genotype and surface trigger receptor as the source iPSC, thereby introducing one or more exogenous surface trigger receptors into the effector cells. 8. Antibodies for immunotherapy

[0166] In some embodiments, in addition to the genome-engineered effector cells provided herein, additional therapeutic agents, including antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication, may be expressed by these effector cells and used 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, the tumor or virus-specific antigen activates the administered iPSC-derived effector cells to enhance their killing ability. In some embodiments, suitable antibodies for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include, but are not limited to, CD20 antibodies (rituximab, bertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), HER2 antibodies (trastuzumab, partuzumab), CD52 antibodies (aremtuzumab), EGFR antibodies (cerltuximab), GD2 antibodies (dinutuximab), PDL1 antibodies (avelumab), CD38 antibodies (daratumumab, isatuximab, MOR202), CD123 antibodies (7G3, CSL362), SLAMF7 antibodies (elotuzumab), MICA / B antibodies (7C6, 6F11, 1C2), and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars. In some embodiments, iPSC-derived effector cells include hematopoietic lineage cells containing the genotypes listed in Table 2. In some embodiments, iPSC-derived effector cells include NK lineage cells containing the genotypes listed in Table 2. In some embodiments, iPSC-derived effector cells include T lineage cells containing the genotypes listed in Table 2.

[0167] In some embodiments of combinations useful for treating humoral or solid tumors, the combination comprises a pre-selected monoclonal antibody and iPSC-derived NK cells or T cells containing at least a CAR with a provided endodomain. In some other embodiments of combinations useful for treating humoral or solid tumors, the combination comprises a pre-selected monoclonal antibody and iPSC-derived NK cells or T cells containing at least hnCD16 with a provided endodomain and a CAR. In some embodiments of combinations useful for treating humoral or solid tumors, the combination comprises a monoclonal antibody and iPSC-derived NK cells or T cells containing at least hnCD16 with a provided endodomain and a CAR. Without being limited by theory, hnCD16 provides an enhanced ADCC of the monoclonal antibody, while the CAR not only targets a specific tumor antigen but also prevents the escape of the tumor antigen using a dual-targeting strategy combined with a monoclonal antibody that targets a different tumor antigen. In some embodiments of combinations useful for treating liquid or solid tumors, the combination comprises iPSC-derived NK cells or T cells containing at least one CD38-CAR containing an endodomain, CD38 null, and a CD38 antibody, as provided herein. In one embodiment, the combination comprises a CD38-CAR containing an iPSC-derived NK cell containing an endodomain, CD38 null, and hnCD16, as provided herein, and one of the CD38 antibodies, daratumumab, isatuximab, or MOR202. In one embodiment, the combination comprises an iPSC-derived NK cell containing an endodomain, CD38 null, and hnCD16, as provided herein, and a CD38-CAR containing daratumumab.In some further embodiments, the iPSC-derived NK cells included in the combination with daratumumab include CD38-CAR, CD38 null, hnCD16, IL15, and a CAR that targets at least one of MICA / B or CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1, where the IL15 signaling complex is co-expressed or separately with the CAR, and IL15 is one of the forms presented in Designs 1-7 described herein. In some specific embodiments, IL15 is in the form of construct 3, 4, or 7 when the signaling complex is expressed concurrently or separately with the CAR. 9. Checkpoint inhibitors

[0168] Checkpoints are cellular molecules, often cell surface molecules, that, if not inhibited, can suppress or downregulate the immune response. It is now clear that tumors utilize certain immune checkpoint pathways, particularly as a primary mechanism for immune resistance to tumor antigen-specific T cells. Checkpoint inhibitors (CIs) are antagonists that can reduce checkpoint gene expression or gene product, or decrease the activity of checkpoint molecules, thereby blocking inhibitory checkpoints and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has changed the landscape of oncology, with these drugs resulting in long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and recurrence remains a significant concern. One aspect of this application provides a therapeutic approach to overcome CI resistance by including genomically engineered functional derived cells, which may be expressed by cells or used in conjunction with cells, as provided in combination therapy with CIs. In one embodiment of the combination therapy, the derived cells are NK cells. In another embodiment of the combination therapy, the derived cells are T cells. In addition to exhibiting direct antitumor capabilities, the derived NK cells provided herein have been shown to be resistant to PDL1-PD1-mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at tumor sites. Thus, T cell tumor infiltration facilitated by functionally potent genome-engineered derived NK cells indicates that these NK cells can synergistically interact with T cell-targeted immunotherapy, including checkpoint inhibitors, to alleviate local immunosuppression and reduce tumor burden.

[0169] In one embodiment, derived NK cells for combination therapy with checkpoint inhibitors comprise a CAR containing an endodomain provided herein, and optionally one, two, three, or more of the following: CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and exogenous cell surface cytokine and / or receptor expression, wherein if B2M is knocked out, at least one of the following may be included: a polynucleotide encoding HLA-G, or a knockout of CD58 or CD54. In some embodiments, derived NK cells comprise one of the genotypes listed in Table 2. In some embodiments, the derived NK cells described above further include deletion or reduced expression of at least one of the following genes in the chromosome 6p21 region: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention further comprises introduced or increased expression in at least one of the following: R, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for binding to bispecific, multispecific, or universal engagers.

[0170] In another embodiment, derived T cells for combination therapy of checkpoint inhibitors include a CAR comprising an endodomain provided herein, and optionally one, two, three, or more of the following: CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and exogenous cell surface cytokine and / or receptor expression, where, if B2M is knocked out, one of the following may be included as needed: a polynucleotide encoding HLA-G, or a knockout of CD58 or CD54. In some embodiments, derived T cells include one of the genotypes listed in Table 2. In some embodiments, the derived T cells described above further include deletion or reduced expression of at least one of the following genes in the chromosome 6p21 region: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention further comprises introduced or increased expression in at least one of the following: R, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for binding to bispecific, multispecific, or universal engagers.

[0171] The derived NK cells or derived T cells described above can be obtained by differentiating an iPSC clone containing a CAR comprising an endodomain provided herein, and optionally all of the following: CD38 knockout, hnCD16 expression, B2M / CIITA knockout, a second CAR, and exogenous cell surface cytokine expression, wherein if B2M is knocked out, at least one of the following is optionally introduced: a polynucleotide encoding HLA-G, or knockout of CD58 and CD54. In some embodiments, the iPSC clone further comprises deletion or reduced expression in at least one of the following genes: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region, or HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention further comprises introduced or increased expression in at least one of the following: R, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for binding to bispecific, multispecific, or universal engagers.

[0172] Checkpoint inhibitors suitable for combination therapy with derived NK cells or T cells provided herein include PD1 (Pdcdl, CD279), PDL-1 (CD274), TIM3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG3 (Lag3, CD223), CTLA4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl), CD47, and CD73 (NT5E). This includes, but is not limited to, antagonists of CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0173] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitor antibody may be a mouse antibody, a human antibody, a humanized antibody, a camel Ig antibody, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or a fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one, two, or three or more checkpoint inhibitors include at least one of the following: 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 their derivatives, functional equivalents, or biosimilars.

[0174] In some embodiments, many miRNAs are found to act as regulators controlling the expression of immune checkpoints, so antagonists that inhibit any of the above checkpoint molecules are microRNA-based (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint antagonist miRNAs include, but are not limited to, miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197, miR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c.

[0175] Some embodiments of the combination therapy with the provided derived NK cells or derived T cells include at least one checkpoint inhibitor targeting at least one checkpoint molecule, and the derived cells have the genotypes listed in Table 2. Some other embodiments of the combination therapy with the provided derived NK cells or T cells include two or more checkpoint inhibitors so that two or three or more checkpoint molecules are targeted. In some embodiments of the combination therapy including at least one checkpoint inhibitor and derived cells having the genotypes listed in Table 2, the checkpoint inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the checkpoint inhibitor is produced by the derived cells by expressing an exogenous polynucleotide sequence encoding the antibody, or the fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the antibody or the fragment or variant that inhibits the checkpoint is co-expressed with the CAR in either a separate construct or a bisistronic construct containing both the CAR and the sequence encoding the antibody or the fragment thereof. In some further embodiments, the sequence encoding the antibody or a fragment thereof may be ligated to either the 5' or 3' end of the CAR expression construct via an autocleaved 2A coding sequence, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences of the checkpoint inhibitor and the CAR reside in a single open reading frame (ORF). When the checkpoint inhibitor is delivered, expressed, and secreted as a payload by derived effector cells capable of infiltrating the tumor microenvironment (TME), it binds to the TME, counteracting the inhibitory checkpoint molecule and enabling the activation of the effector cells by activating modalities such as the CAR or activating receptor.In some embodiments, checkpoint inhibitors co-expressed with CAR inhibit at least one of the following checkpoint molecules: PD1, PDL-1, TIM3, TIGIT, LAG3, CTLA4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIRs. In some embodiments, the checkpoint inhibitors co-expressed with CAR in derived cells having the genotypes listed in Table 2 are selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is atezolizumab, or its humanized or Fc-modified variants, fragments, or their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is nivolumab, or its humanized or Fc-modified variants, fragments, or their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is pembrolizumab, or a humanized or Fc-modified variant, fragment thereof, or a functional equivalent or biosimilar thereof.

[0176] In some other embodiments of the combination therapies provided herein, which include at least one antibody inhibiting derived cells and checkpoint molecules, the antibody is not produced by or within the derived cells, but is administered before, concurrently with, or subsequently to the administration of derived cells having the genotypes listed in Table 2. In some embodiments, the administration of one, two, three or more checkpoint inhibitors in the combination therapy with derived NK cells or T cells provided is simultaneous or sequential. In one embodiment of the combination therapy including derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitors included in the therapy are one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of combination therapy involving derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the therapy is atezolizumab, or its humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of combination therapy involving derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the therapy is nivolumab, or its humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of combination therapy involving derived NK cells or T cells having the genotypes listed in Table 2, the checkpoint inhibitor included in the therapy is pembrolizumab, or its humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. III. Targeted genome editing methods at selected loci in iPSCs

[0177] Genome editing, genomic editing, or gene editing as used interchangeably herein is a type of genetic manipulation in which DNA is inserted, deleted, and / or replaced in the genome of a targeted cell. Targeted genome editing (interchangeable with “targeted genomic editing” or “targeted gene editing”) allows for insertion, deletion, and / or replacement at pre-selected sites within the genome. If an endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down by the deletion of the sequence. Thus, targeted editing can also be used to precisely disrupt the expression of endogenous genes. The term “targeted integration” is used similarly herein and refers to a process involving the insertion of one or more exogenous sequences, with or without the deletion of an endogenous sequence at the insertion site. In comparison, randomly integrated genes are subject to positional effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomere regions are particularly susceptible to transgene silencing. Mutually, newly incorporated genes can affect surrounding endogenous genes and chromatin, potentially altering cellular behavior or supporting cellular transformation. Therefore, inserting exogenous DNA into pre-selected loci, such as safe harbor loci or genome-safe harbors (GSHs), is crucial for safe, efficient, copy number control, and reliable gene response regulation. Alternatively, exogenous DNA may be inserted into pre-selected loci where disruption of gene expression, including knockdown and knockout, is intended.

[0178] Targeted editing can be achieved by either a nuclease-independent or nuclease-dependent approach. In nuclease-independent targeted editing approaches, homologous recombination is induced by a homologous sequence adjacent to the inserted exogenous polynucleotide via an enzymatic mechanism in the host cell.

[0179] Alternatively, targeted editing can be achieved more frequently through the specific introduction of double-strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. In the absence of a donor vector containing exogenous gene material, NHEJ often results in random insertions or deletions (in / del) of a small number of endogenous nucleotides. In comparison, when a donor vector containing exogenous gene material adjacent to a pair of homologous arms is present, the exogenous gene material can be introduced into the genome during homology-directed repair (HDR) by homologous recombination, resulting in "targeted integration." In some cases, because the targeted integration site is intended to be within the coding region of the selected gene, targeted integration can interfere with gene expression, resulting in simultaneous knock-in and knock-out (KI / KO) in a single editing step.

[0180] It is possible to achieve simultaneous gene knockout by inserting one or more transgenes at a selected location of a target gene locus (GOI). Suitable gene loci for simultaneous knock-in and knockout (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. Each site-specific targeted homology arm for site-selective insertion allows the transgene to be expressed either under the endogenous promoter of that site or under the exogenous promoter included in the construct. When two or more transgenes are inserted at a selected location on the CD38 locus, a linker sequence, such as a 2A linker or IRES, is placed between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-I, and TaV (also referred to as "F2A," "E2A," "P2A," and "T2A," respectively), allowing 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 may be CAG, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, including but not limited to CMV, EF1α, PGK, and UBC.

[0181] Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR (clustered regular-arranged palindromic sequence repeat) systems. In addition, DICE (dual integrase cassette exchange) systems utilizing phiC31 and Bxb1 integrases are also promising tools for targeted incorporation.

[0182] ZFNs are targeted nucleases 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 a zinc finger-binding domain, whose structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2, C3H, and C4 zinc fingers. “Engineered” zinc finger domains are domains that do not exist in nature, and whose design / composition arises primarily from the application of reasonable criteria, e.g., substitution rules and computerized algorithms to process information from databases containing information on existing ZFP designs and binding data. See, for example, U.S. Patents 6,140,081, 6,453,242, and 6,534,261. See also International Publications 98 / 53058, 98 / 53059, 98 / 53060, 02 / 016536, and 03 / 016496. “Selected” zinc finger domains are domains not found in nature, and their production arises primarily from empirical processes such as phage display, interaction trapping, or hybrid selection. ZFNs are described in great detail in U.S. Patents 7,888,121 and 7,972,854, the complete disclosure of which is incorporated herein by reference. The most recognized example of a ZFN in the art is the fusion of a FokI nuclease with a zinc finger DNA-binding domain.

[0183] TALEN is a targeted nuclease containing a nuclease fused to the TAL effector DNA-binding domain. “Transcription activator-like effector DNA-binding domain,” “TAL effector DNA-binding domain,” or “TALE DNA-binding domain” refers to the polypeptide domain of the TAL effector protein involved in the binding of the TAL effector protein to DNA. TAL effector proteins are secreted from plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells and bind to effector-specific DNA sequences via their DNA-binding domains, activating gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA-binding domain depends on the effector variable number of incomplete 34-amino acid repeats containing polymorphism at a selective repeat position called a repeat variable two residue (RVD). TALEN is described in great detail in U.S. Patent Publication 2011 / 0145940, incorporated herein by reference. The most well-known example of a TALEN in this field is a fusion polypeptide of the FokI nuclease to the TAL effector DNA-binding domain.

[0184] Another example of a targeted nuclease found to be used in the subjective method is the targeted Spo11 nuclease, which is a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain specific to the DNA sequence of interest, such as a zinc finger DNA-binding domain or a TAL effector DNA-binding domain.

[0185] 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.

[0186] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR-related nucleases from families including Cas, CPF, CSE, CSY, CSN, CSD, CST, CSH, CSA, CSM, and CMR; restriction endonucleases; meganucleases; homing endonucleases, etc.

[0187] Using Cas9 as an example, CRISPR / Cas9 requires two main components: (1) Cas9 endonuclease and (2) a crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to a target DNA sequence containing the PAM and a seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA), which can then induce Cas9 to target a selected sequence. These two components can be delivered to mammalian cells via transfection or transduction.

[0188] DICE-mediated insertion provides unidirectional integration of exogenous DNA, strictly limited to small attB and attP recognition sites within each enzyme itself, using a pair of recombinases, e.g., phiC31 and Bxb1. Since these target att sites are not naturally present in the mammalian genome, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Patent Publication 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0189] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the targeted integration method comprises introducing the construct into a cell to enable site-directed homologous recombination by a cell-host enzymatic mechanism. In another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site into the cell to enable ZFN-mediated insertion. In yet another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site into the cell to enable TALEN-mediated insertion. In another embodiment, a targeted integration method in cells includes introducing a construct containing one or more exogenous polynucleotides into cells, introducing a Cas9 expression cassette, and introducing a gRNA containing a guide sequence specific to a desired integration site into cells to enable Cas9-mediated insertion. In yet another embodiment, a targeted integration method in cells includes introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in cells, introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a DICE recombinase expression cassette to enable DICE-mediated targeted integration.

[0190] Promising sites for targeted integration include, but are not limited to, intragenetic or extragenetic regions of the human genome that, theoretically, can accommodate the predictable expression of newly integrated DNA without adverse effects on the host cell or organism, safe harbor loci or genome-safe harbors (GSHs). A useful safe harbor must allow sufficient expression of the transgene to obtain the desired level of the protein or non-coding RNA encoded by the vector. The safe harbor must also not make cells more susceptible to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it must ideally meet criteria including, but not limited to,: no disruption of regulatory elements or genes as determined by sequence annotation; being an intergeneric region within a densely populated gene region or a convergence site between two genes transcribed in opposite directions; maintaining distance to minimize the possibility of long-range interactions between the vector-encoded transcription activator and adjacent genes, particularly cancer-related genes and microRNA gene promoters; and possessing clearly ubiquitous transcriptional activity, as reflected in broad spatial and temporal expression sequence tag (EST) expression patterns exhibiting ubiquitous transcriptional activity. This latter characteristic is particularly important in stem cells where chromatin remodeling during differentiation typically results in the silencing of some loci and the potential activation of others. Within regions suitable for exogenous insertion, the precise locus selected for insertion should lack repeating elements and conserved sequences, allowing for the easy design of primers for homologous arm amplification.

[0191] Suitable sites for human genome editing, or more specifically, targeted integration, include, but are not limited to, human orthologs of adeno-associated virus site 1 (AAVS1), chemokine (CC motif) receptor 5 (CCR5) locus, and mouse ROSA26 locus. In addition, human orthologs of the mouse H11 locus may also be suitable sites for insertion using the compositions and targeted integration methods disclosed herein. Furthermore, collagen and HTRP loci may also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be necessary, particularly in stem cells for specific integration events, and optimization of the insertion strategy, including promoter selection, exogenous gene sequencing and placement, and construct design, is often required.

[0192] In the case of targeted in / dels, the editing site is often located in an endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted in / del is related to the regulation and control of the immune response. In several other embodiments, the endogenous gene containing the targeted in / del is related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and control, 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.

[0193] Accordingly, one aspect of the present invention provides a targeted integration method at selected loci, including genome-safe harbors, pre-selected loci known or proven to be safe and well-controlled for continuous or transient gene expression such as AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, or other loci that meet the criteria for genome-safe harbors. In some embodiments, the targeted integration is located at one of the loci where knockdown or knockout of the gene as a result of the integration is desired, such loci include, but are not limited to, B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0194] In one embodiment, a targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a construct containing a pair of homologous arms and one or more exogenous sequences specific to a desired integration site to enable site-directed homologous recombination by a cell-host enzyme mechanism, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0195] In another embodiment, a targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a ZFN expression cassette containing a DNA-binding domain specific to a desired integration site into cells to enable ZFN-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells and introducing a TALEN expression cassette containing a DNA-binding domain specific to a desired integration site into cells to enable TALEN-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.In another embodiment, a targeted integration method in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a gRNA containing a CRISPR nuclease expression cassette and a guide sequence specific to a desired integration site into cells to enable CRISPR nuclease-mediated insertion, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In another embodiment, a targeted integration method in cells comprises introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in a cell, introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing a DICE recombinase expression cassette to enable DICE-mediated targeted integration, the desired integration site including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0196] Furthermore, as provided herein, the above method for targeted incorporation in a safe harbor is used to insert any polynucleotide of interest, e.g., safety switch proteins, targeted modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and polynucleotides encoding proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some other embodiments, a construct comprising one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the 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, and for example, genetic modification of T lymphocytes at the B cell molecule CD20 can be eliminated by administration of the mAb rituximab. Furthermore, modified EGFR-containing epitopes recognized by cetuximab can be used to deplete genetically modified cells when the cells are exposed to cetuximab. Thus, one aspect of the present invention provides a targeted method for incorporating one or more suicide genes encoding a safety switch protein selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0197] In some embodiments, one or more exogenous polynucleotides incorporated by the methods herein are driven by an operablely linked exogenous promoter contained in a construct for targeted incorporation. The promoter may be inductive or constitutive, and may be time-specific, tissue-specific, or cell-type-specific. Suitable constitutive promoters for the methods of the present invention include, but are not limited to, cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0198] The exogenous polynucleotides incorporated by the methods herein may be driven at the integration site by an endogenous promoter of the host genome. In one embodiment, the method includes targeted integration of one or more exogenous polynucleotides at the AAVS1 locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the method includes targeted integration at the ROSA26 locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the method includes targeted integration at the H11 locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous H11 promoter. In yet another embodiment, the method includes targeted integration at the collagen locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method includes targeted integration at the HTRP locus of the cell's genome. In one embodiment, at least one incorporated polynucleotide is driven by an endogenous HTRP promoter. Theoretically, only correct insertion at the desired site would enable gene expression of the exogenous gene driven by the endogenous promoter.

[0199] In some embodiments, one or more exogenous polynucleotides included in a construct for a targeted integration method are driven by a single promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to broaden the physical separation between the parts and maximize access to the enzymatic mechanism. The linker peptide of the linker sequence may consist of amino acids selected to make the physical separation between the parts (exogenous polynucleotides, and / or proteins or peptides encoded therefrom) more flexible or more rigid, depending on the relevant function. The linker sequence may be protease-cleavable or chemically cleavable to produce distinct parts. Examples of enzymatic cleavage sites in the linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease is either naturally produced by the host or exogenously introduced. Alternatively, the cleavage sites in the linker may be sites that can be cleaved by selected chemicals, such as cyanide bromide, hydroxylamine, or exposure to low pH. Optional linker sequences may serve purposes other than providing cleavage sites. The linker sequence should allow for the effective positioning of parts relative to other adjacent parts for the parts to function properly. The linker may also be a simple amino acid sequence of sufficient length to prevent steric hindrance between parts. In addition, the linker sequence may provide post-translational modifications, including but not limited to phosphorylation sites, biotinylation sites, sulfated sites, and γ-carboxylation sites. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single undesirable conformation. To provide flexibility, the linker may be primarily composed of amino acids with small side chains, such as glycine, alanine, and serine. In some embodiments, about 80 or 90 percent or more of the linker sequence consists of glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, the G4S linker peptide separates the terminal processing domain and endonuclease domain of the fusion protein. In other embodiments, the 2A linker sequence allows two distinct proteins to be produced from a single translation. A suitable linker sequence can be easily identified empirically. In addition, a suitable size and sequence of the linker sequence can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an internal ribosome entry sequence (IRES). In some embodiments, any two consecutive linker sequences are distinct.

[0200] Methods for introducing constructs containing exogenous polynucleotides for targeted integration into cells can be achieved using known methods of gene transfer into cells. In one embodiment, the construct comprises a viral vector skeleton such as an adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, or Sendai virus vector. In some embodiments, plasmid vectors are used to deliver and / or express exogenous polynucleotides into target cells (e.g., pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo), etc. In some other embodiments, episomal vectors are used to deliver exogenous polynucleotides to target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV is not integrated into the host genome. In addition, episomal rAAV vectors mediate homology-directed gene targeting at a much higher rate compared to transfection of conventional targeted plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the genome of iPSCs. In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions herein contain at least one genotype listed in Table 2. IV. Methods for obtaining and maintaining genetically engineered iPSCs

[0201] The present invention provides a method for obtaining and maintaining a genome-engineered iPSC comprising one or more targeted edits at one or more desired sites, wherein the targeted edits remain intact and functional at each selected edit site in the expanded genome-engineered iPSC or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions, i.e., targeted integrations and / or in / dels, at selected sites into the genome of the iPSC and its derived cells. Compared to directly manipulating primary effector cells derived from patient peripheral blood, the many advantages of obtaining genomically engineered derived 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 are rejuvenated due to elongated telomeres and less depletion; and the effector cell population is homogeneous in terms of editing site, copy number, and lack of allelic mutation, random mutation, and expression diversity, primarily due to the possibility of clonal selection in the engineered iPSCs provided herein.

[0202] In certain embodiments, genome-engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and expanded as single cells for extended periods in cell culture media shown in Table 3 as Fate Maintenance Medium (FMM), and the iPSCs retain the targeted edits and functional modifications at the selected sites. The composition of the medium may be present in the optimal range of amounts shown in Table 3. iPSCs cultured in FMM have been shown to remain undifferentiated, with a basal or naive profile, maintain genomic stability without the need to wash or select the culture, and readily induce in vitro differentiation via all three somatic cell lineages, embryoid bodies or monolayers (without embryoid body formation), and in vivo differentiation via teratoma formation. See, for example, International Publication No. 2015 / 134652, the disclosure of which is incorporated herein by reference. [Table 2]

[0203] In some embodiments, genome-engineered iPSCs containing one or more targeted embedded and / or in / del are maintained, passaged, and expanded in a medium containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not containing or essentially not containing a TGFβ receptor / ALK5 inhibitor, and the iPSCs retain intact and functional targeted edits at selected sites.

[0204] Another aspect of the present invention provides a method for generating genome-engineered iPSCs, either through targeted editing of iPSCs or by first generating genome-engineered non-pluripotent cells by targeted editing, and then reprogramming the selected / isolated genome-engineered non-pluripotent cells to obtain iPSCs containing the same targeted editing as the non-pluripotent cells. A further aspect of the present invention provides genome-engineered non-pluripotent cells that are simultaneously being reprogrammed by introducing targeted embedding and / or targeted in / del into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and the conditions for reprogramming include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genome engineering and reprogramming, targeted embedding and / or targeted in / del can be introduced into non-pluripotent cells before or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally small molecules.

[0205] In some embodiments, to simultaneously manipulate and reprogram non-pluripotent cells genomes, targeted integrations and / or in / dels may also be introduced into non-pluripotent cells after a multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181, and CD30, in which case the construct-carrying vector is introduced.

[0206] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor, and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 3). In some embodiments, genomically engineered iPSCs by any of the above methods are further maintained and expanded using a mixture containing a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 3).

[0207] In some embodiments of a method for generating genetically engineered iPSCs, the method comprises genetically engineering an iPSC by introducing one or more targeted embeddings and / or in / dels into the iPSC to obtain a genetically engineered iPSC having at least one genotype listed in Table 2. Alternatively, a method for generating a genetically engineered iPSC comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genetically engineered non-pluripotent cell containing targeted embeddings and / or in / dels at a selected site, and (b) contacting the genetically engineered non-pluripotent cell with a small molecule composition comprising one or more reprogramming factors and optionally a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to obtain a genetically engineered iPSC containing targeted embeddings and / or in / dels at a selected site. Alternatively, a method for generating genome-engineered iPSCs comprises (a) contacting non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and optionally a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate the reprogramming of the non-pluripotent cells; (b) introducing one or more targeted inclusions and / or in / dels into the reprogrammed non-pluripotent cells for genome engineering; and (c) obtaining a clone genome-engineered iPSC containing targeted inclusions and / or in / dels at a selected site.

[0208] Reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combination thereof, as disclosed in PCT / US2015 / 018801 and PCT / US16 / 57136 (these disclosures are incorporated herein by reference). One or more reprogramming factors may be in the form of polypeptides. Reprogramming factors may also be in the form of polynucleotides and are therefore introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by a plasmid combination that takes into account the stoichiometry of various reprogramming factors. See, for example, International Publication 2019 / 075057, the disclosure of which is incorporated herein by reference.

[0209] In some embodiments, non-pluripotent cells are imported with multiple constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted integration at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding targeted modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of iPSCs or their derived cells. In some embodiments, the exogenous polynucleotides encode RNA, including but not limited to siRNA, shRNA, miRNA, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue or cell type-specific promoters. Thus, the polynucleotides can be expressed under conditions that activate the promoters, for example, in the presence of an inducer, or in a particular differentiated cell type. In some embodiments, the polynucleotides are expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, one or more suicide genes are driven by a constitutive promoter, e.g., capase-9 driven by CAG. These constructs, comprising different exogenous polynucleotides and / or different promoters, can be transferred into non-pluripotent cells either simultaneously or sequentially. Non-pluripotent cells subjected to targeted integration of multiple constructs can be simultaneously exposed to one or more reprogramming factors to initiate reprogramming concurrently with genomic manipulation, thereby obtaining genomically engineered iPSCs containing multiple targeted integrations in the same pool of cells. Thus, this robust method enables simultaneous reprogramming and manipulation strategies to lead to clonally engineered hiPSCs with multiple modalities integrated into one or more selected target sites.In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions described herein include at least one genotype listed in Table 2. V. A method for obtaining genetically engineered effector cells by differentiating genomically engineered iPSCs and CAR end-domain screening cells using an iPSC differentiation platform.

[0210] Further embodiments of the present invention provide a method for in vivo differentiation of genomically engineered iPSCs by teratoma formation, wherein in vivo differentiated cells derived from genomically engineered iPSCs retain intact and functional targeted edits, including targeted integration and / or in / del at desired sites. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma contain one or more inducible suicide genes integrated at one or more desired sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genome-safe harbor. In some other embodiments, in vivo differentiated cells derived from genomically engineered iPSCs via teratoma contain polynucleotides encoding a targeted modality, or polynucleotides encoding proteins that promote the transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some embodiments, in vivo differentiated cells derived from genomically engineered iPSCs via a teratoma containing one or more inducible suicide genes further include one or more in / dels of endogenous genes associated with the regulation and mediation of immune responses. In some embodiments, the in / dels include one or more endogenous checkpoint genes. In some embodiments, the in / dels include one or more endogenous T cell receptor genes. In some embodiments, the in / dels include one or more endogenous MHC class I suppressor genes. In some embodiments, the in / dels include one or more endogenous genes associated with the major histocompatibility complex. In some embodiments, the in / dels include one or more endogenous genes including, but not limited to, B2M, PD1, TAP1, TAP2, tapasin, and TCR genes. In one embodiment, a genomically engineered iPSC containing one or more exogenous polynucleotides at a selected site further includes targeted editing in the gene encoding B2M (beta-2-microglobulin).

[0211] In certain embodiments, genome-engineered iPSCs comprising one or more of the genetic modifications provided herein are used to direct hematopoietic cell lineages or any other specific cell type in vitro, and the derived non-pluripotent cells retain functional genetic modifications including targeted editing at selected sites. In one embodiment, the genome-engineered iPSC-derived cells include, but are not limited to, mesodermal cells with the potential to be secondary hematopoietic endothelium (HE), secondary HE, CD34+ hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPP), T cell progenitor cells, NK cell progenitor cells, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, and these cells derived from the genome-engineered iPSCs retain functional genetic modifications including targeted editing at desired sites.

[0212] The applied differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lineages include, for example, those shown in International Publication No. WO 2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, the methods and compositions for generating hematopoietic cell lineages are in a serum-free, feeder-free, and / or stroma-free condition and in a culture platform that does not require scalable and monolayer EB formation and are via secondary hematopoietic endothelium (HE) derived from pluripotent stem cells, including hiPSCs. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated cells and transdifferentiated cells, and various lineages of cells that have directly transitioned to a hematopoietic fate without passing through pluripotent intermediates. Similarly, the cells produced by differentiating stem cells range from multipotent stem or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0213] Methods for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture involve contacting the pluripotent stem cells with a BMP pathway activator and optionally bFGF. As provided, mesodermal cells derived from pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with the potential to form secondary hematopoietic endothelium (HE) without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF and optionally a ROCK inhibitor and / or a WNT pathway activator causes the mesodermal cells with the potential to form secondary HE to differentiate into secondary HE cells, which also expand during differentiation.

[0214] The methods provided herein for obtaining hematopoietic lineage cells are superior to pluripotent stem cell differentiation via embryoid bodies (EBs) because monolayer culture, which is important for many applications that require moderate to minimal cell expansion and homogeneous expansion, and does not allow for homogeneous differentiation of cells within the population, is difficult and inefficient.

[0215] The provided monolayer differentiation platform facilitates differentiation into secondary hematopoietic endothelium, which gives rise to differentiated progeny such as hematopoietic stem cells and T cells, B cells, NKT cells, NK cells, etc. The monolayer differentiation strategy combines enhanced differentiation efficiency and large-scale expansion to enable the delivery of a therapeutically relevant number of pluripotent stem cell-derived hematopoietic cells for various therapeutic applications. Furthermore, monolayer culture using the methods provided herein yields functional hematopoietic lineage cells that enable the 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 hematopoietic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem and progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0216] A method for initiating the differentiation of pluripotent stem cells into cells of a secondary hematopoietic lineage, comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate the differentiation and expansion of mesodermal cells from the pluripotent stem cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor (the composition optionally does not include a TGFβ receptor / ALK inhibitor) to initiate the differentiation and expansion of mesodermal cells with secondary hematopoietic endothelial potential from the mesodermal cells; and (iii) contacting mesodermal cells with secondary hematopoietic endothelial potential with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator (the composition optionally does not include a TGFβ receptor / ALK inhibitor) to initiate the differentiation and expansion of secondary hematopoietic endothelium from pluripotent stem cell-derived mesodermal cells with secondary hematopoietic endothelial potential.

[0217] In some embodiments, the method further comprises contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (the composition does not contain a TGFβ receptor / ALK inhibitor) to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs containing one or more gene imprints, the one or more gene imprints contained in the iPSCs being retained in hematopoietic cells differentiated therefrom. In some embodiments of the method directed toward the differentiation of pluripotent stem cells into hematopoietic lineage cells, the differentiation of pluripotent stem cells into hematopoietic lineage cells lacks embryoid body formation and is in a monolayer culture form.

[0218] In some embodiments of the above method, the obtained pluripotent stem cell-derived secondary hematopoietic endothelial cells are CD34 + In some embodiments, the resulting secondary hematopoietic endothelial cells are CD34 + CD43 - In some embodiments, secondary hematopoietic endothelial cells are CD34 + CD43 - CXCR4- CD73 - In some embodiments, secondary hematopoietic endothelial cells are CD34 + CXCR4 - CD73 - In some embodiments, secondary hematopoietic endothelial cells are CD34 + CD43 - CD93 - In some embodiments, secondary hematopoietic endothelial cells are CD34 + CD93 - That is the case.

[0219] In some embodiments of the above method, the method further comprises (i) contacting secondary hematopoietic endothelium derived from pluripotent stem cells with a composition comprising a ROCK inhibitor; one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7; and optionally a BMP activator, thereby initiating the differentiation of the secondary hematopoietic endothelium into pre-T cell progenitor cells; and optionally, (ii) contacting the pre-T cell progenitor cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, thereby initiating the differentiation of the pre-T cell progenitor cells into T cell progenitor cells or T cells. In some embodiments of the method, the T cell progenitor cells derived from pluripotent stem cells are CD34 + CD45 + CD7 + In some embodiments of this method, pluripotent stem cell-derived T cell progenitor cells are CD45 + CD7 + That is the case.

[0220] In some further embodiments of the above method for oriented the differentiation of pluripotent stem cells into hematopoietic cell lineage cells, the method further comprises (i) contacting pluripotent stem cell-derived secondary hematopoietic endothelium with a composition comprising one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors; VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, to initiate the differentiation of the secondary hematopoietic endothelium into pre-NK cell progenitor cells; and optionally, (ii) contacting pluripotent stem cell-derived pre-NK cell progenitor cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 (the medium does not contain one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitors), to initiate the differentiation of the pre-NK cell progenitor cells into NK cell progenitor cells or NK cells. In some embodiments, pluripotent stem cell-derived NK cell progenitor cells are CD3 - CD45 + CD56 + CD7 + In some embodiments, NK cells derived from pluripotent stem cells are CD3 - CD45 + CD56 + And, optionally, NKp46 + CD57 + , and CD16 + It is further defined by...

[0221] 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 hematopoietic endothelium (iHE) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iii) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2. (iv) Multipotent progenitor cells (iMPP) using iMPP-A, (v) T cell progenitor cells (ipro-T) using one or more culture media selected from iTC-A2 and iTC-B2, (vi) T cells (iTC) using iTC-B2, (vii) NK cell progenitor cells (ipro-NK) using one or more culture media selected from iNK-A2 and iNK-B2, and / or (viii) NK cell progenitor cells (iNK) and iNK-B2. In some embodiments, the culture media are as follows: a. iCD34-C comprises one or more growth factors and cytokines selected from the group consisting of ROCK inhibitors, bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator, but does not contain a TGFβ receptor / ALK inhibitor; b.iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11; c.iTC-A2 contains ROCK inhibitors; one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7, and optionally, a BMP activator; d.iTC-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7; e.iNK-A2 comprises a ROCK inhibitor, as well as one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15; and f.iNK-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0222] In some embodiments, the genome-modified iPSC-derived cells obtained by the above method contain one or more inducible suicide genes integrated at one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In some other embodiments, genome-engineered iPSC-derived cells include polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transport, homing, viability, self-renewal, persistence, and / or survival of stem cells and / or progenitor cells. In some embodiments, genome-engineered iPSC-derived cells containing one or more suicide genes further include one or more in / dels contained in one or more endogenous genes involved in the regulation and mediation of immune responses, including but not limited to checkpoint genes, endogenous T cell receptor genes, and MHC class I repressor genes. In one embodiment, genome-engineered iPSC-derived cells containing one or more suicide genes further include an in / del in the B2M gene, where B2M is knocked out.

[0223] In addition, applicable dedifferentiation methods and compositions for obtaining second-fate genome-modified hematopoietic cells from first-fate genome-modified hematopoietic cells include, for example, those shown in International Publication No. 2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided herein partially reprogram initiating non-pluripotent cells into non-pluripotent intermediate cells by restricting the expression of the endogenous Nanog gene during reprogramming, and enable the non-pluripotent intermediate cells to be subjected to conditions for differentiation of intermediate cells into desired cell types. In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions herein include at least one genotype listed in Table 2. VI. Therapeutic use of derived immune cells with exogenous functional modalities differentiated from genetically engineered iPSCs

[0224] The present invention provides compositions comprising, in some embodiments, an isolated population or subpopulation of functionally enhanced derived immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSCs comprise one or more targeted gene edits retainable in the iPSC-derived immune cells, and the genetically engineered iPSCs and their derived cells are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived CD34 cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived proT or T lineage cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived proNK or NK lineage cells. In one embodiment, the isolated population or subpopulation of genetically engineered immune cells comprises iPSC-derived immunoregulatory cells or bone marrow-derived suppressor cells (MDSCs). In some embodiments, genetically engineered immune cells derived from iPSCs are further modified ex vivo for improved therapeutic potential. In one embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In another embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of type I NKT cells. In yet another embodiment, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of adaptive NK cells. In some embodiments, an isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cell lineage cells, NK cell lineage cells, or bone marrow-derived suppressor cells derived from iPSCs is allogeneic. In some other embodiments, an isolated population or subpopulation of genetically engineered CD34 cells, HSC cells, T cells, NK cells, NKT cells, or MDSCs derived from iPSCs is autologous.

[0225] In some embodiments, iPSCs for differentiation include selected gene imprints to convey desired therapeutic attributes in effector cells, provided that the cell developmental biology during differentiation is not disrupted and the gene imprints are retained and functional in differentiated effector cells derived from the iPSCs.

[0226] In some embodiments, the gene imprinting of pluripotent stem cells includes (i) one or more genetically modified modalities obtained by genomic insertions, deletions, or substitutions in the genome of pluripotent cells during or after reprogramming non-pluripotent cells into iPSCs, or (ii) one or more retainable therapeutic attributes of source-specific immune cells that are donor-specific, disease-specific, or treatment response-specific, wherein the pluripotent cells are reprogrammed from source-specific immune cells, and the iPSCs retain the source therapeutic attributes that are also present in iPSC-derived hematopoietic lineage cells.

[0227] In some embodiments, the genetic modification modality includes one or more safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, regulation and adjustment of immune responses, and / or viability of iPSCs or their derived cells. In some embodiments, the genetically modified iPSCs and their derived cells include the genotypes listed in Table 2. In several other embodiments, genetically modified iPSCs and their derived cells, including the genotypes listed in Table 2, further include additional genetic modification modalities, including (1) deletion or reduction of expression of one or more genes in the chromosome 6p21 region, such as TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and (2) introduced or increased expression of surface trigger receptors for coupling with HLA-E, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, antigen-specific TCR, Fc receptor, or bispecific, multispecific, or universal engagers.

[0228] In some other embodiments, the hematopoietic lineage cells include therapeutic properties of source-specific immune cells relating to at least two combinations of the following: (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) resistance to the tumor microenvironment, (iv) recruitment and immunomodulation of bystander immune cells, (v) improved target specificity by reducing extratumor effects, and (vi) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0229] In some embodiments, iPSC-derived hematopoietic cells include the genotypes listed in Table 2, and these cells express at least one cytokine and / or its receptor, or any modified protein thereof, including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, and express at least CAR. In some embodiments, the engineered expression of cytokines and CAR is NK cell-specific. In some other embodiments, the engineered expression of cytokines and CAR is T cell-specific. In one embodiment, the CAR includes a MICA / B binding domain. In some embodiments, the iPSC-derived hematopoietic effector cells are antigen-specific. In some embodiments, the antigen-specific derived effector cells target humoral tumors. In some embodiments, the antigen-specific derived effector cells target solid tumors. In some embodiments, the antigen-specific iPSC-derived hematopoietic effector cells can rescue tumor antigen escapes.

[0230] By introducing the immune cells of the present invention into subjects suitable for adoptive cell therapy, various diseases can be cured. In some embodiments, the iPSC-derived hematopoietic cells provided are for allogeneic adoptive cell therapy. In addition, in some embodiments, the present invention provides therapeutic use of the above-mentioned therapeutic composition by introducing the composition into subjects suitable for adoptive cell therapy, the subjects having autoimmune disorders, hematological malignancies, solid tumors, or infections related to HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus. Examples of hematological malignancies include, but are not limited to, acute and chronic leukemias (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, non-Hodgkin lymphoma (NHL), Hodgkin's disease, multiple myeloma, and myelodysplastic syndromes). Examples of solid tumors include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune disorders include alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), several forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, several forms of myocarditis, and multiple sclerosis. Examples of conditions include, but are not limited to, thyroiditis, bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, several forms of thyroiditis, several forms of uveitis, vitiligo, and granulomatous disease 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.

[0231] Treatment using the hematopoietic lineage cells derived from the embodiments disclosed herein can be carried out according to the symptoms or for preventing recurrence. The terms "treating", "treatment", etc. are generally used herein to mean obtaining the desired pharmacological and / or physiological effects. The effects can be preventive in terms of completely or partially preventing the disease, and / or can be therapeutic with respect to the partial or complete cure of the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any intervention in a disease in a subject, including: preventing the occurrence of a disease in a subject who is susceptible to the disease but has not yet been diagnosed as having it, inhibiting the disease, i.e., preventing its onset, or alleviating the disease, i.e., causing the disease to regress. The therapeutic agent or composition can be administered before, during, or after the onset of the disease or injury. Treatment of an ongoing disease where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient is also of particular interest. In certain embodiments, the subject requiring treatment has a disease, condition, and / or injury that can be suppressed, restored, and / or improved by cell therapy with respect to at least one related symptom. Certain specific embodiments contemplate that the subject requiring cell therapy includes, but is not limited to, a candidate for bone marrow or stem cell transplantation, a subject who has received chemotherapy or radiotherapy, a subject having or at risk of having a hyperproliferative disorder or cancer, such as a hematopoietic hyperproliferative disorder or cancer, a subject having or at risk of developing a tumor, such as a solid tumor, and a subject having or at risk of having a viral infection or a disease associated with a viral infection.

[0232] When evaluating the responsiveness to a treatment comprising the hematopoietic lineage cells derived from the embodiments disclosed herein, the response can be measured by criteria including at least one of clinical benefit rate, survival until death, pathologic complete response, semi - quantitative measurement of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, relapse - free survival, metastasis - free survival, disease - free survival, reduction of circulating tumor cells, circulating marker response, and RECIST (Response Evaluation Criteria In Solid Tumors) criteria.

[0233] The therapeutic compositions comprising the disclosed hematopoietic lineage cells may be administered to a subject before, during, and / or after other therapies. Therefore, combination therapy methods may involve the administration or preparation of iPSC-derived immune cells before, during, and / or after the use of additional therapeutic agents. As provided above, one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors containing one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive portions, or immunomodulatory agents (IMiDs). The administration of iPSC-derived immune cells can be separated from the administration of additional therapeutic agents by time units of hours, days, or even weeks. In addition, or alternatively, the administration may be combined with other bioactive agents or modalities, such as antitumor agents, non-pharmacological therapies such as surgery, etc., but are not limited to these.

[0234] In some embodiments of combination cell therapy, the therapeutic combination comprises iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent, which is an antibody or antibody fragment. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody may be a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability. In some embodiments, suitable antibodies for combination therapy as additional therapeutic agents to administered iPSC-derived hematopoietic lineage cells include CD20 antibodies (e.g., rituximab, bertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab), HER2 antibodies (e.g., trastuzumab, bertuzumab), CD52 antibodies (e.g., alemtuzumab), EGFR antibodies (e.g., cerltuximab), and GD2 antibodies (e.g., dinutuzumab). Examples include, but are not limited to, ximab, PDL1 antibodies (e.g., avelumab), CD38 antibodies (e.g., daratumumab, isatuximab, MOR202), CD123 antibodies (e.g., 7G3, CSL362), SLAMF7 antibodies (e.g., elotuzumab), MICA / B antibodies (e.g., 7C6, 6F11, 1C2), and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars.

[0235] In some embodiments, additional therapeutic agents include one or more checkpoint inhibitors. Checkpoints are cellular molecules, often cell surface molecules, that, if not inhibited, can suppress or downregulate the immune response. Checkpoint inhibitors are antagonists that can reduce checkpoint gene expression or gene product, or decrease the activity of checkpoint molecules. Checkpoint inhibitors provided herein that are suitable for combination therapy with derived effector cells, including NK cells or T cells, include PD1 (Pdcdl, CD279), PDL-1 (CD274), TIM3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG3 (Lag3, CD223), CTLA4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2aR, BATE, BTLA, CD39 (Entpdl), CD47, and CD73. This list includes, but is not limited to, antagonists of retinoic acid receptor alpha (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0236] Some embodiments of the combination therapy, including the provided derived effector cells, further include at least one inhibitor that targets a checkpoint molecule. Some other embodiments of the combination therapy with the provided derived effector cells include two or more inhibitors such that two or three or more checkpoint molecules are targeted. In some embodiments, the effector cells for the combination therapy described herein are the provided derived NK cells. In some embodiments, the effector cells for the combination therapy described herein are derived T cells. In some embodiments, the derived NK cells or T cells for the combination therapy are functionally enhanced as provided herein. In some embodiments, two or more checkpoint inhibitors may be administered in the combination therapy before or after administration, together with the administration of the derived effector cells. In some embodiments, two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

[0237] In some embodiments, the antagonist that inhibits any of the above checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitor antibody may be a mouse antibody, a human antibody, a humanized antibody, a camel Ig antibody, a shark heavy chain-only antibody (VNAR), an Ig NAR, a chimeric antibody, a recombinant antibody, or a fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be cost-effective to produce, easier to use, or more sensitive than the whole antibody. In some embodiments, one, two, or three or more checkpoint inhibitors include at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents.

[0238] Combination therapy including derived effector cells and one or more check inhibitors is used to treat cutaneous T-cell lymphoma, non-Hodgkin lymphoma (NHL), mycosis fungoides, Paget's reticular plaque, Sézary syndrome, granulomatous lasiocarcinoma, lymphomatoid papulosis, chronic lichenoid pityriasis, acute lichenoid plaque, and CD30 + Cutaneous T-cell lymphoma, secondary cutaneous CD30 +This treatment is applicable to the treatment of humoral and solid tumors, including but not limited to large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Renat lymphoma, subcutaneous T-cell lymphoma, vascular central lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, Hodgkins lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyosarcoma, Lewis lung cancer (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, small cell neuroendocrine carcinoma of the prostate (SCNC), liver cancer, glioblastoma, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal cancer.

[0239] In some embodiments, in addition to the derived effector cells provided herein, the combination for therapeutic use includes one or more additional therapeutic agents comprising chemotherapeutic agents or radioactive moieties. Chemotherapeutic agents refer to cytotoxic antitumor agents, i.e., chemical agents found to preferentially kill tumor cells, disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the growth of neoplastic cells. Chemotherapeutic agents are also sometimes referred to as antitumor or cytotoxic drugs or agents, and are well known in the art.

[0240] In some embodiments, the chemotherapeutic agent includes anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, folic acid analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxins, platinum-containing drugs, interferons, and interleukins. Exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (cyclophosphamide, mechloretamine, mephalin, chlorambucil, hairmethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), animitabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisanthren, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, meitansine, and amsacrine.Additional medications include amine glutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, carsterone, capecitabine, celecoxib, and cetonia. Simab, cladribine, cloflavin, cytarabine, dacarbazine, denileukin difutitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinylestradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, Idarubicin, Ifosfamide, Imatinib, Interferon alfa (2a, 2b), Irinotecan, Letrozole, Leucovorin, Leuprolide, Lebamisol, Mechloretamine, Megestrol, Melphalin, Mercaptopurine, Methotrexate, Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone, Nofetumomab, Oxaliplatin, Paclitaxel, Pamidronate, Pemetrexed, Pegademase, These include pegasparagase, pentostatin, pipobromane, plicamycin, polyfeprosan, porfimer, procarbazine, quinacrine, rituximab, salglamostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, barrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those known in the art and approved as chemotherapeutic or radiotherapeutic agents.Such drugs can be found in one of several standard physician and oncologist references (for example, Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Ninth Edition, McGraw-Hill, NY, 1995) or through the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated regularly.

[0241] Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide stimulate both NK cells and T cells. As provided herein, IMiDs may be used in conjunction with iPSC-derived therapeutic immune cells for cancer treatment.

[0242] In addition to the isolated population of iPSC-derived hematopoietic cells contained in the therapeutic composition, a composition suitable for administration to a patient may further include one or more pharmaceutically acceptable carriers (additives) and / or diluents (e.g., pharmaceutically acceptable media, e.g., cell culture media), or other pharmaceutically acceptable components. The pharmaceutically acceptable carriers and / or diluents are determined in part by the specific composition administered and the specific method used to administer the therapeutic composition. Thus, a wide variety of suitable formulations of the therapeutic composition of the present invention exist (e.g., Remington's Pharmaceutical Sciences, 17). th See ed. 1985, the disclosure thereof (which is incorporated herein in its entirety by reference).

[0243] In one embodiment, the therapeutic composition comprises pluripotent T cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent NK cells produced by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent CD34 cells produced by the methods and compositions disclosed herein. +The therapeutic composition includes HE cells. In one embodiment, the therapeutic composition includes pluripotent cell-derived hematopoietic stem cells (HSCs) prepared by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition includes pluripotent cell-derived hematopoietic stem cells (MDSCs) prepared by the methods and compositions disclosed herein. The therapeutic composition, comprising a population of iPSC-derived hematopoietic lineage cells disclosed herein, can be administered separately or in combination with other suitable compounds by intravenous, intraperitoneal, enteral, or tracheal administration to influence a desired therapeutic target.

[0244] These pharmaceutically acceptable carriers and / or diluents may be present in amounts sufficient to maintain the pH of the therapeutic composition between about 3 and about 10. Therefore, the buffer may be about 5% by weight relative to the total composition. Electrolytes, but not limited to sodium chloride and potassium chloride, may also be included in the therapeutic composition. In one embodiment, the pH of the therapeutic composition is in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition includes a buffer having a pH within one of these pH ranges. In yet another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0245] The present invention also provides, in part, the use of pharmaceutically acceptable cell culture media in certain compositions and / or cultures of the present invention. Such compositions are suitable for administration to human subjects. Generally speaking, any medium supporting the maintenance, growth, and / or health of iPSC-derived immune cells according to embodiments of the present invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, the serum-free medium is free of animal matter and may optionally be free of protein. Optionally, the medium may contain recombinant proteins acceptable for biological formulations. A medium free of animal matter refers to a medium whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in a medium free of animal matter, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, a protein-free medium is defined as substantially protein-free. Those skilled in the art will understand that the above examples of media are illustrative and in no way limit the formulation of media suitable for use in the present invention, and that there are many suitable media available that are known to those skilled in the art.

[0246] Isolated pluripotent stem cell-derived hematopoietic cell lines may contain at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34+HE cells, HSCs, B cells, bone marrow-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, isolated pluripotent stem cell-derived hematopoietic cell lines contain approximately 95% to approximately 100% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or bone marrow-derived suppressor cells (MDSCs). In some embodiments, the present invention provides therapeutic compositions having purified T cells or NK cells, such as compositions having an isolated population of about 95% T cells, NK cells, proT cells, proNK cells, CD34+HE cells, or bone marrow-derived suppressor cells (MDSCs), for treating subjects requiring cell therapy.

[0247] In one embodiment, the combination cell therapy comprises a therapeutic protein or peptide and a population of NK cells derived from a genome-engineered iPSC, the derived NK cells being modified with one or more small compound treatments as described herein. In several further embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202 and a population of NK or T cells derived from a genome-engineered iPSC including the genotypes listed in Table 2, the derived NK or T cells comprising a first CAR having the provided endodomain, CD38 null, hnCD16, a second CAR, and one or more exogenous cytokines.

[0248] As those skilled in the art will understand, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs can be used in the cell therapies described herein based on the methods and compositions herein. In the case of autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA-matched with that of the patient. In another embodiment, the derived hematopoietic lineage cells are not HLA-matched with that of the subject, and the derived hematopoietic lineage cells are NK cells or T cells having HLA I and HLA II nulls.

[0249] 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 5 x 10 6 Cells, at least 1 x 10 7 Cells, at least 5 x 10 7 Cells, at least 1 x 10 8 Cells, at least 5 x 10 8 Cells, at least 1 x 10 9 Cells, or at least 5x10 9 These are cells. In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1 x 10⁶ per dose. 5 cells ~ approx. 1x10 6 Cells, approximately 0.5 x 10⁶ per dose 6 cells ~ approx. 1x10 7Cells, about 0.5x10 per dose 7 Cells ~ about 1x10 8 Cells, about 0.5x10 per dose 8 Cells ~ about 1x10 9 Cells, about 1x10 per dose 9 Cells ~ about 5x10 9 Cells, about 0.5x10 per dose 9 Cells ~ about 8x10 9 Cells, about 3x10 per dose 9 Cells ~ about 3x10 10 Cells, or any range therebetween. Generally, for a 60 kg patient, 1x10 8 Cells / dose is converted to 1.67x10 6 Cells / kg.

[0250] In one embodiment, the number of hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a partial or single umbilical cord of blood, or at least 0.1x10 5 Cells / kg body weight, at least 0.5x10 5 Cells / kg body weight, at least 1x10 5 Cells / kg body weight, at least 5x10 5 Cells / kg body weight, at least 10x10 5 Cells / kg body weight, at least 0.7x10 6 Cells / kg body weight, at least 1.25x10 6 Cells / kg body weight, at least 1.5x10 6 Cells / kg body weight, at least 1.75x10 6 Cells / kg body weight, at least 2x10 6 Cells / kg body weight, at least 2.5x10 6 Cells / kg body weight, at least 3x10 6 Cells / kg body weight, at least 4x10 6 Cells / kg body weight, at least 5x10 6 Cells / kg body weight, at least 10x10 6 Cells / kg body weight, at least 15x10 6 Cells / kg body weight, at least 20x10 6 Cells / kg body weight, at least 25x10 6 Cells / kg body weight, at least 30x106 cells / kg body weight, 1x10 8 cells / kg body weight, 5x10 8 cells / kg body weight, or 1x10 9 cells / kg body weight.

[0251] In one embodiment, a certain dose of hematopoietic progenitor cells is delivered to a subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2x10 6 cells / kg, at least 3x10 6 cells / kg, at least 4x10 6 cells / kg, at least 5x10 6 cells / kg, at least 6x10 6 cells / kg, at least 7x10 6 cells / kg, at least 8x10 6 cells / kg, at least 9x10 6 cells / kg, or at least 10x10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0252] In another exemplary embodiment, the effective amount of cells provided to the subject is about 2x10 6 cells / kg, about 3x10 6 cells / kg, about 4x10 6 cells / kg, about 5x10 6 cells / kg, about 6x10 6 cells / kg, about 7x10 6 cells / kg, about 8x10 6 cells / kg, about 9x10 6 cells / kg, or about 10x10 6 cells / kg, or more cells / kg (including all intervening cell doses).

[0253] In another exemplary embodiment, the effective amount of cells provided to the subject is about 2x10 6 cells / kg to about 10x10 6 cells / kg, about 3x10 6 cells / kg to about 10x10 6 cells / kg, about 4x10 6cells / kg ~ approx. 10x10 6 cells / kg, approximately 5x10 6 cells / kg ~ approx. 10x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 2x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 3x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 4x10 6 cells / kg ~ approx. 8x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 6x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 7x10 6 cells / kg, 5x10 6 cells / kg ~ approx. 8x10 6 cells / kg, or 6x10 6 cells / kg ~ approx. 8x10 6 This is expressed as cells / kg (including all intervening cellular doses).

[0254] In some embodiments, the therapeutic use of derived hematopoietic lineage cells is a single-dose therapy. In some embodiments, the therapeutic use of derived hematopoietic lineage cells is a multiple-dose therapy. In some embodiments, the multiple-dose therapy is administered daily, 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 at any number of days in between.

[0255] A composition comprising a population of derived hematopoietic lineage cells of the present invention may be sterile, suitable for administration to human patients, and ready for administration (i.e., can be administered without further treatment). A cell-based composition ready for administration means that the composition does not require any further treatment or manipulation before transplantation or administration to a subject. In other embodiments, the present invention provides an isolated population of derived hematopoietic lineage cells that are expanded and / or regulated before administration of one or more agents. In the case of derived hematopoietic lineage cells genetically engineered to express recombinant TCR or CAR, the cells can be activated and expanded using, for example, the method described in U.S. Patent No. 6,352,694.

[0256] In certain embodiments, the primary stimulatory and co-stimulatory signals of the derived hematopoietic lineage cells may be provided by different protocols. For example, the agents providing each signal may be in solution or conjugated to the surface. If conjugated to the surface, the agents may be conjugated to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent may be conjugated to the surface while the other agent is present in solution. In one embodiment, the agent providing the co-stimulatory signal may be conjugated to the cell surface, and the agent providing the primary activation signal may be in solution or conjugated to the surface. In certain embodiments, both agents may be in solution. In another embodiment, the agent is in a soluble form and can then be crosslinked to the surface of an antibody or other binder that conjugates to an agent, such as those disclosed in U.S. Patent Applications Publication Nos. 2004 / 0101519 and 2006 / 0034810, which are intended for use in activating and expanding cells expressing Fc receptors or artificial antigen-presenting cells (aAPCs) intended for use in embodiments of the present invention.

[0257] Some variation in dosage, frequency, and protocol is inevitable depending on the condition of the patient being treated. The person responsible for administration will, in any case, determine the appropriate dosage, frequency, and protocol for each individual patient. Examples

[0258] The following examples are provided for illustrative purposes only and are not limiting. Example 1 - Materials and Method

[0259] Using the applicant's proprietary hiPSC platform, which enables single-cell passage and high-throughput 96-well plate-based flow cytometry sorting to effectively select and test suicide systems in combination with different safe harbor locus integration strategies under the control of various promoters, it enables the induction of clonal hiPSCs by single or multiple gene regulation.

[0260] Maintenance of hiPSCs in small molecule cultures: When the culture reached confluence at 75%–90%, hiPSCs were routinely passaged as single cells. For single-cell dissociation, hiPSCs were washed once with PBS (Mediatech), treated with acetylase (Millipore) at 37°C for 3–5 minutes, and then pipetted to ensure single-cell dissociation. The single-cell suspension was then mixed with an equal volume of conventional medium, centrifuged at 225xg for 4 minutes, resuspended in FMM, and plated onto a Matrigel-coated surface. Passaging was typically done at a ratio of 1:6–1:8, at 37°C for 2–4 hours, transferred to a pre-coated tissue culture plate with Matrigel, and supplied with FMM every 2–3 days. Cell cultures were maintained in a humidified incubator set to 37°C and 5% CO2.

[0261] 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, 2 million iPSCs were transfected with a mixture of 2.5 ug of ZFN-L (FTV893), 2.5 ug of ZFN-R (FTV894), and 5 ug of donor construct for AAVS1-targeted insertion. For CRISPR-mediated genome editing, 2 million iPSCs were transfected with a mixture of 5 ug of ROSA26-gRNA / Cas9 (FTV922) and 5 ug of donor construct for ROSA26-targeted insertion. Transfection was performed using the Neon transfection system (Life Technologies) with parameters 1500 V, 10 ms, and 3 pulses. Transfection efficiency was measured using flow cytometry on day 2 or 3 after transfection, if the plasmid contained an artificial promoter driver GFP and / or RFP expression cassette. Four days after transfection, puromycin was added to the culture medium at a concentration of 0.1 ug / ml for the first seven days, and then at a concentration of 0.2 ug / ml thereafter, to select targeted cells. During puromycin selection, cells were passaged into new wells coated with Matrigel on day 10. From day 16 onward of puromycin selection, viable cells were GFP-treated. + The proportion of iPS cells was analyzed using flow cytometry.

[0262] Bulk and clonal sorting of genome-edited iPSCs: iPSCs with genome-targeted editing using ZFN or CRISPR-Cas9 are selected 20 days after puromycin selection, followed by GFP selection. + SSEA4 + TRA181 +Bulk and clonal sorting of iPSCs was performed. Targeted iPSC pools dissociated as single cells were resuspended in a newly prepared chilled staining buffer containing Hanks equilibrium salt solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1x penicillin / streptomycin (MediaTech), and 10 mM Hepes (MediaTech) for optimal performance. Conjugated primary antibodies, including SSEA4-PE and TRA181-Alexa Fluor-647 (BD Biosciences), were added to the cell solution and incubated on ice for 15 minutes. All antibodies were used at a rate of 7 μL per 1 million cells in 100 μL of staining buffer. The solution was washed once with staining buffer, spun down at 225 g for 4 minutes, resuspended in staining buffer containing 10 μM thiazovibone, and maintained on ice for flow cytometry sorting. Flow cytometry sorting was performed using FACS Aria II (BD Biosciences). In bulk sorting, GFP + SSEA4 + TRA181 +Cells were gated and sorted into 15 ml standard tubes filled with 7 ml of FMM. For clonal sorting, sorted cells were dispensed directly into a 96-well plate using a 100 μM nozzle at a concentration of 3 events per well. Each well was pre-filled with 200 μL of FMM supplemented with 5 μg / mL of fibronectin and 1x penicillin / streptomycin (Mediatech) and pre-coated overnight with 5x Matrigel. The 5x Matrigel pre-coating involved adding one aliquot of Matrigel to 5 mL of DMEM / F12, then incubating overnight at 4°C to allow proper resuspension, and finally adding 50 μL per well to the 96-well plate, followed by incubation overnight at 37°C. The 5x Matrigel was aspirated immediately before adding medium to each well. Once sorting was complete, the 96-well plate was centrifuged at 225 g for 1–2 minutes before incubation. The plates were left standing for 7 days. On day 7, 150 μL of medium was removed from each well and replaced with 100 μL of FMM. On day 10 after sorting, an additional 100 μL of FMM was resupplied to the wells. Colony formation was detected as early as day 2, and most colonies expanded within 7–10 days after sorting. For the first passaging, the wells were washed with PBS and dissociated with 30 μL of acetylene ester (Accutase) at 37°C for approximately 10 minutes. The need for extended Accutase treatment reflects the compactness of colonies that had been idling in long-term culture. After the cells were confirmed to be dissociated, 200 μL of FMM was added to each well and the colonies were disrupted by pipetting several times. The dissociated colonies were transferred to another well in a 96-well plate pre-coated with 5x Matrigel and then centrifuged at 225 g for 2 minutes before incubation. This 1:1 passaging is performed to expand the initial colonies before they expand. Subsequent passaging was routinely performed with 3–5 minutes of accutase treatment and 1:4–1:8 expansion at 75–90% confluence into larger wells pre-coated with 1x Matrigel in FMM. Each clonal cell line was analyzed for GFP fluorescence levels and TRA1-81 expression levels. Near 100% GFP + and TRA181 +Clonal strains were selected for further PCR screening and analysis. Flow cytometry analysis was performed using Guava EasyCyte 8 HT (Millipore) and analyzed using Flowjo (FlowJo, LLC). Example 2 - In vitro and in vivo functional profiling of iPSC-derived NK cells treated with small compounds.

[0263] iNK cells expressing a CD19-specific chimeric antigen receptor were treated with dexamethasone or dexamethasone and IL-7 for the last 5 days of post-differentiation proliferation without supplementing the growth medium with IL-15 or IL-2. Granzyme B levels were then compared to untreated control iNK cells by flow cytometry staining. The geometric mean fluorescence intensity (GMFI) in Figure 1A shows that granzyme B protein levels in iNK cells treated with dexamethasone or dexamethasone and IL-7 were reduced compared to the untreated control. Similar functional suppression, reflected by decreased granzyme B expression, was observed using primary NK cells treated with dexamethasone. As shown in Figure 1B, peripheral blood NK cells were treated with either IL-15, known to upregulate granzyme levels, or 1 or 10 μM dexamethasone. Additional dexamethasone concentration levels were tested, demonstrating that the effect was not particularly concentration-dependent. Granzyme B levels were determined by flow cytometry staining, and the geometric mean fluorescence intensity (GMFI) is shown in Figure 1B, indicating decreased granzyme B expression and thus suppressed cellular function of treated primary NK cells.

[0264] iNK cells expressing CD19-CAR were treated for 5 days with dexamethasone, lenalidomide, rapamycin, or a combination of dexamethasone and lenalidomide, and then cryopreserved. The cells were thawed and Nalm6(CD19) +Cytotoxicity was assessed by immediately using the cells as effectors in a 4-hour cytotoxicity assay against CAR-expressing iNK cells or Nalm6 CD19 knockout (19ko) cells. EC50 was determined by nonlinear regression, where EC50 = 50% is the E:T ratio required to achieve specific cytotoxicity, and a lower EC50 indicates higher cytotoxicity. As shown in Figure 2A, small molecule treatment of CAR-expressing iNK cells improved antigen-specific recognition, with dexamethasone showing the best discrimination between antigen-positive and negative targets. It remains unclear whether the reduced nonspecific interactions in vitro promote better in vivo distribution of effector cells, which is beneficial to cell efficacy.

[0265] The cytotoxicity of iNK cells was further evaluated using CD19-CAR-expressing iNK cells that were treated for 5 days with dexamethasone, lenalidomide, rapamycin, or a combination of dexamethasone and lenalidomide, then cryopreserved, thawed, and rested overnight before initiating cytotoxicity assays. Similarly, EC50 was determined by nonlinear regression, where EC50 = 50% is the E:T ratio required to achieve specific cytotoxicity, and a lower EC50 indicates higher cytotoxicity. As shown in Figure 2B, treatment with small compounds during post-differentiation iNK cell proliferation resulted in better functional recovery over time, with dexamethasone being superior to rapamycin treatment, and the dexamethasone / lenalidomide combination treatment being superior to untreated control cells before cryopreservation.

[0266] To perform a long-range killing assay, CAR-iNK cells were treated with specified compounds, cryopreserved, thawed, and used as effectors in a 24-hour cytotoxicity assay against Raji B-cell lymphoma lines. The results are shown in Figure 3A, where the normalized number of target cells remaining at each time point is 100 for target alone, and a lower number of targets indicates higher cytotoxicity. The area on the curve (AOC) was also calculated for cytotoxicity against Raji and Raji CD19 knockout (CD19KO) cells (Figure 3B). A larger AOC corresponds to increased cytotoxicity. As shown in Figures 3A and 3B, dexamethasone alone or in combination with lenalidomide showed the best tumor-killing performance in CAR-iNK cells, followed by lenalidomide-treated and AQX-treated cells. Consistent with the cytotoxicity assays in 2A and 2B, dexamethasone treatment resulted in better differentiation between antigen-positive and negative targets (see Figure 3B). In comparison, CAR-iNK cells cryopreserved and thawed without pretreatment do not effectively control tumor cell proliferation.

[0267] To evaluate the effect of compound treatment on the in vivo efficacy of cells, CAR-iNK cells were treated with the indicated compounds for the last 5 days of culture prior to cryopreservation. Next, cryopreserved control cells or compound-treated CD19-CAR iNK cells were thawed from the frozen stock and used to treat NSG mice transplanted with 1E5Nalm6-luciferase cells 1 day prior. Tumor progression was monitored by bioluminescence imaging on days 7 and 14. As shown in Figure 4A, compound treatment of iNK cultures with dexamethasone alone or in combination with dexamethasone and lenalidomide improved in vivo efficacy.

[0268] CD19-CAR hnCD16iNK cells treated with dexamethasone for the last 5 days of culture prior to cryopreservation were tested in an in vivo model of B-cell lymphoma using NSG mice transplanted with 1E5 Raji luciferase cells 1 day prior. Cryopreserved CD19-CAR hnCD16iNK cells treated with dexamethasone during culture and prior to cryopreservation were thawed and injected into Raji luciferase-inoculated mice on days 1, 4, and 7 post-inoculation in combination with rituximab (0.3 ug / mouse) 1 day post-inoculation. Tumor progression was monitored by bioluminescence imaging on days 2, 7, and 15. As shown in Figure 4B, the combination of dexamethasone-treated rituximab and CD19-CAR hnCD16iNK cells provided improved control of tumor growth compared to rituximab alone without the iNK cells.

[0269] MICA / B-CAR iNK cells treated with dexamethasone for the last 5 days of culture prior to cryopreservation were tested in an in vivo model of solid tumor metastasis using NSG mice that had been IV transplanted 1 day prior with B16 melanoma cells engineered to express MICA. Cryopreserved MICA / B-CAR iNK cells treated with dexamethasone during culture and prior to cryopreservation were thawed and injected into tumor-transplanted mice. Fourteen days after tumor transplantation, the number of tumor nodules in the lungs (Figure 4C) and the number of iNK cells present in the spleen (Figure 4D) were quantified. As shown in Figures 4C and 4D, compound treatment of iNK cultures with dexamethasone alone improved tumor growth control and iNK persistence compared to iNK cells that were not treated prior to cryopreservation.

[0270] To further demonstrate the persistence of iNK cells in vivo, approximately 1.2E7 cells were injected into tumor-free NSG mice three times a week (days 1, 8, and 15) using peripheral blood NK cells grown in vitro, iNK cells cultured without dexamethasone, or iNK cells cultured with dexamethasone. The persistence of cells injected into peripheral blood was assessed by flow cytometry on days 8, 15, 16, 22, 29, 36, and 43. As shown in Figure 4E, compound treatment of iNK cultures with dexamethasone alone improved the persistence of iNK cells throughout the study period.

[0271] Differential gene analysis was performed using RNAseq™ to compare gene expression profiles between untreated control iNK cells and iNK cells treated with dexamethasone or a dexamethasone / lenoridamide combination during post-differentiation proliferation. As shown in Figure 5, treatment of iNK cells with dexamethasone or dexamethasone and lenoridamide promotes specific gene expression profiles. In the case of dexamethasone-treated iNK cells, the most differentially expressed genes compared to untreated iNK cells include at least upregulated genes such as SPOCK2, PTGDS, IL7R, LCNL1, RASGRP2, and SMAP2, as well as downregulated genes such as JCHAIN, KLF3, KLRB1, IGFBP4, and NUCB2. Known information regarding these genes is briefly described in Table 4. [Table 3] Example 3 - In vitro and in vivo functional profiling of iPSC-derived T cells treated with small compounds.

[0272] iT cells expressing CD19-specific chimeric antigen receptors are differentiated from iPSCs and then subjected to 41BBL and IL21 or CD19 lowUsing feeder cells expressing IL-7, treatment with dexamethasone (with or without IL-7) was performed for the last 5 days of proliferation. Control CAR-iT cells cultured with dexamethasone or CAR-iT cell cultures were evaluated by RNA-seq and differential gene set enrichment analysis was performed. Treatment of iT cells with dexamethasone promotes unique gene expression profiles. As shown in Figure 6, IL6ST, IL-7R, and IL2RA were highly induced by dexamethasone treatment, while CXCR6 and CSF2RB were highly expressed in iT cells without dexamethasone treatment. Dexamethasone treatment with or without IL-7 did not show a significant difference in the proliferation rate of CAR-iT cells (Figure 7A). Next, various cell surface markers were evaluated by flow cytometry. The absence of IL7 during dexamethasone treatment did not affect the phenotype (Figure 7B-7C), and furthermore, it indicates that there is no need to supplement IL7 with dexamethasone during CAR-iT cell proliferation.

[0273] CAR-iT cells proliferated during the last 5 days of culture prior to cryopreservation, regardless of whether or not they were treated with dexamethasone. Cryopreserved control or dexamethasone-treated CD19-CAR iT cells were thawed from the frozen stock and administered intravenously on days 3, 6, and 9 to NSG female mice (N=5 in each group) that had been intravenously injected with 1E5 Nalm6-luciferase cells 3 days prior on day 0. Tumor progression was monitored by bioluminescence imaging on days 7, 14, and 20 after tumor injection. Dexamethasone-treated CAR-iT cells (Figure 8B) were compared with untreated control cells in culture (Figure 8A) for iv / iv in vivo efficacy. As shown, dexamethasone treatment improved iT in vivo efficacy. Figures 9A–9B show that dexamethasone-treated CAR-iT cells performed better than primary CAR-T cells in in vivo tumor control and clearance. The mice used to generate the data in Figures 9A-9B were sacrificed on days 24, 31, and 35 after tumor injection for analysis of human and tumor cells by flow cytometry. As shown in Figures 10A-10B, CAR-iT cells treated with dexamethasone survived in the mouse bone marrow tissue of the systemic xenography mouse model of lymphocytic leukemia, and their survival rate was extended compared to primary CAR-T cells (survival days > 80 days, p > 0.1), further confirming the effect of dexamethasone treatment on the in vivo efficacy of iPSC-derived effector cells.

[0274] In further cytokine withdrawal studies, CAR-iT cells were cultured with dexamethasone alone (no cytokines), dexamethasone and cytokine IL7 alone (without IL2 or IL15), or various combinations of cytokines including IL2, IL7, and IL15 (data not shown). After 7 days of culture, cell proliferation and expansion were evaluated. As shown in Figures 11A-11B, CAR-iT cells treated with dexamethasone showed a similar cellular phenotype to those treated with dexamethasone + IL7. As shown in Figure 11C, dexamethasone treatment in the absence of cytokines resulted in significantly lower CAR-iT cell proliferation compared to dexamethasone treatment with various combinations of IL2, IL7, and IL15. CAR-iT cells treated with dexamethasone alone and dexamethasone + IL7 were administered intravenously on day 0 to NSG female mice (N=5 in each group) that had been intravenously injected with 1E5 Nalm6-luciferase cells 3 days prior. These were administered intravenously on days 3, 6, and 9 after tumor transplantation. Tumor progression was monitored by bioluminescence imaging on days 2, 7, 14, 21, 28, and 35 after tumor injection. As shown in Figure 11D, CAR-iT cells treated with dexamethasone alone (no cytokines) and dexamethasone + IL7 were compared with untreated control cells in culture for iv / iv in vivo efficacy. Despite their low in vitro proliferation rate, CAR-iT cells treated with dexamethasone exhibited in vivo efficacy, at least surprisingly, similar to CAR-iT cells treated with dexamethasone + IL7, with both treated CAR-iT cells showing improved efficacy compared to untreated cells (without dexamethasone or cytokines).

[0275] Those skilled in the art will readily understand that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended to limit the scope of the invention. It will also be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosures disclosed herein without departing from the scope and spirit of the invention.

[0276] All patents and publications referenced herein represent the level of expertise of those skilled in the art. All patents and publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0277] The disclosures described herein as exemplary may be implemented without any elements or limitations not specifically disclosed herein. For example, in each case herein, any of the terms “including,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms. The terms and expressions used are for illustrative purposes only and are not limiting; in the use of such terms and expressions, there is no intention to exclude any equivalents of the exhibited and described features or parts thereof, but it is recognized that various modifications are possible within the scope of the claimed disclosure. Therefore, although the disclosure is specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be conceived by those skilled in the art, and such modifications and variations should be understood as falling within the scope of the invention as defined by the appended claims.

Claims

1. A method for producing immune cells or a population thereof, (1) Treat the immune cells with a small compound including dexamethasone, and (2) A method for producing immune cells or a population thereof, wherein the immune cells subjected to the small compound treatment are cryopreserved, thereby obtaining immune cells with enhanced cytotoxicity after thawing compared to corresponding immune cells without the same small compound treatment, wherein the small compound treatment is performed during the in vitro proliferation of the immune cells.

2. The method according to claim 1, wherein the immune cells are derived effector immune cells differentiated from induced pluripotent stem cells (iPSCs), and the effector immune cells include derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional characteristics not present in the corresponding primary T, NK, NKT, and / or B cells.

3. The aforementioned iPSC, (i) A first chimeric antigen receptor (CAR) having first target specificity, (ii) CD38 Knockout, (iii) HLA-I deficient and / or HLA-II deficient cells compared to the corresponding natural cells. (iv) Introduction of HLA-G or non-cleavable HLA-G expression, (v) CD16, (vi) A second CAR having second target specificity, (vii) A signaling complex comprising a partial or complete peptide of an exogenous cytokine expressed on the cell surface and / or its receptor, (viiii) Deletion or reduced expression of at least one of the following compared to the corresponding natural cells: B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ The method according to claim 2, comprising at least one of introduced or increased expression of at least one of R, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for binding to a bispecific or multispecific or universal engager, wherein the effector immune cells differentiated from the iPSC contain the same one or more edits as the iPSC.

4. The first CAR is, (i) An ectodomain comprising at least one antigen recognition region, a transmembrane domain, and an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is derived from the cytoplasmic domain of a signal transduction protein specific to the activation or function of T and / or NK cells. (ii) an antigen recognition domain that specifically binds to an antigen associated with a disease, pathogen, humoral tumor, or solid tumor, or (iii) The following: (a) Any one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1, or (b) ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigens of cytomegalovirus (CMV) infected cells, epithelial glycoprotein 2 EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase ( hTERT), ICAM-1, Integrin B7, Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, Melanoma antigen family A1 (MAGE-A1), MICA / B, Mucin 1 (Muc-1), Mucin 16 (Muc-16), Mesothelin (MSLN), NKCSI The method according to claim 3, comprising an antigen recognition domain specific to one of the following: NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryonic antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1).

5. The first CAR is, (1) A partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface, wherein the exogenous cytokine or its receptor is (a) IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and at least one of their respective receptors, (b) (i) Co-expression of IL15 and IL15Rα using self-cleaving peptides, (ii) A fusion protein of IL15 and IL15Rα, (iii) IL15 / IL15Rα fusion protein having the intracellular domain of IL15Rα that has been cleaved or excluded, (iv) A fusion protein of the membrane-bound Sushi domains of IL15 and IL15Rα, (v) A fusion protein of IL15 and IL15Rβ, (vi) A fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either natural or modified. (vii) A partial or complete peptide of a cell surface-expressed exogenous cytokine or its receptor, comprising at least one of the homodimer of IL15Rβ and (2) Antibodies or fragments thereof (3) Engeja, or (4) The method according to claim 3, comprising a bicistronic construct co-expressing a checkpoint inhibitor.

6. The method according to claim 1, further comprising cryopreserving the immune cells and then treating the immune cells with the small compound.

7. The method according to claim 6, wherein the cryopreservation does not include, or substantially does not include, one or more of the minor compounds of the treatment.

8. The enhanced post-thaw cytotoxicity includes enhanced in vivo efficacy of immune cells thawed after cryopreservation, and the post-thaw immune cells having the small compound treatment exhibit the following characteristics compared to the corresponding post-thaw immune cells without the same small compound treatment: (i) Enhanced ability in tumor control, tumor clearance, and / or reduction of tumor recurrence, (ii) Improvement of tumor penetration, or (iii) Improved ability to move to the bone marrow and / or tumor site The method according to claim 1, comprising at least one of the following.

9. The aforementioned treatment of the small compound (i) Optionally, the cytokine IL-7 is not included or is essentially not included, wherein the immune cells during the treatment are T cells. (ii) Optionally, the preparation does not contain cytokine IL2 and / or cytokine IL15, or is essentially free from cytokine IL2, wherein the immune cells during the treatment are NK cells. (iii) Contains dexamethasone but does not contain cytokine IL-7, (iv) Does not contain cytokines, or does not contain them in nature, (v) During cell culture and / or before or after cryopreservation, (vi) Immune cells are proliferating after being differentiated from iPSCs, and / or (vii) The method according to claim 1, wherein the freezing and storage is carried out for 1 to 12 days prior to the freezing and storage.

10. The method according to claim 9, wherein the dexamethasone is present in a concentration range of 10 nM to 20 μM.

11. (a) cells or a population thereof, and (b) frozen A composition comprising a preservation medium, (i) The cells are immune cells that have been treated with one or more small compounds containing an effective amount of dexamethasone, and which exhibit enhanced cytotoxicity after thawing compared to corresponding immune cells that have not been treated with the same small compounds. (ii) The composition is stored by freezing, composition.

12. (iii) The immune cells are derived effector immune cells differentiated from induced pluripotent stem cells (iPSCs), (iv) The composition according to claim 11, wherein the effector immune cells include derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional features not present in the corresponding primary T, NK, NKT, and / or B cells.

13. The aforementioned immune cells, edited as follows, (i) A first chimeric antigen receptor (CAR) having first target specificity, (ii) CD38 Knockout, (iii) HLA-I deficient and / or HLA-II deficient cells compared to their natural corresponding cells, (iv) Introduction of HLA-G or non-cleavable HLA-G expression, (v) CD16, (vi) A second CAR having second target specificity, (vii) A signaling complex comprising a partial or complete peptide of an exogenous cytokine expressed on the cell surface and / or its receptor, (viiii) deletion or reduced expression of at least one of the following compared to the corresponding native cells: B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ The composition according to claim 12, comprising at least one of R, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and introduced or increased expression of at least one of surface trigger receptors for binding to bispecific or multispecific or universal engagers, wherein the effector immune cells differentiated from the iPSC contain the same one or more edits as the iPSC.

14. The first and second CARs operate independently, (i) An ectodomain comprising at least one antigen recognition region, a transmembrane domain, and an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is derived from the cytoplasmic domain of a signal transduction protein specific to the activation or function of T and / or NK cells. (ii) an antigen recognition domain that specifically binds to an antigen associated with a disease, pathogen, humoral tumor, or solid tumor, or (iii) (a) Any one of CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, EGFR, GD2, MICA / B, MSLN, VEGF-R2, PSMA, and PDL1, or (b) ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV) infected cells, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine-protein kinase erb- B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A The composition according to claim 13, comprising an antigen recognition domain specific to one of the following: 1 (MAGE-A1), MICA / B, mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor embryo 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), and Wilms tumor protein (WT-1).

15. The first CAR is, (1) A partial or complete peptide of an exogenous cytokine or its receptor expressed on the cell surface, wherein the exogenous cytokine or its receptor is (a) IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and at least one of their respective receptors, (b) (i) Co-expression of IL15 and IL15Rα using self-cleaving peptides, (ii) A fusion protein of IL15 and IL15Rα, (iii) IL15 / IL15Rα fusion protein having the intracellular domain of IL15Rα that has been cleaved or excluded, (iv) A fusion protein of the membrane-bound Sushi domains of IL15 and IL15Rα, (v) A fusion protein of IL15 and IL15Rβ, (vi) A fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either natural or modified. (vii) A partial or complete peptide of a cell surface-expressed exogenous cytokine or its receptor, comprising at least one of the homodimer of IL15Rβ and (2) Antibodies or fragments thereof, (3) The composition according to claim 13, comprising a bicistronic construct co-expressing a checkpoint inhibitor.

16. The composition according to claim 11, wherein dexamethasone is present at a concentration in the range of 10 nM to 20 μM.

17. The enhanced post-thaw cytotoxicity includes enhanced in vivo efficacy of immune cells thawed after cryopreservation, and further features the following: (i) Enhanced ability in tumor control, tumor clearance, and / or reduction of tumor recurrence, (ii) Improvement of tumor penetration, or (iii) Improved ability to move to the bone marrow and / or tumor site The composition according to claim 11, comprising at least one of the following.

18. The aforementioned treatment of the small compound (i) Optionally, the cytokine IL-7 is not included or is essentially not included, wherein the immune cells during the treatment are T cells. (ii) Optionally, the preparation does not contain cytokine IL2 and / or cytokine IL15, or is essentially free from cytokine IL2, wherein the immune cells during the treatment are NK cells. (iii) Does not contain cytokines, or does not contain them in nature. The composition according to claim 11.

19. The aforementioned immune cells, compared to the corresponding immune cells without the same small compound treatment, (i) Upward control of SPOCK2, PTGDS, IL7R, LCNL1, RASGRP2, SMAP2, IL6ST, IL-7R, and IL2RA, or (ii) The composition according to claim 11, comprising one or more differentially expressed genes, including at least one of the downregulations of JCHAIN, KLF3, KLRB1, IGFBP4, NUCB2, CSF2RB, and CXCR6.

20. The one or more small compound treatments are (i) The cytokine IL7 is not included, and optionally the immune cell is a T cell, (ii) Not containing cytokine IL2 or cytokine IL15, and optionally the immune cells being NK cells, (iii) The composition according to claim 11, which does not contain cytokines or is essentially free from them.

21. A method for producing immune cells or a population thereof, (a) Differentiating genetically modified iPSCs to obtain the immune cells, wherein the iPSCs are as follows: (i) A first chimeric antigen receptor (CAR) having first target specificity, (ii) CD38 Knockout, (iii) HLA-I deficient and / or HLA-II deficient cells compared to their natural corresponding cells, (iv) Introduction of HLA-G or non-cleavable HLA-G expression, (v) CD16, (vi) A second CAR having second target specificity, (vii) A signaling complex comprising a partial or complete peptide of an exogenous cytokine expressed on the cell surface and / or its receptor, (viiii) Deletion or reduced expression of at least one of the following compared to the corresponding native cells: B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. (ix) HLA-E, 41BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ The at least one of the following is introduced or increased expression of at least one of R, antigen-specific TCR, Fc receptor, antibody or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for binding to bispecific or multispecific or universal engager, The immune cells differentiated from the iPSC contain the same one or more edits as the iPSC, (b) Providing the immune cells for treatment with a small compound containing dexamethasone, (c) The immune cells subjected to the small compound treatment are cryopreserved, A method for producing immune cells or populations thereof, comprising obtaining immune cells with enhanced cytotoxicity after thawing compared to corresponding immune cells without the same small compound treatment, wherein the small compound treatment is performed during the in vitro proliferation of the immune cells.

22. The further method comprises genomically manipulating a cloned iPSC to knock in a polynucleotide encoding the first CAR, optionally, (i) Knock out CD38, (ii) Knock out B2M and CIITA, and / or (iii) The method according to claim 21, further comprising introducing the expression of HLA-G or an incleavable HLA-G, CD16, a second CAR, and / or a partially or completely peptide of a cell surface-expressed exogenous cytokine or its receptor.

23. The method according to claim 21, wherein the immune cells differentiated from the induced pluripotent stem cells (iPSCs) include derived CD34 cells, derived hematopoietic stem and progenitor cells, derived hematopoietic pluripotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells, derived B cells, or derived effector cells having one or more functional characteristics not present in the corresponding primary T, NK, NKT, and / or B cells.

24. A therapeutic composition comprising the composition according to any one of claims 11 to 20 and one or more therapeutic agents.

25. The therapeutic composition according to claim 24, wherein the one or more therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors containing one or more target polynucleic acids, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulators (IMiDs).

26. (i) The checkpoint inhibitor is (a) One or more antagonists for 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 the following: atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents, or (c) comprising at least one of atezolizumab, nivolumab, and pembrolizumab, or (ii) The therapeutic composition according to claim 25, wherein the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide.

27. The aforementioned 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 the following: rituximab, vertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cerutuximab, dinutuzumab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, and their functional equivalents and biosimilars; or (c) The composition according to claim 25, comprising daratumumab, wherein the immune cells are derived NK cells or derived T cells containing CD38 knockout, and optionally expressing CD16.

28. Use of cells or populations thereof according to any one of claims 11 to 19 or later for the manufacture of pharmaceuticals for the treatment of autoimmune disorders, hematological malignancies, solid tumors, cancer, or viral infections.

29. (i) Thawing cryopreserved immune cells, (ii) The method according to claim 21, further comprising preparing the thawed immune cells for administration to a subject.

30. The method according to claim 29, wherein the immune cells are iPSC-derived NK cells, iPSC-derived T cells, or iPSC-derived effector cells having one or more functional characteristics not present in the corresponding primary T, NK, NKT, and / or B cells.

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