Protected effector cells for allogeneic adoptive cell therapy and uses thereof

Abstract

Methods and compositions are provided for obtaining functionally enhanced derived effector cells obtained from directed differentiation of genomically engineered iPSCs. The derivative cell embodiments provided herein have stable and functional genome editing that results in improved or enhanced therapeutic effects. Therapeutic compositions and uses thereof are also provided that include the functionally enhanced derived effector cells alone or in combination therapy with antibodies or checkpoint inhibitors.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 218,204, filed July 2, 2021, U.S. Provisional Patent Application No. 63 / 265,190, filed December 9, 2021, and U.S. Provisional Patent Application No. 63 / 341,943, filed May 13, 2022, the disclosures of each of which are incorporated by reference in their entireties herein.

[0002] Incorporation by reference of sequence listing The Sequence Listing, entitled 184143-635601_SequenceListing.xml, was created on July 1, 2022, is 16,198 bytes in size, and is incorporated by reference in its entirety.

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

[0004] The field of adoptive cell therapy is currently focused on using patient- and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapies and to provide therapy to all patients who may benefit from them. There is also a need to improve the efficacy and persistence of adoptively transferred lymphocytes to promote better patient outcomes. Lymphocytes, such as T cells and natural killer (NK) cells, are potent antitumor effectors that play a key role in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains challenging and there is an unmet need for improvement. Thus, significant opportunities remain to fully exploit the potential of T cells and NK cells, or other lymphocytes, in adoptive immunotherapy. Summary of the Invention

[0005] There is a need for functionally improved effector cells that address a variety of issues, including response rate, cell attrition, loss of infused cells (viability and / or persistence), tumor escape due to target loss or lineage switching, precision of tumor targeting, off-target toxicity, off-tumor effects, efficacy against solid tumors, i.e., the tumor microenvironment and associated immune suppression, recruitment, trafficking, and invasion.

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

[0007] The iPSC-derived non-pluripotent cells of the present application contain CD34 +These include, but are not limited to, hematopoietic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, and B cells. The non-pluripotent cells derived from iPSCs of the present application contain one or several genetic modifications in their genomes through differentiation from iPSCs containing the same genetic modifications. In some embodiments, the engineered clonal iPSC differentiation strategy to obtain genetically engineered derivative cells benefits from the fact that the developmental potential of iPSCs in directed differentiation is not significantly adversely affected by the engineered modality of iPSCs, and that the engineered modality functions as intended in the derivative cells. Furthermore, this strategy overcomes the current barriers in engineering primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely, that such cells are often difficult to engineer, resulting in cells that lack reproducibility and uniformity and exhibit poor cell persistence with high cell death and low cell proliferation.

[0008] Thus, in one aspect, the invention provides a cell or population thereof, wherein (i) the cell is an induced pluripotent cell (iPSC), a clonal iPSC, an iPS cell line cell, or a derived cell obtained by differentiation of an iPSC, and (ii) the cell comprises (a) HLA-I deficient, (b) CD38 knockout, and optionally (c) an exogenous polynucleotide encoding CD16 or a variant thereof. In various embodiments, the cells or populations thereof further comprise one or more of: (i) an exogenous polynucleotide encoding a cytokine signaling complex comprising a cell surface expressed exogenous cytokine and / or a partial or complete peptide of its receptor; (ii) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (iii) an HLA-II deficiency; and (iv) an exogenous polynucleotide encoding HLA-G, HLA-E, or a variant thereof, wherein the cells are suitable for CD38 conditioning, and the cells have improved persistence in the presence of alloreactive host cells in adoptive cell therapy incorporating CD38 conditioning. In various embodiments, the cells (i) comprise at least one of the genotypes listed in Table 1; (ii) comprise a knockout of one or both of CD58 and CD54; (iii) comprise a disruption of at least one of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; and (iv) comprise a disruption of 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D, D, E, F ... 2Aand / or (v) does not comprise an exogenous polynucleotide encoding HLA-G, HLA-E, or a variant thereof. In some embodiments, the HLA-I deficiency comprises a disruption of at least one of B2M, TAP1, TAP2, and tapasin. In some other embodiments, the HLA-II deficiency comprises a disruption of at least one of CIITA, RFX5, RFXAP, and RFXANK.

[0009] In various embodiments of the cells or populations thereof, the derived cells include (a) derived CD34 + (b) are used as allogeneic effector cells, derived hematopoietic stem progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T cells, derived NKT cells, derived NK cells, or derived B cells, or (c) are used as allogeneic effector cells, which are derived NK cells or derived T cells having at least one of the following characteristics compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue, including: (i) improved persistence and / or survival, (ii) increased resistance to activated recipient immune cells, (iii) increased cytotoxicity, (iv) improved tumor penetration, (v) enhanced or acquired ADCC, (vi) enhanced ability to migrate and / or activate or recruit bystander immune cells to tumor sites, (vii) enhanced ability to reduce tumor immune suppression, (viii) improved ability to rescue tumor antigen escape, and (ix) reduced fratricide.

[0010] In various embodiments of the cells or populations thereof, the CD16 or variant thereof comprises at least one of: (a) high affinity non-cleavable CD16 (hnCD16) or a variant thereof; (b) F176V and S197P in the ectodomain of CD16; (c) a complete or partial ectodomain derived from CD64; (d) a non-native (or non-CD16) transmembrane domain; (e) a non-native (or non-CD16) intracellular domain; (f) a non-native (or non-CD16) signaling domain; (g) a non-native stimulatory domain; and (h) a transmembrane domain, signaling domain, and stimulatory domain not derived from CD16 but derived from the same or a different polypeptide. In certain embodiments, (a) the non-native transmembrane domain is derived from a CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide; or (b) the non-native stimulatory domain is derived from a CD27, CD28, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor (TCR) polypeptide. (c) the non-native signaling domain is derived from a CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide; or (d) the non-native transmembrane domain is derived from NKG2D, the non-native stimulatory domain is derived from 2B4, and the non-native signaling domain is derived from CD3ζ.

[0011] In various embodiments of the cells or populations thereof, the CAR is (i) T cell specific or NK cell specific, (ii) bispecific antigen binding CAR, (iii) switchable CAR, (iv) dimerized CAR, (v) split CAR, (vi) multi-chain CAR, (vii) inducible CAR, (viii) co-expressed, optionally in a separate construct or in a bicistronic construct, with a cytokine signaling complex comprising a cell surface expressed exogenous cytokine and / or a partial or complete peptide of its receptor, (ix) optionally in a separate construct or in a bicistronic construct, with a checkpoint inhibitor, and / or (x) optionally inserted into the TRAC or TRBC locus and / or driven by the endogenous promoter of the TCR and / or the TCR is knocked out by the CAR insertion, inserted into a safe harbor locus, or inserted into a locus intended for disruption.In various embodiments of the cells or populations thereof, the CAR is (i) specific for CD19, BCMA, B7H3, MICA / B, or MR1, and / or (ii) specific for 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, cytomegalovirus (CMV)-infected cell antigen, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside IgE receptor (GII) receptor (GIG2), folate receptor-α, ... Lysine G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), mucin 1 (Muc-1), mucin 16 (Mu c-16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigens.

[0012] In various embodiments of the cells or populations thereof, the cytokine signaling complex is a cell surface expressed exogenous cytokine and / or a partial or complete peptide of its receptor, including at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors, or (b) a cytokine signaling complex that is a fusion protein comprising (i) co-expressed IL15 and IL15Rα with an intervening 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 truncated. (iv) a fusion protein of IL15 and the membrane-binding Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and common receptor γC, where common receptor γC is native or modified, and (vii) a homodimer of IL15Rβ, wherein any one of (i)-(vii) is optionally co-expressed with the CAR in a separate construct or in a bicistronic construct, and optionally (c) is expressed transiently.

[0013] In various embodiments of the cells or populations thereof, the cells are derived NK cells or derived T cells, the derived NK cells are capable of recruiting and / or migrating T cells to tumor sites, and the derived NK cells or derived T cells are capable of reducing tumor immunosuppression in the presence of one or more checkpoint inhibitors. In some embodiments, the one or more checkpoint inhibitors are PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2Aand antagonists against one or more checkpoint molecules, including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In certain embodiments, the one or more checkpoint inhibitors comprise (a) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (b) at least one of atezolizumab, nivolumab, and pembrolizumab.

[0014] In various embodiments of the cells or populations thereof, the cells comprise (i) one or more exogenous polynucleotides integrated into one safe harbor locus or locus intended for disruption, or (ii) three or more exogenous polynucleotides integrated into different safe harbor loci or loci intended for disruption. In some embodiments, the safe harbor loci comprise at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1, or the loci intended for disruption comprise B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0015] In various embodiments of the cells or populations thereof, the CD38 conditioning is: (i) via a CD38 antagonist, including an anti-CD38 antibody or a CAR that specifically binds to CD38 (CD38-CAR); (ii) via daratumumab, isatuximab, or MOR202; (iii) via daratumumab; (iv) comprising administering to a subject in need of adoptive cell therapy a CD38 antagonist before, during, or after infusion of the cells or populations thereof for therapy; (v) comprising pre-loading the cells or populations thereof in vitro with a CD38 antagonist followed by infusion of the cells or populations thereof; (vi) eliminating or reducing the number of alloreactive host cells; (vii) delaying host immune reconstitution; and / or (viii) prolonging the viability and persistence of the cells or populations thereof in the presence of alloreactive host cells in a subject in need of adoptive cell therapy. In various embodiments of the cell or population thereof, the alloreactive host cells (i) comprise primary T cells, primary B cells and / or primary NK cells that are allogeneic to the cell or population thereof, (ii) are sensitized to CD38 conditioning by the cell or population thereof, and / or (iii) are dose-dependently eliminated by CD38 conditioning via a CD38 antagonist.

[0016] In another aspect, the invention provides a composition comprising a CD38 antagonist and a cell or population thereof as described herein. In various embodiments, the composition further comprises one or more therapeutic agents. In certain embodiments, the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a supplement thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In some embodiments, (i) the checkpoint inhibitor is (a) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AOne or more of the following checkpoint molecules: R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof; (c) at least one of atezolizumab, nivolumab, and pembrolizumab; or (ii) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide. In some embodiments, the antibody is (a) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, an anti-CD25 antibody, an anti-CD69 antibody, an anti-CD71 antibody, or an anti-CD44 antibody; or (b) rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daclizumab, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, bivatuzumab, RG7356, G44-26, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof and their functional equivalents and biosimilars.

[0017] In various embodiments of the composition, the CD38 antagonist (i) comprises an anti-CD38 antibody or a CD38-CAR, (ii) comprises daratumumab, isatuximab, or MOR202, (iii) comprises daratumumab, or (iv) is provided to a subject in need of adoptive cell therapy before, during, or after infusion of the cells or populations thereof. In another aspect, the invention provides for the therapeutic use of a composition described herein by introducing the composition into a subject in need of adoptive cell therapy, wherein the subject has an autoimmune disorder, hematological malignancy, solid tumor, cancer, or viral infection.

[0018] In yet another aspect, the invention provides a method of reducing or preventing alloreactivity of host cells to allogeneic effector cells in an adoptive cell therapy provided to a subject in need thereof, the allogeneic effector cells comprising a cell or population thereof described herein, and the method comprising CD38 conditioning. In various embodiments, the host cells comprise alloreactive immune cells comprising primary T cells, primary B cells, and / or primary NK cells. In various embodiments, the CD38 conditioning comprises (i) administering a CD38 antagonist to the subject before, during, or after infusion of allogeneic effector cells into the subject, or (ii) preloading the allogeneic effector cells in vitro with a CD38 antagonist followed by infusion of the allogeneic effector cells into the subject, wherein the CD38 conditioning (a) eliminates or reduces the number of alloreactive host cells, (b) prolongs the viability and persistence of the allogeneic effector cells to an extent controllable by a given dose of the CD38 antagonist, and / or (c) delays host immune reconstitution. In some embodiments, the CD38 antagonist comprises (i) an anti-CD38 antibody or CD38-CAR, (ii) daratumumab, isatuximab, or MOR202, and / or (iii) daratumumab. In some embodiments, the alloreactive host cells comprise upregulated CD38 expression.

[0019] In various embodiments of the method of reducing or preventing alloreactivity of host cells to allogeneic effector cells in adoptive cell therapy, the method further comprises administering a therapeutic agent to the subject. In some embodiments, the therapeutic agent comprises a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a supplement thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In certain embodiments, (i) the checkpoint inhibitor is selected from the group consisting of (a) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2A one or more of the checkpoint molecules including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR or (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or (ii) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide. In some embodiments, the method does not require or minimally requires lymphodepletion with a combination of cyclophosphamide and fludarabine (Cy / Flu). In some embodiments, the method does not include lymphodepletion with Cy / Flu.

[0020] In another aspect, the present invention provides a method of treating a subject in need of adoptive cell therapy, comprising administering to the subject a CD38 antagonist for CD38 conditioning and injecting the cells or populations thereof described herein. In various embodiments, CD38 conditioning (i) reduces or prevents host cell alloreactivity to allogeneic effector cells, (ii) eliminates or reduces the number of alloreactive host cells, (iii) extends the survival and persistence of allogeneic effector cells, (iv) delays host immune reconstitution, (v) prevents leakage of protection of allogeneic effector cells against host cell alloreactivity via overexpression of HLA-G or HLA-E, and / or (vi) increases nicotinamide adenine dinucleotide (NAD) availability, reduces NAD consumption-related cell death, and supports cell rejuvenation. In some embodiments, the method does not require or minimally requires lymphodepletion with a combination of cyclophosphamide and fludarabine (Cy / Flu). In some embodiments, the method does not include lymphodepletion with Cy / Flu.

[0021] Various objects and advantages of the compositions and methods provided herein will become apparent from the following description, taken in conjunction with the accompanying drawings, in which are set forth by way of illustration and example certain embodiments of the invention. [Brief description of the drawings]

[0022] [Figure 1A] 1 shows the phenotypic similarities between B2M WT and B2M KO effector cell lines. [Figure 1B] 1 shows similar levels of antibody-dependent cellular cytotoxicity between B2M WT and B2M KO effector cell lines. [Figure 2A] We show that iNK cells were successfully engineered and all engineered elements were assessed by flow cytometry. [Figure 2B]Figure 1 shows that HLA-I and HLA-II deficiency (dKO) is protective against allogeneic T cell reactivity. [Diagram 3] We show that inhibitory ligand overexpression does not protect against all subsets of NK cells and that subsets of NK cells are resistant to inhibitory pathways involving CD47 and HLA-E signaling. [Figure 4] 1 shows recognition of B2M KO effector cell lines by pbNK and the protective effect on effector cells mediated by anti-CD38 antibody conditioning. [Figure 5A] Figure 1 shows that anti-CD38 antibody conditioning protects iNK cells from pbNK allorejection in vitro. Groups of bars from left to right are indicated by the legends from top to bottom, respectively. [Figure 5B] Figure 1 shows that anti-CD38 antibody conditioning protects iNK cells from pbNK allorejection in vitro. Groups of bars from left to right are indicated by the legends from top to bottom, respectively. [Figure 6A] CD38 expression levels in donor PBMCs cultured alone and after priming with allogeneic iNK cells are shown. [Figure 6B] CD38 expression levels in donor PBMCs cultured alone and after priming with allogeneic iNK cells are shown. [Figure 7A] FIG. 1 shows the dose-dependent effect of anti-CD38 antibody conditioning on the sensitivity of iPSC-derived B2M KO cells to allogeneic host cells (PBMCs). [Figure 7B] FIG. 1 shows the dose-dependent effect of anti-CD38 antibody conditioning on the sensitivity of iPSC-derived B2M KO cells to allogeneic host cells (PBMCs). [Figure 8A] We show that iNK cells deficient in both CD38 and B2M are resistant to allogeneic T cell and NK cell attack in vitro. [Figure 8B]We show that iNK cells deficient in both CD38 and B2M are resistant to allogeneic T cell and NK cell attack in vitro. [Figure 8C] We show that iNK cells deficient in both CD38 and B2M are resistant to allogeneic T cell and NK cell attack in vitro. [Figure 8D] We show that iNK cells deficient in both CD38 and B2M are resistant to allogeneic T cell and NK cell attack in vitro. [Figure 8E] We show that iNK cells deficient in both CD38 and B2M are resistant to allogeneic T cell and NK cell attack in vitro. [Figure 8F] We show that iNK cells deficient in both CD38 and B2M are resistant to allogeneic T cell and NK cell attack in vitro. [Figure 9A] We show that both B2M KO and anti-CD38 antibody conditioning attenuate the activated PBMC signature in co-culture with iNK cells. [Figure 9B] We show that both B2M KO and anti-CD38 antibody conditioning attenuate the activated PBMC signature in co-culture with iNK cells. [Figure 9C] We show that both B2M KO and anti-CD38 antibody conditioning attenuate the activated PBMC signature in co-culture with iNK cells. [Figure 9D] We show that both B2M KO and anti-CD38 antibody conditioning attenuate the activated PBMC signature in co-culture with iNK cells. [Figure 10A] Exemplary results are shown for cells with the B2M / CIITA dKO combination. [Figure 10B] Exemplary results are shown for cells with the B2M / CIITA dKO combination. [Figure 10C] Exemplary results are shown for cells with the B2M / CIITA dKO combination. [Figure 11A] 14 shows that CD38 knockout in iT cells eliminates anti-CD38 ADCC when combined with peripheral blood NK cells. [Figure 11B] 14 shows that CD38 knockout in iT cells eliminates anti-CD38 ADCC when combined with peripheral blood NK cells. [Figure 12] We show that anti-CD38 antibody conditioning effectively depletes pbNK cells in the NSG-IL15 transgenic mouse model. [Figure 13A] We show that anti-CD38 antibodies protect iNK cells from pbNK allorejection in vivo using IL15 transgenic NSG mice. Figure 13A shows circulating pbNK levels when injected alone with or without anti-CD38 antibodies, while Figure 13B shows circulating WT, B2M KO, and B2M / CIITA dKO iNK levels when injected without pbNK. Figure 13C shows circulating WT iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13D shows circulating B2M KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13E shows circulating B2M / CIITA KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. (n=5 mice / group, P values ​​*<0.05, **<0.001, ****<0.0001). [Figure 13B]We show that anti-CD38 antibodies protect iNK cells from pbNK allorejection in vivo using IL15 transgenic NSG mice. Figure 13A shows circulating pbNK levels when injected alone with or without anti-CD38 antibodies, while Figure 13B shows circulating WT, B2M KO, and B2M / CIITA dKO iNK levels when injected without pbNK. Figure 13C shows circulating WT iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13D shows circulating B2M KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13E shows circulating B2M / CIITA KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. (n=5 mice / group, P values ​​*<0.05, **<0.001, ****<0.0001). [Figure 13C] We show that anti-CD38 antibodies protect iNK cells from pbNK allorejection in vivo using IL15 transgenic NSG mice. Figure 13A shows circulating pbNK levels when injected alone with or without anti-CD38 antibodies, while Figure 13B shows circulating WT, B2M KO, and B2M / CIITA dKO iNK levels when injected without pbNK. Figure 13C shows circulating WT iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13D shows circulating B2M KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13E shows circulating B2M / CIITA KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. (n=5 mice / group, P values ​​*<0.05, **<0.001, ****<0.0001). [Figure 13D]We show that anti-CD38 antibodies protect iNK cells from pbNK allorejection in vivo using IL15 transgenic NSG mice. Figure 13A shows circulating pbNK levels when injected alone with or without anti-CD38 antibodies, while Figure 13B shows circulating WT, B2M KO, and B2M / CIITA dKO iNK levels when injected without pbNK. Figure 13C shows circulating WT iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13D shows circulating B2M KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13E shows circulating B2M / CIITA KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. (n=5 mice / group, P values ​​*<0.05, **<0.001, ****<0.0001). [Figure 13E] We show that anti-CD38 antibodies protect iNK cells from pbNK allorejection in vivo using IL15 transgenic NSG mice. Figure 13A shows circulating pbNK levels when injected alone with or without anti-CD38 antibodies, while Figure 13B shows circulating WT, B2M KO, and B2M / CIITA dKO iNK levels when injected without pbNK. Figure 13C shows circulating WT iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13D shows circulating B2M KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. Figure 13E shows circulating B2M / CIITA KO iNK levels when co-injected with pbNK in the presence and absence of daratumumab. (n=5 mice / group, P values ​​*<0.05, **<0.001, ****<0.0001). [Figure 14A]Figure 1 shows that pbNK numbers were significantly reduced in the presence of daratumumab, which resulted in persistence of B2M KO and B2M / CIITA KO iNK in blood, spleen and bone marrow in IL15 transgenic NSG mice. Cell numbers were normalized to the group without daratumumab (n=5 mice / group, P-values ​​*<0.05, **<0.001, ****<0.0001). [Figure 14B] Figure 1 shows that pbNK numbers were significantly reduced in the presence of daratumumab, which resulted in persistence of B2M KO and B2M / CIITA KO iNK in blood, spleen and bone marrow in IL15 transgenic NSG mice. Cell numbers were normalized to the group without daratumumab (n=5 mice / group, P-values ​​*<0.05, **<0.001, ****<0.0001). [Figure 14C] Figure 1 shows that pbNK numbers were significantly reduced in the presence of daratumumab, which resulted in persistence of B2M KO and B2M / CIITA KO iNK in blood, spleen and bone marrow in IL15 transgenic NSG mice. Cell numbers were normalized to the group without daratumumab (n=5 mice / group, P-values ​​*<0.05, **<0.001, ****<0.0001). [Figure 15] We show that the addition of anti-CD38 antibodies to lymphodepleting chemotherapy (LDC) delays host immune reconstitution and extends the therapeutic window for adoptive cell therapy in patients treated with engineered CD38KO hnCD16 iNK cells in combination with daratumumab. [Figure 16] FIG. 1 shows Uniform Manifold Approximation and Projection (UMAP) visualization of the lymphocyte profile in one subject treated with engineered iNK cells in combination with daratumumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Genome modification of iPSCs (induced pluripotent stem cells) includes polynucleotide insertion, deletion, and replacement. Exogenous gene expression in genome-engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal expansion of the original genome-engineered iPSCs, after cell differentiation, and in dedifferentiated cell types from cells derived from genome-engineered iPSCs. Meanwhile, it is difficult to directly manipulate primary immune cells such as T cells or NK cells, which poses obstacles to the preparation and delivery of engineered immune cells for adoptive cell therapy. In various embodiments, the present invention provides an efficient and reliable targeted approach to stably integrate one or more exogenous genes, including suicide genes and other functional modalities, which confer improved therapeutic properties with respect to engraftment, trafficking, homing, migration, cytotoxicity, viability, maintenance, proliferation, longevity, self-renewal, persistence, and / or survival to iPSC-derived cells, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells.

[0024] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0025] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0026] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0027] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0028] The term "and / or" should be understood to mean either one or both of the alternatives.

[0029] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0030] As used herein, the term "substantially" or "essentially" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "substantially the same" or "essentially the same" refers to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range that is about the same as the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0031] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that is free of a particular substance or source thereof, such as, for example, 95% free, 96% free, 97% free, 98% free, 99% free, etc., of a particular substance or source thereof, or is undetectable as measured by conventional means. The term "free" or "essentially free" of a particular component or substance in a composition also means that such component or substance is (1) not included in the composition at any concentration, or (2) included in the composition at a low concentration that is functionally inactive. A similar meaning may be applied to the term "absent," which refers to the absence of a particular substance or source thereof in a composition.

[0032] Throughout this specification, unless the context requires otherwise, "comprise", "comprises", and "comprising" will be understood to mean the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "include", "having", "contain", and "comprise" are used interchangeably.

[0033] "Consisting of" means inclusive of and limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present.

[0034] "Consisting essentially of" means the inclusion of any elements listed following the phrase, limited to other elements that do not interfere with or contribute to the activity or operation specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or operation of the recited elements.

[0035] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "particular embodiment," "additional embodiment," or "further embodiment," or combinations thereof, means that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0036] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements performed in or on living tissue in an artificial environment outside of the living organism, preferably with minimal alteration of natural conditions. In certain embodiments, "ex vivo" procedures include live cells or tissues taken from a living organism and cultured in a laboratory setup, usually under sterile conditions, typically for a few hours or up to about 24 hours, but up to 48 hours or 72 hours or more depending on the circumstances. In certain embodiments, such tissues or cells may be collected and frozen, and later thawed for ex vivo processing. Tissue culture experiments or procedures lasting longer than a few days using live cells or tissues are typically considered to be "in vitro", although in certain embodiments, the term may be used interchangeably with ex vivo.

[0037] The term "in vivo" generally refers to activities that take place inside a living organism.

[0038] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to a method of increasing the potential of a cell or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in a non-reprogrammed state.

[0039] As used herein, the term "differentiation" refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell, such as, for example, a blood cell or a muscle cell. A differentiated or differentiation-induced cell is a cell that assumes a more specialized ("committed") position within the lineage of a cell. The term "committed" as applied to the process of differentiation refers to a cell that has progressed down a differentiation pathway to a position where it will continue to differentiate into a particular cell type or subset of cell types under normal circumstances, and cannot, under normal circumstances, differentiate into a different cell type and revert to a less differentiated cell type. As used herein, the term "pluripotency" refers to the ability of a cell (i.e., the embryo itself) to form all lineages of an organism or somatic cells. For example, an embryonic stem cell is a type of pluripotent stem cell that can form cells from each of the three germ layers, ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs), which are unable to give rise to an entire organism, to more primitive, more pluripotent cells (e.g., embryonic stem cells), which are able to give rise to an entire organism.

[0040] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to stem cells produced in vitro from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed, using reprogramming factors and / or small molecule chemical drive methods into cells capable of differentiating into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. Generated iPSCs do not refer to naturally occurring cells.

[0041] As used herein, the term "embryonic stem cells" refers to naturally occurring pluripotent stem cells of the inner cell mass of blastocysts. Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers during development, namely ectoderm, endoderm, and mesoderm. They do not contribute to the extraembryonic membranes or placenta (i.e., they are not totipotent).

[0042] As used herein, the term "pluripotent stem cell" refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (i.e., ectoderm, mesoderm, and endoderm), but not all three. Thus, pluripotent cells can also be referred to as "partially differentiated cells." Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotency" indicates that a cell can form many types of cells in a given lineage, but not cells of other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelets, etc.), but cannot form neurons. Thus, the term "multipotency" refers to a state of a cell that has a degree of developmental potential that is less than totipotency and pluripotency.

[0043] Pluripotency can be determined, in part, by assessing the pluripotency characteristics of the cells, including, but not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) expression of pluripotent stem cell markers, including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50, (iv) the ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation composed of the three somatic lineages, and (vi) formation of embryoid bodies composed of cells from the three somatic lineages.

[0044] Two types of pluripotency have been described so far: a "primed" or "metastable" state of pluripotency similar to epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "ground" state similar to the inner cell mass of pluripotent early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or ground state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and survival in single cell culture, (iii) overall reduction in DNA methylation, (iv) reduced H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters, and (v) reduced expression of differentiation markers compared to primed state pluripotent cells. Standard methodologies of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then either silenced or removed from the resulting pluripotent cells, generally appear to have the characteristics of the primed state of pluripotency. Under standard pluripotent cell culture conditions, such cells remain in a primed state and characteristics of the ground state are observed unless exogenous transgene expression is maintained.

[0045] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape with a high nuclear to cytoplasmic ratio, prominent presence of nucleoli, and typical intercellular spacing.

[0046] As used herein, the term "subject" refers to any animal, preferably a human patient, livestock, or other domesticated animals.

[0047] "Pluripotency factor" or "reprogramming factor" refers to an agent that can increase the developmental potential of a cell, alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0048] "Culture" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium", "culture medium" (in each case singular "medium"), "supplement" and "medium supplement" refer to a nutritional composition in which a cell culture is cultivated.

[0049] "Culturing" or "maintaining" refers to sustaining, propagating (growing), and / or differentiating a tissue or cells outside the body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culturing" or "maintaining" can utilize culture medium as a source of nutrients, hormones, and / or other factors that aid in the growth and / or maintenance of the cells.

[0050] As used herein, the term "mesoderm" refers to one of three germ layers that emerge during early embryonic development and give rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

[0051] As used herein, the term "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refers to a subset of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process called endothelial-hematopoietic conversion. Hematopoietic cell development in the embryo progresses sequentially from lateral plate mesoderm through hemangioblasts to definitive hemogenic endothelial cells and hematopoietic precursors.

[0052] The terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells", or "hematopoietic progenitor cells" refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including multipotent hematopoietic stem cells (blood cells), myeloid progenitors, megakaryocyte progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells) lineages. As used herein, the term "secondary hematopoietic stem cells" refers to cells that are CD34+ / CD35+ / CD36+ / CD37+ / CD38+ / CD39 ... + Refers to hematopoietic cells, which also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0053] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to a major type of white blood cell that has completed maturation in the thymus and has a variety of roles in the immune system, including identifying specific foreign antigens in the body and activating and inactivating other immune cells in an MHC class I-restricted manner. A T cell can be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. T cells are CD3 + T cells can be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 +The T cells can be of any type and at any stage of development, including, but not limited to, T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), and the like. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). The term "T cells" can also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also be differentiated from stem or progenitor cells ("derived T cells" or "derived T cell-like effector cells", or collectively "derived T lineage cells"). Derived T cell-like effector cells may have T cell lineage in some respects, but at the same time have one or more functional attributes not present in primary T cells. In this application, T cells, T cell-like effector cells, derived T cells, derived T cell-like effector cells, or derived T lineage cells are collectively referred to as "T lineage cells".

[0054] "CD4 +"CD4 cells" refers to a subset of T cells that express CD4 on their surface and are associated with cellular immune responses. They are characterized by their secretion profile after stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. The "CD4" molecule is a 55 kD glycoprotein originally defined as a differentiation antigen for T lymphocytes, but is also found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as the relevant recognition element of the MHC (major histocompatibility complex) class II-restricted immune response. In T lymphocytes, it defines the helper / inducer subsets.

[0055] "CD8 + "CD8 cells" refers to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is the associated recognition element of major histocompatibility complex class I restricted interactions.

[0056] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of the T cell receptor (CD3). The NK cells can be any NK cell, for example, cultured NK cells (e.g., primary NK cells), or NK cells derived from cultured or expanded NK cells, or cell line NK cells (e.g., NK-92), or NK cells obtained from a mammal with a healthy or diseased state. As used herein, the terms "adaptive NK cells" and "memory NK cells" are interchangeable and are phenotypically CD3+ / CD4+ / CD56+ / CD16+ / CD3 ... - and CD56 + and refers to a subset of NK cells that express at least one of NKG2C and CD57, and optionally CD16, but lack expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, CD56 +The isolated subpopulation of NK cells includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. + may be dim or bright expression. NK cells or NK cell-like effector cells may be differentiated from stem or progenitor cells ("derived NK cells" or "derived NK cell-like effector cells" or collectively "derived NK lineage cells"). Derived NK cell-like effector cells may have NK cell lineage in some respects, but at the same time have one or more functional attributes not present in primary NK cells. In this application, NK cells, NK cell-like effector cells, derived NK cells, derived NK cell-like effector cells, or derived NK lineage cells are collectively referred to as "NK lineage cells."

[0057] As used herein, the term "NKT cells" or "natural killer T cells" refers to CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by the non-classical MHC molecule, CD1d. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire - a canonical α chain (Vα24-Jα18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, presents a more heterogeneous use of TCRαβ. Type I NKT cells are considered to be suitable for immunotherapy. Adaptive or invariant (Type I) NKT cells can be identified by expression of one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0058] The term "effector cell" generally refers to a specific cell in the immune system that performs a specific activity in response to stimulation and / or activation, or a cell that provides a specific function upon activation. As used herein, the term "effector cell" includes, and in some contexts is interchangeable with, immune cells, "differentiated immune cells," and primary or differentiated cells that have been edited and / or regulated to perform a specific activity in response to stimulation and / or activation. Non-limiting examples of effector cells include primary or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0059] As used herein, terms such as "isolated" refer to a cell or population of cells that has been separated from its original environment, i.e., the environment of the isolated cell is substantially free of at least one component found in the environment in which the "non-isolated" reference cell resides. The term includes cells that have been removed from some or all components as found in their natural environment, e.g., isolated from a tissue or biopsy sample. The term also includes cells that have been removed from at least one, some, or all components as they are found in a non-native environment, e.g., isolated from a cell culture or cell suspension. Thus, an "isolated cell" is partially or completely separated from at least one component, including other substances, cells, or cell populations, as found in nature, or as grown, stored, or persisted in a non-native environment. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in a medium that does not occur in nature. Isolated cells can be obtained by isolating a desired cell or population thereof from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0060] 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%.

[0061] As used herein, the term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to "encode" the protein or other product of that gene or cDNA.

[0062] A "construct" refers to a complex of polymers or molecules, including polynucleotides, that are delivered to a host cell, either in vitro or in vivo. As used herein, a "vector" refers to any nucleic acid construct that can direct the delivery or transfer of foreign genetic material to a target cell and can replicate and / or express in the target cell. Thus, the term "vector" includes the construct that is delivered. A vector can be a linear or circular molecule. A vector can be integrating or non-integrating. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, and the like.

[0063] "Integration" means that one or more nucleotides of the construct are stably inserted into the cell genome, i.e., covalently linked to a nucleic acid sequence in the chromosomal DNA of the cell. "Targeted integration" means that the nucleotides of the construct are inserted into the chromosomal or mitochondrial DNA of the cell at a preselected site or "integration site". As used herein, the term "integration" further refers to a process that includes the insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of the endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" can further include the replacement of the deleted nucleotide with the endogenous sequence or one or more inserted nucleotides.

[0064] As used herein, the term "exogenous" is intended to mean that the referenced molecule or activity is introduced into the host cell or is non-native to the host cell. The molecule can be introduced, for example, by introducing an encoding nucleic acid into the genetic material of the host, for example, by integration into a chromosome of the host, or as non-chromosomal genetic material, for example, a plasmid. Thus, the term used in reference to expression of an encoding nucleic acid refers to introducing the encoding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a referenced molecule or activity present in the host cell. Similarly, when used in reference to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid that is contained within the cell and not exogenously introduced.

[0065] As used herein, a "gene of interest" or a "polynucleotide sequence of interest" is a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo. A gene or polynucleotide of interest may include, but is not limited to, a prokaryotic sequence, a cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a gene of interest may code for an miRNA, an shRNA, a natural polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, a variant polypeptide (i.e., a mutant of a natural polypeptide having less than 100% sequence identity with the natural polypeptide) or a fragment thereof, an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selection marker, etc.

[0066] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The sequence of a polynucleotide is composed of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T); if the polynucleotide is RNA, thymine is uracil (U). Polynucleotides can include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. "Polynucleotide" also refers to both double-stranded and single-stranded molecules.

[0067] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also generally referred to in the art as peptides, oligopeptides and oligomers, and longer chains, generally referred to in the art as polypeptides or proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0068] As used herein, the term "subunit" refers to each separate polypeptide chain of a protein complex, each separate polypeptide chain being capable of forming a stable folded structure by itself. Many protein molecules are composed of two or more subunits, where the amino acid sequence can be either identical, similar, or completely different for each subunit. For example, the CD3 complex is composed of CD3α, CD3ε, CD3δ, CD3γ, and CD3ζ subunits, which form CD3ε / CD3γ, CD3ε / CD3δ, and CD3ζ / CD3ζ dimers. Within a single subunit, contiguous portions of the polypeptide chains are often folded into compact, localized, semi-independent units called "domains". Many protein domains may further comprise independent "structural subunits", also called subdomains, that contribute to a common function of the domain. Thus, as used herein, the term "subdomain" refers to a protein domain inside a larger domain, for example, a binding domain in the ectodomain of a cell surface receptor, or a stimulatory or signaling domain in the endodomain of a cell surface receptor.

[0069] "Operably-linked" or "operatively linked" are interchangeable with "operably connected" or "operatively connected" and refer to the association of nucleic acid sequences (or amino acids in a polypeptide having multiple domains) on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA if it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. As a further example, a receptor binding domain can be operably connected to an intracellular signaling domain such that binding of the receptor to a ligand transduces a signal in response to said binding.

[0070] A "fusion protein" or "chimeric protein," as used herein, is a protein created through genetic engineering to join two or more partial or complete polynucleotides encoding sequences coding for separate proteins, such that expression of these joined polynucleotides results in a single peptide or multiple polypeptides having functional properties derived from each of the original proteins or fragments thereof. A linker (or spacer) peptide can be added between the two adjacent polypeptides of different sources in the fusion protein.

[0071] As used herein, the term "genetic imprint" refers to genetic or epigenetic information that contributes to the preferential therapeutic properties of source cells or iPSCs and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, a "source cell" is a non-pluripotent cell that can be used to generate iPSCs through reprogramming, which can be further differentiated into a specific cell type, including any hematopoietic lineage cell. Source cell-derived iPSCs, and cells differentiated therefrom, can be collectively referred to as "derived" cells or "derivative" cells, depending on the context. For example, derived effector cells, or derived NK cells or derived T cells, as used throughout this application, are cells differentiated from iPSCs, when compared to their primary counterparts obtained from natural / native sources, such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, genetic imprints conferring preferential therapeutic properties are incorporated into iPSCs by reprogramming selected source cells that are donor, disease, or therapeutic response specific, or by introducing genetic modification modalities into iPSCs using genome editing. In aspects of source cells obtained from specifically selected donors, diseases, or therapeutic settings, genetic imprints that contribute to preferential therapeutic properties may include context-specific genetic or epigenetic modifications that represent a retainable phenotype, i.e., preferential therapeutic property, that is passed on to derived cells of the selected source cells, regardless of whether the underlying molecular events have been identified.The source cells that are donor, disease, or therapeutic response specific may contain genetic imprints that can be retained in iPSCs and derived hematopoietic lineage cells, including, but not limited to, pre-positioned monospecific TCRs, e.g., from virus-specific T cells or invariant natural killer T (iNKT) cells, traceable and desirable genetic polymorphisms, e.g., homozygosity for a point mutation encoding a high affinity CD16 receptor in selected donors, and selected HLA-matched donor cells that exhibit a predefined HLA requirement, i.e., haplotype, in an increased population. As used herein, preferential therapeutic properties include improved engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modification, survival, and cytotoxicity of derived cells. Preferential therapeutic properties are also manifested by antigen-targeting receptor expression, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modification, improved on-target specificity with reduced extratumoral effects, and resistance to treatments such as chemotherapy. When derived cells with one or more therapeutic properties are obtained from differentiating iPSCs that incorporate genetic imprints that confer preferential therapeutic properties, such derived cells are also referred to as "synthetic cells". For example, synthetic effector cells, or synthetic NK cells or synthetic T cells, as used throughout this application, are cells differentiated from genomically modified iPSCs, compared to their primary counterparts obtained from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissues. In some embodiments, synthetic cells have one or more non-native cell functions when compared to their closest corresponding primary cells.

[0072] As used herein, the term "enhanced therapeutic properties" refers to therapeutic properties of a cell that are enhanced compared to typical immune cells of the same general cell type. For example, NK cells with "enhanced therapeutic properties" have enhanced, improved, and / or increased therapeutic properties compared to typical unmodified and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modification, viability, and cytotoxicity. Therapeutic properties of immune cells are also manifested by antigen-targeting receptor expression, HLA presentation or lack thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immune modifications, improved on-target specificity with reduced extratumoral effects, and resistance to treatments such as chemotherapy.

[0073] As used herein, the term "engager" refers to a molecule, e.g., a fusion polypeptide, that can form a link between an immune cell (e.g., T cell, NK cell, NKT cell, B cell, macrophage, neutrophil) and a tumor cell and activate the immune cell. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs), or multispecific killer cell engagers, or universal engagers that may be compatible with multiple immune cell types.

[0074] As used herein, the term "surface triggering receptor" refers to a receptor that can induce or initiate an immune response, e.g., a cytotoxic response. Surface triggering receptors can be engineered and expressed on effector cells, e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, the surface triggering receptor promotes bispecific or multispecific antibody binding between an effector cell and a specific target cell (e.g., tumor cell) regardless of the effector cell's native receptor and cell type. Using this approach, it is possible to generate iPSCs that contain a universal surface triggering receptor and differentiate such iPSCs into a population of various effector cell types that express the universal surface triggering receptor. "Universal" means that the surface triggering receptor can be expressed and activated on any effector cell regardless of cell type, and all effector cells that express the universal receptor can bind or link to an engager that has the same epitope that can be recognized by the surface triggering receptor, regardless of the tumor-binding specificity of the engager. In some embodiments, an engager with the same tumor-targeting specificity is used to bind to the universal surface triggering receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to the universal surface triggering receptor. Thus, one or more effector cell types may be used to kill one specific type of tumor cell or two or more types of tumors. Surface triggering receptors generally contain a costimulatory domain for effector cell activation and an anti-epitope specific to the epitope of the engager. Bispecific engagers are specific to the anti-epitope of the surface triggering receptor on one end and specific to the tumor antigen on the other end.

[0075] As used herein, the term "safety switch protein" refers to an engineered protein designed to prevent possible toxicity or other adverse effects of a cell therapy. In some examples, expression of the safety switch protein is conditionally controlled to address safety concerns of transplanted engineered cells that have a gene encoding the safety switch protein permanently integrated into their genome. This conditional regulation can vary and can include control by post-translational activation via small molecules and tissue-specific and / or transient transcriptional regulation. The safety switch protein can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation and / or antibody-mediated depletion. In some examples, the safety switch protein is activated by an exogenous molecule, e.g., a prodrug, and upon activation, induces apoptosis and / or cell death of the therapeutic cell. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered upon the occurrence of an adverse event is activated by the suicide gene product and kills the transduced cells.

[0076] As used herein, the term "pharmaceutical active protein or peptide" refers to a protein or peptide capable of achieving a biological and / or pharmaceutical effect on an organism. A pharmaceutical active protein has curative or palliative properties against a disease and can be administered to improve, relieve, alleviate, reverse or lessen the severity of the disease. A pharmaceutical active protein also has prophylactic properties and is used to prevent the onset of a disease or to lessen the severity of such a disease or pathological condition once it appears. A "pharmaceutical active protein" includes an entire protein or peptide or a pharmaceutical active fragment thereof. The term also includes pharmaceutical active analogs of a protein or peptide or analogs of a fragment of a protein or peptide. The term pharmaceutical active protein also refers to multiple proteins or peptides that act cooperatively or synergistically to produce a therapeutic effect. Examples of pharmaceutical active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressor proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0077] As used herein, the term "signaling molecule" refers to any molecule that modifies, participates in, inhibits, activates, reduces, or enhances cell signaling. "Signaling" refers to the transmission of a molecular signal in the form of a chemical modification by the recruitment of protein complexes along a pathway that ultimately leads to a biochemical event in the cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, toll gate signaling, ligand-gated ion channel signaling, ERK / MAPK signaling pathway, Wnt signaling pathway, cAMP-dependent pathway, and IP3 / DAG signaling pathway.

[0078] As used herein, the term "targeting modality" refers to molecules (e.g., polypeptides) that are genetically incorporated into cells to promote antigen and / or epitope specificity, including, but not limited to: i) antigen specificity when associated with a unique chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when associated with a monoclonal antibody or bispecific engager; iii) transformed cell targeting; iv) cancer stem cell targeting; and v) other targeting strategies in the absence of a specific antigen or surface molecule.

[0079] As used herein, the terms "specific" or "specificity" can 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 non-specific or non-selective binding.

[0080] As used herein, the term "adoptive cell therapy" refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes (e.g., T cells, B cells, and / or NK cells), either genetically modified or unmodified, that are expanded ex vivo prior to infusion.

[0081] As used herein, "lymphodepletion" and "lymphatic conditioning" are used interchangeably to refer to the destruction of lymphocytes and T cells, typically prior to immunotherapy. The purpose of lymphatic conditioning prior to administration of adoptive cell therapy is to promote homeostatic proliferation of effector cells, as well as to eliminate regulatory immune cells and other competing elements of the immune system that compete for homeostatic cytokines. Thus, lymphatic conditioning is typically accomplished by administering one or more chemotherapeutic agents to the subject prior to the first dose of adoptive cell therapy. In various embodiments, lymphatic conditioning is performed hours to days prior to the first dose of adoptive cell therapy. Exemplary chemotherapeutic agents useful for lymphatic conditioning include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below. However, sufficient lymphodepletion with anti-CD38 mAb may provide an alternative conditioning process for the present iNK cell therapy without or at a minimum the need for a CY / FLU-based lymphoid conditioning procedure, as further described herein.

[0082] As used herein, "homing" or "trafficking" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate the interaction of a cell to a target site.

[0083] As used herein, a "therapeutically sufficient amount" includes within its meaning a non-toxic but sufficient and / or effective amount of the particular therapeutic agent and / or pharmaceutical composition to which it refers to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient's overall health, the patient's age, and the stage and severity of the condition being treated. In certain embodiments, a "therapeutically sufficient amount" is sufficient and / or effective to ameliorate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0084] Differentiation of pluripotent stem cells requires changes in the culture system, including changes in stimuli in the medium and the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a common and important intermediate for initiating lineage-specific differentiation. "Embryoid bodies" are three-dimensional clusters that have been shown to mimic embryonic development, giving rise to multiple lineages within a three-dimensional area. Throughout the differentiation process, which typically takes hours to days, simple EBs (e.g., aggregated pluripotent stem cells induced to differentiate) continue to mature and grow into cystic EBs, at which point they are further processed to continue differentiation, typically for days to weeks. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multi-layered cluster of cells. Typically, this is accomplished by one of several methods, including settling the pluripotent cells in droplets, settling the cells in "U" bottom well plates, or by mechanical agitation. Because aggregates maintained in pluripotency culture maintenance medium do not form proper EBs, pluripotent stem cell aggregates require further differentiation cues to promote EB growth. Therefore, pluripotent stem cell aggregates need to be transferred to differentiation medium that provides cue induction to the selected lineage. EB-based culture of pluripotent stem cells typically generates differentiated cell populations (i.e., ectodermal, mesodermal, and endodermal germ layers) with moderate proliferation within the EB cell clusters. Although EBs have been proven to promote cell differentiation, they give rise to heterogeneous cells with various differentiation states due to inconsistent exposure of the cells in the three-dimensional structure to differentiation cues in the environment. In addition, EBs are laborious to generate and maintain. Furthermore, cell differentiation by EBs is accompanied by moderate cell proliferation, which also leads to reduced differentiation efficiency.

[0085] In contrast, "aggregate formation", unlike "EB formation", can be used to expand a population of pluripotent stem cell-derived cells. For example, during aggregate-based pluripotent stem cell expansion, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of the aggregates forming larger aggregates that can be mechanically or enzymatically dissociated into smaller aggregates to maintain cell proliferation and increase cell number in culture. Unlike EB culture, cells cultured within aggregates in maintenance culture medium maintain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation cues to induce differentiation.

[0086] As used herein, "monolayer differentiation" refers to a differentiation method that is different from the differentiation of three-dimensional multi-layered clusters of cells, i.e. "EB formation".Monolayer differentiation, among other advantages disclosed herein, avoids the need for EB formation to initiate differentiation.Since monolayer culture does not mimic embryonic development as in the case of EB formation, differentiation into specific lineages is considered minimal compared to the differentiation of all three germ layers in EB formation.

[0087] As used herein, "dissociated cells" or "single dissociated cells" refers to cells that are substantially separated or purified from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be enzymatically or mechanically dissociated from each other, for example, by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells can be dissociated from a culture plate or other surface. Thus, dissociation includes disrupting the extracellular matrix (ECM) and cellular interactions with the substrate (such as the culture surface) or disrupting the ECM between cells.

[0088] As used herein, "master cell bank" or "MCB" refers to a clonal master engineered iPSC line that is a clonal population of iPSCs that have been engineered to contain one or more therapeutic properties, characterized, tested, qualified, expanded, and shown to reliably serve as starting cell material for the production of cell-based therapeutics by directed differentiation in a manufacturing environment. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic mutations and / or potential contamination by reducing and / or eliminating the total number of times the iPS cell line is passaged, thawed, or handled during the manufacturing process.

[0089] As used herein, "feeder cells" or "feeders" is a term that refers to 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, proliferate, or differentiate, where the feeder cells provide stimuli, growth factors, nutrients, and support the second cell type. Feeder cells may be derived 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 proliferation and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with antimitotic agents such as mitomycin to prevent them from outgrowing the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the foregoing, one particular feeder cell type can be a human feeder, such as human dermal fibroblasts. Another feeder cell type can be mouse embryonic fibroblast (MEF). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation to specific lineages, enhance proliferation capacity, and promote maturation into specialized cell types such as effector cells.

[0090] As used herein, a "feeder-free" (FF) environment refers to an environment, such as culture conditions, cell culture or culture medium, that is essentially free of feeder or stromal cells and / or has not been preconditioned by culturing feeder cells. A "preconditioned" medium refers to a medium that has been collected after feeder cells have been cultured in the medium for a period of time, e.g., at least one day, and thus 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, nor has it been preconditioned by culturing feeder cells.

[0091] "Functional" as used in the context of genome editing or modification of iPSCs and derived non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and derived iPSCs reprogrammed therefrom, means (1) at the genetic level, successful transgenic or controlled gene expression, such as inducible or transient expression, at a desired stage of cellular development, achieved by knock-in, knock-out, knock-down gene expression, direct genome editing or modification, or by "passaging" through differentiation or reprogramming from an initially genomically engineered starting cell, or (2) at the cellular level - (i) the genetic modification or alteration achieved in that cell through direct genome editing. (ii) gene expression modifications that are maintained in the cell through "passage" via differentiation or reprogramming from the original genomically engineered starting cell; (iii) downstream gene regulation in the cell as a result of gene expression modifications that are only apparent in earlier developmental stages of the cell or only in the starting cell that gives rise to the cell via differentiation or reprogramming, or (iv) successful removal, addition or modification of a cellular function / characteristic by enhanced or newly achieved cellular function or attribute exhibited in the mature cell product originally derived from genomic editing or modifications performed on an iPSC, precursor or de-differentiated cellular source.

[0092] "HLA-deficient," including HLA class I-deficient, HLA class II-deficient, or both, refers to cells that lack or no longer maintain or have reduced levels of surface expression of complete MHC complexes comprising HLA class I protein heterodimers and / or HLA class II heterodimers, the reduced or decreased levels being lower than those naturally detectable by other cells or synthetic methods.

[0093] As used herein, "modified HLA-deficient iPSCs" refers to HLA-deficient iPSCs that are further modified by introducing genes expressing proteins associated with improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to inhibition, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as, but not limited to, non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 4-1BB, DAP10, DAP12, CD24, CD3zeta, 4-1BBL, CD47, CD113, and PDL1. "Modified HLA-deficient" cells also include cells other than iPSCs.

[0094] The term "ligand" refers to a substance that forms a complex with a target molecule and generates a signal by binding to a site on the target. A ligand may be a natural or artificial substance that can specifically bind to a target. A ligand may be in the form of a protein, peptide, antibody, antibody complex, conjugate, nucleic acid, lipid, polysaccharide, monosaccharide, small molecule, nanoparticle, ion, neurotransmitter, or any other molecular entity that can specifically bind to a target. The target to which the ligand binds may be a protein, nucleic acid, antigen, receptor, protein complex, or cell. A ligand that binds to a target and changes its function, eliciting a response, is called "agonistic" or "agonist". A ligand that binds to a target and blocks or reduces a signaling response is "antagonistic" or "antagonist".

[0095] The term "antibody" is used herein in the broadest sense and generally refers to an immune response generating molecule that contains at least one binding site that specifically binds to a target, which may be an antigen or a receptor that can interact with a particular antibody. For example, NK cells can be activated by the binding of an antibody or the Fc region of an antibody to its Fc-gamma receptor (FcγR), thereby triggering ADCC (antibody-dependent cellular cytotoxicity)-mediated effector cell activation. The particular fragment or portion of an antigen or receptor that an antibody binds to, or generally the target, is known as an epitope or antigenic determinant. The term "antibody" includes, but is not limited to, antibody mimetics that mimic the structure and / or function of an antibody or a particular fragment or portion thereof, including natural antibodies and variants thereof, fragments of natural antibodies and variants thereof, peptibodies and variants thereof, and single chain antibodies and fragments thereof. The antibody may be a murine antibody, a human antibody, a humanized antibody, a camelid IgG, a single variable novel antigen receptor (VNAR), a shark heavy chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single domain antibody (dAb), an anti-idiotypic antibody, a bispecific, multispecific, or multimeric antibody, or an antibody fragment thereof. An anti-idiotypic antibody is specific for binding to the idiotope of another antibody, an idiotope being an antigenic determinant of an antibody. A bispecific antibody may be a BiTE (bispecific T cell engager) or BiKE (bispecific killer cell engager), and a multispecific antibody may be a TriKE (trispecific killer cell engager).Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fabc, pFc, Fd, single chain variable fragment (scFv), tandem scFv (scFv)2, single chain Fab (scFab), disulfide stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen binding fragments (sdAb), camelid heavy chain IgG and Nanobody® fragments, recombinant heavy-chain-only antibodies (VHH), and other antibody fragments which maintain the binding specificity of the whole antibody.

[0096] "Fc receptors", abbreviated as FcR, are classified based on the type of antibody they recognize. For example, those that bind the most common class of antibody IgG are called Fc-gamma receptors (FcγR), those that bind IgA are called Fc-alpha receptors (FcαR), and those that bind IgE are called Fc-epsilon receptors (FcεR). Classes of FcR are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling properties of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and therefore different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0097] "Chimeric receptor" is a general term used to describe engineered artificial or hybrid receptor protein molecules that are made to contain two or more portions of amino acid sequence from at least two different proteins. Chimeric receptor proteins are engineered to confer the cell the ability to initiate signal transduction and perform downstream functions upon binding of an agonist ligand to the receptor. Exemplary "chimeric receptors" include, but are not limited to, chimeric antigen receptors (CARs), chimeric fusion receptors (CFRs), chimeric Fc receptors (CFcRs), as well as fusions of two or more receptors.

[0098] "Chimeric Fc receptor", abbreviated as CFcR, is a term used to describe engineered Fc receptors in which the native transmembrane domain and / or intracellular signaling domain have been modified or replaced with a non-native transmembrane domain and / or intracellular signaling domain. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane and signaling domains being non-native, one or more stimulatory domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance receptor-induced cell activation, proliferation, and function. Unlike chimeric antigen receptors (CARs), which contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to Fc fragments, or Fc regions of antibodies, or Fc regions contained in engagers or binding molecules, and bind to molecules to activate cell functions, with or without bringing the target cell into close proximity. For example, Fcγ receptors can be engineered to contain selected transmembrane domains, stimulatory domains, and / or signaling domains in the intracellular region that respond to binding of IgG at the extracellular domain, thereby generating CFcR. In one example, CFcR is produced by engineering the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domains. To further improve the binding affinity of CD16-based CFcR, the extracellular domain of CD64 or a high affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcRs that include a high affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is removed or the extracellular domain of the receptor is replaced to be non-cleavable, i.e., not shed, thereby obtaining hnCD16-based CFcR.

[0099] Two isoforms of the FcγR receptor CD16 have been identified: Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells to activate NK cells and promote antibody-dependent cellular cytotoxicity (ADCC). As used herein, "high affinity CD16", "non-cleavable CD16", or "high affinity non-cleavable CD16" (abbreviated as hnCD16) refers to natural or non-natural variants of CD16. Wild-type CD16 has low affinity and upon NK cell activation is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane proximal region (positions 189-212) has been altered or eliminated are not shed. The cleavage site and membrane proximal region are described in detail in WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. The S197P variant of CD16 is a non-cleavable version of CD16. CD16 variants containing both F158V and S197P are high affinity and non-cleavable. Another exemplary high affinity non-cleavable CD16 (hnCD16) variant is an engineered CD16 that contains an ectodomain derived from one or more of the three exons of the CD64 ectodomain.

[0100] "T cell receptor", abbreviated as "TCR", generally refers to a protein complex found on the surface of T cells and is responsible for the recognition of fragments of antigenic peptides bound to major histocompatibility complex (MHC) molecules. Binding of the TCR to an antigenic peptide initiates TCR-CD3 intracellular activation, recruitment of multiple signaling molecules, and branching and integration of signaling pathways, leading to the recruitment of transcription factors important for gene expression and typical T cell proliferation and acquisition of function. A typical TCR contains two highly variable protein chains (α and β), each of which contains a constant region adjacent to the cell membrane and a variable region (i.e., a binding domain) that binds peptide / MHC.

[0101] I. Cells and Compositions Useful for Adoptive Cell Therapy with Enhanced Properties Provided herein is a strategy to systematically manipulate the regulatory circuitry of clonal iPSCs without affecting the differentiation potential and cell developmental biology of iPSCs and their derived cells while enhancing the therapeutic properties of derived cells differentiated from iPSCs. iPSC-derived cells are functionally improved and suitable for adoptive cell therapy after a combination of selective modalities is introduced into the cells at the iPSC level through genomic manipulation. Previously, it was unclear whether modified iPSCs containing one or more gene edits provided would still have the ability to enter cell development and / or mature to generate functional differentiated cells while retaining the modified activity and / or properties. Unexpected failures during directed cell differentiation from iPSCs are due to aspects including, but not limited to, developmental stage specific gene expression or lack thereof, requirement for HLA complex presentation, protein shedding of introduced surface expression modalities, and the need for reconstitution of differentiation protocols to allow for alteration of cell phenotype and / or function. The present application has demonstrated that one or more selected genomic modifications provided herein do not adversely affect iPSC differentiation potential, and that functional effector cells derived from engineered iPSCs have enhanced and / or acquired therapeutic properties resulting from individual or combined genomic modifications that are retained in the effector cells following iPSC differentiation. Furthermore, all genomic modifications and combinations thereof that may be described in the context of iPSCs and iPSC-derived effector cells are applicable to primary derived cells, including primary immune cells such as T cells, NK cells, or immune-modulating cells, whether cultured or expanded, which modifications result in engineered immune cells useful for adoptive cell therapy.

[0102] Also provided herein is a solution for allorejection control in a ready-made allogeneic adoptive cell therapy setting using effector cells derived from engineered iPSCs. To avoid the problem of allorejection, it is believed that multiple HLA class I and class II proteins must be matched for the histocompatibility of the allogeneic recipient. One approach that has been investigated in allogeneic adoptive cell therapy without MHC matching is to eliminate or substantially reduce the expression of both HLA class I and HLA class II proteins. HLA class I deficiency can be achieved by functional deletion or disruption of any region of the HLA class I locus (chromosome 6p21), or by deleting or disrupting, or reducing the expression level of, HLA class I-associated genes, including but not limited to the beta 2 microglobulin (B2M) gene, the TAP1 gene, the TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit that is essential for cell surface expression of all HLA class I heterodimers. B2M-negative cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion, disruption, or reduction of HLA II-associated genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-negative cells are HLA-II deficient.

[0103] However, lack of HLA class I expression increases susceptibility to lysis by NK cells. Moreover, deficiencies in both HLA-I and HLA-II still do not prevent allorejection mediated by alloantigens other than MHC of allogeneic adoptive cells. Furthermore, HLA-I-dependent NK cell education processes, such as licensing, arming, or disarming, are thought to affect innate immune responsiveness to allogeneic cells, which may result in reactivity, or partial reactivity, of recipient NK cells to allogeneic donor cells even when the allogeneic donor cells are HLA-I sufficient.

[0104] The present application provides a strategy for allorejection control by eliminating or substantially reducing expression of one or both of HLA class I and HLA class II proteins on allogeneic effector cells and modifying these cells for CD38 conditioning. Additionally, the present application addresses technical problems presented in exogenous or increased expression of HLA-E, HLA-G, or other non-classical HLA-I proteins with the goal of avoiding recipient primary NK cell lysis of HLA-I-deficient allogeneic adoptive effector cells. Because the inhibitory receptors that recognize HLA-E and HLA-G are stochastically expressed on primary NK cells (i.e., they are not expressed by all primary NK cells), it has been discovered that HLA-E / G does not provide complete protection from primary NK cell-based recognition to HLA-I-deficient allogeneic cells, resulting in leakage of the protection against NK cell lysis that HLA-E / G provides. In addition, there are corresponding activating receptors on primary NK cells that recognize HLA-E (and possibly HLA-G), which may cause accelerated rejection of HLA-I-deficient and HLA-E / G-expressing adoptive cells. As shown in the present application, modified HLA-I-deficient effector cells amenable / suitable for CD38 conditioning (e.g., by using anti-CD38 antibodies or CD38 antagonists such as CD38-CAR) are better protected against allorejection and therefore have higher therapeutic value in adoptive cell therapy. The strategies for allorejection control provided herein avoid the need for increased or exogenous expression of HLA-E / G in HLA-I-deficient allogeneic effector cells for improved and / or more complete protection against allorejection in adoptive cell therapy. In addition, the present application provides further genome engineering aspects to achieve enhanced functionality of effector cells, as detailed herein.

[0105] 1. HLA-I and HLA-II deficiency As mentioned above, to avoid the problem of allogeneic rejection, multiple HLA class I and class II proteins must be matched for histocompatibility with the allogeneic recipient. Provided herein is an iPSC cell line in which expression of one or both of HLA class I and HLA class II proteins is eliminated or substantially reduced. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21) or deletion, disruption, or reduction of expression levels of HLA class I associated genes, including but not limited to beta 2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers. B2M negative cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion, disruption, or reduction of HLA-II associated genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator and functions through activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-negative cells are HLA-II deficient. Provided herein are iPSC lines and their derived cells with deficiencies in HLA-I and optionally HLA-II, e.g., via B2M knockout and optionally CIITA knockout, and the derived effector cells obtained enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching to avoid recognition and killing by the host's (allogeneic) T cells.

[0106] In some cell types, lack of HLA class I expression leads to lysis by NK cells. To overcome this "loss of self" response, HLA-G or HLA-E can be optionally knocked in to avoid NK cell recognition and killing of HLA-I-deficient effector cells derived from engineered iPSCs. Alternatively, knockout of one or both of CD58 (or LFA-3) and CD54 (or ICAM-1), adhesion proteins that initiate signal-dependent cell interactions and promote migration of cells (including immune cells), has been shown to reduce allogeneic NK cell activation. Thus, in one embodiment, the HLA-I-deficient iPSCs and derived cells thereof provided further comprise an HLA-G knock-in. In one embodiment, the HLA-I-deficient iPSCs and derived cells thereof provided further comprise an HLA-E knock-in. However, as presented herein, the inhibitory receptors that recognize HLA-E and HLA-G are stochastically expressed, i.e., they are not expressed by all cells, and therefore knock-in of HLA-E / G does not provide complete protection from primary NK cell-based recognition. Additionally, there are corresponding activating receptors that can recognize HLA-E (and possibly HLA-G), which may lead to accelerated rejection of HLA-I-deficient effector cells.

[0107] Thus, in some embodiments, the present invention provides strategies to enhance effector cell persistence and / or survival by reducing or preventing allorejection by creating HLA-I and / or HLA-II deficiency without adversely affecting the differentiation potential of iPSCs and the function of derived effector cells (including derived T cells and derived NK cells). In some embodiments, the effector cells have increased persistence and / or survival in vivo in the presence of and / or after exposure to various therapeutic agents described herein. As provided, the strategies include generating iPSC lines containing a B2M knockout and directing differentiation of the engineered iPSC lines to B2M negative (B2M - / - and obtaining derived effector cells comprising the

[0108] In some embodiments, the effector cells have increased persistence and / or survival in vivo in the presence of and / or after exposure to a therapeutic agent. Thus, in some embodiments, iPSCs and derived cells thereof are HLA-I deficient (e.g., B2M negative (B2M - / - In some embodiments, the iPSCs and derived cells are HLA-I-deficient and HLA-II-deficient (e.g., B2M - / - and CIITA negative (CIITA - / - In some embodiments, B2M - / - In some embodiments, the effector cells comprising B2M are NK cells derived from iPSCs. - / - CIITA - / - In some embodiments, the effector cells comprising B2M are NK cells derived from iPSCs. - / - In some embodiments, the effector cells comprising B2M are T cells derived from iPSCs. - / - CIITA - / - In some embodiments, the iPSCs and derived cells thereof comprise one or more additional genome edits as described herein, including but not limited to CD38 negativity, exogenous CD16 or variants thereof, CAR expression, cytokine / cytokine receptor expression, and additional modalities, without adversely affecting the differentiation potential of the iPSCs and the function of the derived effector cells, including derived T cells and derived NK cells.

[0109] 2.CD38 knockout The cell surface molecule CD38 is highly upregulated in multiple hematological malignancies of both lymphoid and myeloid origin, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapy to deplete cancer cells. Antibody-mediated cancer cell depletion usually results from a combination of direct induction of cell apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cellular cytotoxicity). In addition to ADCC, immune effector mechanisms in conjunction with therapeutic antibodies may also include antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0110] In addition to being highly expressed on malignant cells, CD38 is also expressed on plasma cells, as well as on NK cells, activated T cells, and B cells. During hematopoiesis, CD38 is expressed in association with CD34 + It is expressed in stem cells and progenitor cells committed to the lymphoid, erythroid, and myeloid lineages, as well as in the final stages of maturation up to the plasma cell stage. CD38, a type II transmembrane glycoprotein, serves cellular functions both as a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 mediates the synthesis and upregulation of NAD + CD38 catalyzes the hydrolysis of cadmium to ADP-ribose, thereby generating the second messengers CADPR and NAADP, which stimulate calcium release from the endoplasmic reticulum and lysosomes, which is calcium-dependent and important for the process of cell adhesion. CD38 recognizes CD31 as a receptor and regulates cytokine release and cytotoxicity of activated NK cells. CD38 associates with cell surface proteins in lipid rafts and mediates the release of cytoplasmic Ca. 2+ It has also been reported to regulate flux and mediate signaling in lymphoid and myeloid cells.

[0111] In the treatment of malignancies, the systemic use of T cells transduced with the CD38 antigen-binding receptor has been shown to inhibit the expression of CD34 +CD38 on hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells + Fractions are lysed and recipient immune effector cell function is impaired, resulting in incomplete therapeutic responses and reduced or eliminated efficacy. Furthermore, in multiple myeloma patients treated with the CD38-specific antibody daratumumab, a reduction in NK cells was observed in both bone marrow and peripheral blood, whereas other immune cell types such as T and B cells were unaffected despite CD38 expression (Casneuf et al., Blood Advances. 2017;1(23):2105-2114). Without being limited by theory, the present application provides a strategy to maximize the potential of CD38-targeted cancer therapy by reducing allorejection of allogeneic effector cells through HLA deficiency and CD38 conditioning, thereby increasing effector cell survival and persistence. Thus, the present application also provides strategies to enhance effector cell persistence and / or survival through reducing or preventing allorejection by using CD38 antagonists such as anti-CD38 antibodies or CD38-CARs (chimeric antigen receptors) for activation of recipient T and B cells, which in some embodiments can be used as an alternative to lymphodepletion using chemotherapy such as Cy / Flu (cyclophosphamide / fludarabine) prior to adoptive cell transfer. Also disclosed in the present application is that in some embodiments, when hnCD16a+ / CD38- effector cells are used to target CD38+ T and pbNK cells in the presence of anti-CD38 antibodies or CD38 inhibitors, depletion of CD38+ alloreactive cells increases NAD (nicotinamide adenine dinucleotide, a substrate for CD38) availability and reduces NAD consumption-associated cell death, which, among other benefits, boosts effector cell responses in immunosuppressive tumor microenvironments and supports cell rejuvenation in aging, degenerative or inflammatory diseases.

[0112] Therefore, the strategies provided herein also support B2M - / - , CD38 knockout, and optionally CIITA - / -generating an iPSC line comprising B2M iPSCs, generating a master cell bank comprising sorted single cells and expanded clonal iPSCs, and directing differentiation of the engineered iPSC line to produce a B2M iPSC line comprising B2M iPSCs. - / - CD38 negative (CD38 - / - ) derived effector cells or B2M - / - CIITA - / - CD38 - / - and obtaining derived effector cells comprising: a CD38-targeted therapeutic moiety that is protected against fratricide and allorejection when used with the effector cells, among other advantages including improved metabolic fitness, increased resistance to oxidative stress, and induction of a protein expression program in the effector cells that enhances cell activation and effector function. 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 derivative cells have the ability to resist CD38 antibody-mediated depletion and can be effectively administered in combination with anti-CD38 antibodies or CD38-CARs without the use of toxic conditioning agents, thus reducing and / or replacing chemotherapy-based lymphodepletion. In one embodiment, the CD38 knockout in the iPSC line is a biallelic knockout.

[0113] As disclosed herein, B2M - / - CD38 - / - , and optionally CIITA - / - The provided iPSC lines, which undergo directed differentiation to produce mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 + Functional derived hematopoietic cells can be produced, including, but not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, when anti-CD38 antibodies are used to induce ADCC or CD38-CARs are used for targeted cell killing, B2M- / - CD38 - / - iPSCs and / or B2M - / - CIITA - / - CD38 - / - The iPSCs and / or their derived effector cells comprising are not eliminated by anti-CD38 antibodies or CD38-CARs, thereby increasing the persistence and / or survival of the iPSCs and their effector cells in the presence and / or after exposure to such therapeutic moieties. In some embodiments, the effector cells have increased persistence and / or survival in vivo in the presence and / or after exposure to such therapeutic moieties. In some embodiments, the derived effector cells are NK cells derived from the iPSCs. In some embodiments, the B2M - / - CD38 - / - In some embodiments, the effector cells comprising B2M are T cells derived from iPSCs. - / - CIITA - / - CD38 - / - In some embodiments, the effector cells comprising B2M are T cells derived from iPSCs. - / - CD38 - / - iPSCs and / or B2M - / - CIITA - / - CD38 - / - The iPSCs and / or derived cells thereof comprise one or more additional genome edits described herein, including, but not limited to, exogenous CD16 expression, CAR expression, cytokine / cytokine receptor expression, and additional modalities.

[0114] 3.CD16 knock-in CD16 has been identified as two isoforms, the Fc receptors 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 to activate NK cells and promote antibody-dependent cellular cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, "high affinity CD16", "non-cleavable CD16", or "high affinity non-cleavable CD16" refer to various CD16 variants. Wild-type CD16 has low affinity and upon NK cell activation is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V (also referred to as F158V in some publications) is an exemplary CD16 polymorphic variant with high affinity, while the S197P variant is an example of an engineered non-cleavable version of CD16. Engineered CD16 variants including both F176V and S197P have high affinity and are non-cleavable, as described in more detail in WO 2015 / 148926, the full disclosure of which is incorporated herein by reference. In addition, a chimeric CD16 receptor in which the ectodomain of CD16 is essentially replaced with at least a portion of the ectodomain of CD64 can also achieve the desirable high affinity and non-cleavable properties of a CD16 receptor capable of performing ADCC. In some embodiments, the replaced ectodomain of the chimeric CD16 comprises one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or an isoform or polymorphic variant thereof).

[0115] Thus, 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, includes F176V and eliminates the cleavage region. In some other embodiments, hnCD16 includes a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween identity when compared to any of the exemplary sequences SEQ ID NOs: 1, 2, and 3, each of which includes at least a portion of the CD64 ectodomain. As used herein and throughout this application, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be performed using art-recognized mathematical algorithms.

[0116] [Table 1]

[0117] [Table 2]

[0118] [Table 3]

[0119] Thus, provided herein, among other editing contemplated and described herein, are clonal iPSCs genetically engineered to contain exogenous CD16, i.e., the high affinity non-cleavable CD16 receptor (hnCD16), where the genetically engineered iPSCs are capable of differentiating into effector cells that contain hnCD16 introduced into the iPSC. In some embodiments, B2M - / - CD38 - / - and the derived effector cells comprising exogenous CD16 are NK cells. - / - CIITA - / - CD38 - / - and the derived effector cells comprising exogenous CD16 are NK cells. - / - CD38 - / - and the derived effector cell comprising exogenous CD16 is a T cell. - / - CIITA - / - CD38 - / - and the derived effector cells comprising exogenous CD16 are T cells. In some embodiments, the derived NK cells are preloaded with an antibody. In some embodiments, the derived NK cells are used in a combination therapy with an antibody. In some embodiments, the antibody in the combination therapy or the antibody with which the derived NK cells are preloaded specifically targets CD38. In some embodiments, the antibody in the combination therapy or the antibody with which the derived NK cells are preloaded specifically targets an antigen different from CD38. In some embodiments, the anti-CD38 antibody is daratumumab.

[0120] The exogenous hnCD16 expressed on iPSCs or derived cells thereof exhibits high affinity binding not only to ADCC antibodies or fragments thereof, but also to bispecific, trispecific or multispecific engagers or binders that recognize the extracellular binding domain of CD16 or CD64 of said hnCD16. Bispecific, trispecific or multispecific engagers or binders are further described below in this application. Thus, the application provides derived effector cells or cell populations thereof preloaded with one or more preselected ADCC antibodies via high affinity binding to the extracellular domain of hnCD16 expressed on the derived effector cells, said hnCD16 comprising the extracellular binding domain of CD64 or of CD16 with F176V and S197P, in an amount sufficient for therapeutic use in the treatment of a condition, disease or infection as further described below.

[0121] In some other embodiments, the exogenous CD16 expressed in iPSCs or its derived cells comprises a CFcR based on CD16 or a variant thereof. Chimeric Fc receptors (CFcRs) are produced to include a non-native transmembrane domain, a non-native stimulatory domain and / or a non-native signaling domain by modifying or replacing the native CD16 transmembrane and / or intracellular domain. As used herein, the term "non-native" means that the transmembrane domain, stimulatory domain or signaling domain is derived from a different receptor than the receptor that provides the extracellular domain. Exemplary herein, a CFcR based on CD16 or a variant thereof does not have a transmembrane domain, stimulatory domain or signaling domain derived from CD16. In some embodiments, the exogenous CD16-based CFcR comprises a non-native transmembrane domain derived from CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-native stimulatory / inhibitory domain derived from a CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some embodiments, the exogenous CD16-based CFcR comprises a non-native signaling domain derived from a CD3zeta, 2B4, DAP10, DAP12, DNAM1, CD137(4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In some embodiments of the CD16-based CFcR, the chimeric Fc receptor provided comprises a transmembrane domain and a signaling domain that are both derived from one of an IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide.One specific exemplary embodiment of a CD16-based chimeric Fc receptor comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, where the extracellular domain of CFcR is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, where the extracellular domain of CD16 comprises F176V and S197P. Another exemplary embodiment of a CD16-based chimeric Fc receptor comprises the transmembrane domain and signaling domain of CD3ζ, where the extracellular domain of CFcR is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, where the extracellular domain of CD16 comprises F176V and S197P.

[0122] Various embodiments of CD16-based chimeric Fc receptors as described above can bind with high affinity to the Fc region of an antibody or fragment thereof, or to a bispecific, trispecific, or multispecific engager or binder. Upon binding, the stimulatory and / or signaling domains of the chimeric receptor allow for effector cell activation and cytokine secretion, as well as tumor cell killing targeted by the antibody, or by the bispecific, trispecific, or multispecific engager or binder described above having a tumor antigen binding moiety and an Fc region. Without being limited by theory, through the non-native transmembrane, stimulatory, and / or signaling domains of the CD16-based chimeric Fc receptor, or through the binding of the engager to the ectodomain, the CFcR can contribute to the killing capacity of the effector cell, increasing the proliferation and / or growth potential of the effector cell. Antibodies and engagers can bring antigen-expressing tumor cells and CFcR-expressing effector cells into close proximity, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, trispecific, or multispecific engagers or binders include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, trispecific, multispecific engagers or binders suitable for binding effector cells expressing CD16-based CFcR in attacking tumor cells include CD16 (or CD64)-CD30, CD16 (or CD64)-BCMA, CD16 (or CD64)-IL15-EPCAM, and CD16 (or CD64)-IL15-CD33.

[0123] Unlike endogenous CD16 expressed by primary NK cells, which is cleaved from the cell surface following NK cell activation, the various non-cleavable versions of CD16 on derived NK cells avoid CD16 shedding and maintain constant expression. In induced NK cells, non-cleavable CD16 increases the expression of TNFα and CD107a, indicators of improved cell function. Non-cleavable CD16 also enhances antibody-dependent cellular cytotoxicity (ADCC) and the binding of bi-, tri-, or multi-specific engagers. ADCC is a mechanism of NK cell-mediated lysis via binding of CD16 to antibody-coated target cells. The additional high affinity properties of hnCD16 introduced into derived NK cells also allow in vitro loading of ADCC antibodies via hnCD16 prior to administration of the cells to a subject in need of cell therapy. As provided herein, in some embodiments, hnCD16 may comprise F176V and S197P, or may comprise a full-length or partial-length ectodomain derived from CD64, or may further comprise at least one of a non-native transmembrane domain, a stimulatory domain, and a signaling domain. As disclosed, the present application also provides derived NK cells or cell populations thereof preloaded with one or more preselected ADCC antibodies in an amount sufficient for therapeutic use in treating a condition, disease, or infection, as further described herein. In some embodiments, the preloaded antibody is an anti-CD38 antibody. In one particular embodiment, the anti-CD38 antibody is daratumumab.

[0124] Unlike primary NK cells, mature T cells derived from primary sources (i.e., native / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs with expressed exogenous uncleavable CD16 could differentiate into functional induced T lineage cells that not only express exogenous CD16 but also can perform functions through acquired ADCC mechanisms without compromising the developmental biology of T cells. This acquired ADCC in derived T lineage cells could further be used as an approach to rescue antigen escape, which often occurs in dual targeting and / or CAR-T cell therapy, where tumors relapse with reduced or lost expression of CAR-T target antigens, or mutated antigens that avoid recognition by CAR (chimeric antigen receptor). If the induced T lineage cells contain ADCC acquired through exogenous CD16 (including functional variants and CD16-based CFcR) expression, and the antibody targets a tumor antigen different from the tumor antigen targeted by the CAR, the antibody can be used to rescue CAR-T antigen escape and reduce or prevent the recurrence or relapse of the targeted tumor that is common with CAR-T therapy. Such a strategy to reduce and / or prevent antigen escape while achieving dual targeting is similarly applicable to NK cells expressing one or more CARs. Various CARs that can be used in this antigen escape reduction and prevention strategy are further described below.

[0125] 4. Chimeric Antigen Receptor (CAR) Expression Applicable to engineered iPSCs and their derived effector cells can be any CAR design known in the art. CARs are fusion proteins that generally include an ectodomain, a transmembrane domain, and an endodomain, which includes an antigen recognition domain. In some embodiments, the ectodomain can further include a signal peptide or leader sequence and / or a spacer. In some embodiments, the endodomain can further include a signaling peptide that activates the effector cell expressing the CAR. In some aspects, the endodomain can include a signaling domain, which is derived from a cytoplasmic domain of a signaling protein specific for T cell and / or NK cell activation or function. In some embodiments, the antigen recognition domain can specifically bind to an antigen. In some embodiments, the antigen recognition domain can specifically bind to an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, and the tumor can be a liquid tumor or a solid tumor. In some embodiments, the CAR is suitable for activating either T lineage cells or NK lineage cells expressing the CAR. In some embodiments, the CAR is NK cell specific, which includes an NK-specific signaling component. In certain embodiments, the T cells are derived from CAR-expressing iPSCs, and the derived T lineage cells can include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or combinations thereof. In certain embodiments, the NK cells are derived from CAR-expressing iPSCs.

[0126] In certain embodiments, the antigen recognition region / domain comprises a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a single variable novel antigen receptor (VNAR), a shark heavy chain antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single chain antigen binding fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single domain antigen binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody. In some embodiments, the antigen recognition region of the CAR is derived from the binding domain of a T cell receptor (TCR) that targets a tumor associated antigen (TAA).

[0127] Non-limiting examples of antigens that can be targeted by a CAR include ADGRE2, B7H3, 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, cytomegalovirus (CV)-associated leukemia (IM ... antigen of uterine malaria virus (CMV)-infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (EGFR-VIII), HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, MR1, mucin 1 (Muc-1), mucin 16 (Muc-16) , mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, NY-ESO-1, oncofetal antigen (h5T4), PDL1, PRAME, prostate stem cell antigen (PSCA), PRAME prostate specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0128] Thus, in some embodiments, the genetically engineered iPSCs and their derived cells comprise an exogenous polynucleotide encoding a CAR, wherein the CAR comprises a CD19-CAR, a BCMA-CAR, a B7H3-CAR, a MICA / B-CAR, a HER2-CAR, or an MR1-CAR.

[0129] In some embodiments, the transmembrane domain of the CAR comprises the full length or at least a portion of the native or modified transmembrane region of a CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide.

[0130] In some embodiments, the signaling peptide of the internal domain (or intracellular domain) is selected from the group consisting of 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain), CD3ζ1XX (CD3ζ variants), DAP10 (hematopoietic cell signaling receptor), DAP12 (TYRO protein tyrosine kinase binding protein), DNAM1 (CD226 antigen), FcERIγ (high affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta), IL-2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D The polypeptides include full length or at least a portion of the polypeptides of type II integral membrane protein), NKp30 (natural cytotoxicity triggering receptor 3), NKp44 (natural cytotoxicity triggering receptor 2), NKp46 (natural cytotoxicity triggering receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain).

[0131] In some embodiments, the endodomain of the CAR further comprises a second signaling domain and optionally a third signaling domain, wherein each of the first signaling domain, the second signaling domain, and the third signaling domain are different. In certain embodiments, the second signaling domain and / or the third signaling domain comprises the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3zeta, DAP10, DAP12, DNAM1, FcERIgammaIL21R, IL-2Rbeta (IL-15Rbeta), IL-2Rgamma, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3zeta1XX, CS1, or CD8. In certain embodiments, the endodomain further comprises at least one costimulatory signaling region. The costimulatory signaling region can include a full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.

[0132] In some embodiments, a CAR applicable to the cells provided herein comprises a costimulatory domain derived from CD28 and a signaling domain comprising a native or modified ITAM1 of CD3ζ represented by an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 4. In further embodiments, a CAR comprising a costimulatory domain derived from CD28 and a native or modified ITAM1 of CD3ζ also comprises a hinge domain and a transmembrane domain derived from CD28, and the scFv can be connected to the transmembrane domain via the hinge, and the CAR comprises an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 5.

[0133] [Table 4]

[0134] [Table 5]

[0135] In various embodiments, a CAR applicable to the cells provided herein comprises a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3ζ represented by an amino acid sequence at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 6. Such a CAR comprising a transmembrane domain derived from NKG2D, a costimulatory domain derived from 2B4, and a signaling domain comprising native or modified CD3ζ may further comprise a CD8 hinge, the amino acid sequence of such structure being at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 7.

[0136] [Table 6]

[0137] [Table 7]

[0138] Non-limiting CAR strategies include heterodimeric conditionally activated CARs through dimerization of a pair of intracellular domains (see, e.g., U.S. Pat. No. 9,587,020); split CARs (homologous recombination of antigen-binding, hinge, and endodomains to generate a CAR (see, e.g., U.S. Pat. App. Pub. No. 2017 / 0183407); multi-chain CARs that allow for non-covalent association between two transmembrane domains connected to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Pat. App. Pub. No. 2014 / 0134142); CARs with bispecific antigen-binding domains (see, e.g., U.S. Pat. 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. Pat. No. 8,409,577), or tandem CARs (see, e.g., Hegde et al., J Clin. Invest. 2016;126(8):3036-3052); inducible CARs (see, e.g., U.S. Patent Application Publication Nos. 2016 / 0046700, 2016 / 0058857, and 2017 / 0166877); switchable CARs (see, e.g., U.S. Patent Application Publication No. 2014 / 0219975); and any other designs known in the art.

[0139] Thus, aspects of the invention provide derivative cells obtained from differentiating genomically engineered iPSCs, where both the iPSCs and the derivative cells comprise one or more CARs with additional modified modalities, as provided in Table 1. In some embodiments, B2M - / - CD38 - / - The effector cells comprising the CAR are NK cells derived from iPSCs. In some embodiments, B2M - / - CIITA - / - CD38 - / - The effector cells comprising the CAR are NK cells derived from iPSCs. In some embodiments, B2M - / - CD38 - / - The effector cells comprising the CAR are T cells derived from iPSCs. In some embodiments, B2M- / - CIITA - / - CD38 - / - The effector cells comprising the CAR are T cells derived from the iPSCs. In some embodiments, the iPSCs and their derived cells contain one or more additional genome edits as described herein, including but not limited to exogenous CD16 expression and / or cytokine / cytokine receptor expression, and additional modalities, without adversely affecting the differentiation potential of the iPSCs and the function of the derived effector cells, including derived T cells and derived NK cells.

[0140] 5. Exogenously Introduced Cytokine Signaling Complexes By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicity from such actions is reduced and cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional administration of soluble cytokines, a cytokine signaling complex comprising partial or complete peptides of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors is introduced into cells to allow cytokine signaling with or without expression of the cytokine itself, thereby reducing the risk of cytokine toxicity and maintaining or improving cell growth, proliferation, expansion, and / or effector function. In some embodiments, the introduced cytokine and / or its respective native or modified receptor (signaling complex) for cytokine signaling is expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or temporary.

[0141] Various construct designs are provided herein for introducing cytokine signaling complexes into cells for signaling cytokines, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In embodiments where the cytokine signaling complex is for IL15, the transmembrane (TM) domain may be native to the IL15 receptor or may be modified or replaced with the transmembrane domain of any other membrane-bound protein. In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics trans-presentation of IL15 without eliminating cis-presentation of IL15. In other embodiments, IL15Rα is fused to IL15 at the C-terminus via a linker, mimicking trans-presentation without eliminating cis-presentation of IL15, ensuring that IL15 is membrane-bound. In another embodiment, IL15Rα with a truncated intracellular domain is fused to IL15 at its C-terminus via a linker to mimic trans-presentation of IL15, maintain membrane association of IL15, and eliminate cis-presentation and / or other potential signaling pathways mediated by normal IL15R via its intracellular domain.

[0142] Such truncated constructs comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8. In one embodiment of a truncated IL15 / IL15Rα, the construct does not include the last four amino acid residues (KSRQ) of SEQ ID NO: 8 and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 9.

[0143] [Table 8]

[0144] [Table 9]

[0145] In yet other embodiments, the cytoplasmic domain of IL15Rα can be omitted without adversely affecting the autonomous properties of IL15-equipped effector cells. In other embodiments, the entire IL15Rα is removed, with the exception of the Sushi domain, which is fused to IL15 at one end and to a transmembrane domain at the other (mb-Sushi), with optional 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 the membrane-bound protein. Unwanted signaling through IL15Rα, including cis-presentation, is eliminated when only the desired trans-presentation of IL15 is retained. In some embodiments, the component comprising IL15 fused to a Sushi domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:10.

[0146] [Table 10]

[0147] In other embodiments of the cytokine signaling complex, native or modified IL15Rβ is fused to IL15 at the C-terminus via a linker to allow constitutive signaling and maintain IL15 membrane binding and trans presentation. In other embodiments, native or modified common receptor γC is fused to IL15 at the C-terminus via a linker for constitutive signaling and membrane-bound trans presentation of cytokines. Common receptor γC is also called common gamma chain or CD132, and is known as IL2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to the receptor complexes of many interleukin receptors, including but not limited to IL2, IL4, IL7, IL9, IL15, and IL21 receptors. In other embodiments, engineered IL15Rβ, which forms homodimers in the absence of IL15, is useful for generating constitutive signaling of cytokines.

[0148] Those skilled in the art will understand that the signal peptides and linker sequences described above are exemplary and in no way limit the variations thereof suitable for use as signal peptides or linkers. There are many suitable signal peptide or linker sequences known and available to those skilled in the art, and those skilled in the art will understand that the signal peptide and / or linker sequence may be substituted with another sequence without altering the activity of the functional peptide directed by the signal peptide or linked by the linker.

[0149] In iPSCs and derived cells thereof that contain both a CAR and an exogenous signaling complex ("IL") that includes cytokine and / or cytokine receptor signaling, the CAR and IL can be expressed in separate constructs or can be co-expressed in a bicistronic construct that includes both the CAR and IL. In some embodiments, B2M - / - , C.I.T.A. - / - , CD38 - / - , CD16 + , C.A.R. + , and I.L. + iPSCs and derived effector cells thereof comprising a genotype comprising one or more attributes including may further comprise any one of the additional attributes in Table 1.

[0150] In some embodiments, the B2M - / - CD38 - / - IL + In some embodiments, the effector cells comprising B2M are NK cells derived from iPSCs. - / - CIITA - / - CD38 - / - IL + In some embodiments, the effector cells comprising B2M are NK cells derived from iPSCs. - / - CD38 - / - IL + In some embodiments, the effector cells comprising B2M are T cells derived from iPSCs. - / - CIITA - / - CD38 - / - IL +are T cells derived from iPSCs. In some embodiments, the iPSCs and their derived cells include one or more additional genome edits described herein without adversely affecting the differentiation potential of the iPSCs and the function of the derived effector cells, including derived T cells and derived NK cells.

[0151] Thus, in various embodiments, the cytokine IL15 and / or its receptor may be introduced into iPSCs using one or more of the construct designs described above, or into their derived cells upon iPSC differentiation. In addition to induced pluripotent cells (iPSCs), clonal iPSCs, clonal iPS cell lines, or iPSC-derived cells comprising at least one engineered modality disclosed herein are provided. Also provided are master cell banks comprising sorted single cells and expanded clonally engineered iPSCs with at least an exogenously introduced signaling complex comprising cytokine and / or cytokine receptor signaling as described in this section, which provide a platform for further iPSC manipulation and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products that are compositionally defined and uniform and can be mass-produced at scale in a cost-effective manner.

[0152] 6. Engager Engagers are fusion proteins consisting of two or more single chain variable fragments (scFvs), or other functional variants, of different antibodies or fragments thereof, with at least one scFv that binds to an effector cell surface molecule or a surface triggering receptor, and at least another that binds to a target cell via a target cell-specific surface molecule. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs), multispecific killer cell engagers, or universal engagers that can be compatible with multiple immune cell types. Engagers can be bispecific or multispecific. Such bispecific or multispecific engagers can direct effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils) to tumor cells and activate immune effector cells, showing great potential to maximize the benefits of CAR-T cell therapy.

[0153] In some embodiments, the engager is used in combination with a population of effector cells described herein, by simultaneous or sequential administration, where the effector cells comprise a surface molecule or surface triggering receptor that is recognized by the engager. In some other embodiments, the engager is a bispecific antibody that is expressed by derived effector cells through genetic engineering of iPSCs as described herein and directed differentiation of the engineered iPSCs. Exemplary effector cell surface molecules or surface triggering receptors that can be used for bispecific or multispecific engager recognition or coupling include, but are not limited to, CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, and chimeric Fc receptors disclosed herein. In some embodiments, the exogenous CD16 expressed on the surface of the derived effector cells for engager recognition is hnCD16 comprising the CD16 (including F176V and optionally S197P) or CD64 extracellular domain, and native or non-native transmembrane, stimulatory and / or signaling domains, as described herein. In some embodiments, the CD16 expressed on the surface of the effector cells for engager recognition is a CD16-based chimeric Fc receptor (CFcR). In some embodiments, the CD16-based CFcR comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, and the extracellular domain of CD16 is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and optionally S197P.

[0154] In some embodiments, the target cell for the engager is a tumor cell. Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include, but are not limited to, B7H3, BCMA, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, ROR1. In one embodiment, the bispecific engager is a bispecific antibody specific for CD3 and CD19 (CD3-CD19). In another embodiment, the bispecific antibody is CD16-CD30 or CD64-CD30. In another embodiment, the bispecific antibody is CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody is CD3-CD33.

[0155] In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and the tumor cell antigen binding domain. For example, modified IL15 can be used as a linker for effector NK cells to promote cell proliferation (referred to in some publications as TriKE, or trispecific killer engager). 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. The IL15 in TriKE can also be derived from other cytokines, including, but not limited to, IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21.

[0156] In some embodiments, the surface triggering receptor for a bispecific or multispecific engager can be endogenous to the effector cell, sometimes depending on the cell type. In some other embodiments, the methods and compositions provided herein can be used to further manipulate iPSCs, e.g., comprising the genotypes listed in Table 1, to direct the differentiation of iPSCs into T cells, NK cells, or other effector cells that comprise the same genotype and surface triggering receptor as the source iPSCs, and to introduce one or more exogenous surface triggering receptors into the effector cells.

[0157] 7. Antibodies for immunotherapy In some embodiments, in addition to the genomically engineered effector cells provided herein, additional therapeutic agents including antibodies or antibody fragments targeting antigens associated with a condition, disease, or indication can be used in combination therapy with these effector cells. In some embodiments, antibodies are used in combination with a population of effector cells described herein by simultaneous or sequential administration to a subject. In other embodiments, such antibodies or fragments thereof can be expressed by effector cells by genetically engineering iPSCs with an exogenous polynucleotide sequence encoding the antibody or fragment thereof and directing differentiation of the engineered iPSCs. In some embodiments, the effector cells express an exogenous CD16 variant and the cytotoxicity of the effector cells is enhanced by the antibody via ADCC. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, tumor- or virus-specific antigens activate the administered iPSC-derived effector cells, enhancing their killing capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-derived effector cells include, but are not limited to, anti-CD20 (rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-HER2 (trastuzumab, pertuzumab), anti-CD52 (alemtuzumab), anti-EGFR (certuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and humanized or Fc-engineered variants or fragments thereof, or functional equivalents and biosimilars thereof.

[0158] In some embodiments, the iPSC-derived effector cells comprise hematopoietic lineage cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise NK cells comprising a genotype listed in Table 1. In some embodiments, the iPSC-derived effector cells comprise T cells comprising a genotype listed in Table 1. In some embodiments of the combination useful for treating liquid or solid tumors, the combination comprises iPSC-derived NK cells or T cells comprising at least CD38 negative and B2M negative. In one embodiment, the combination comprises iPSC-derived NK cells comprising CD38 negative, B2M negative and exogenous CD16 and one of the anti-CD38 antibodies daratumumab, isatuximab, and MOR202. In one embodiment, the combination comprises iPSC-derived NK cells comprising B2M negative, CD38 negative, exogenous CD16 and daratumumab. In some further embodiments, the iPSC-derived NK cells included in combination with daratumumab comprise one or more of B2M negative, CD38 negative, exogenous CD16, IL15, and optionally CIITA negative, and a CAR, where IL15 is co-expressed with the CAR or expressed separately, and IL15 is in any one of the forms described herein. In some particular embodiments, IL15 is co-expressed with the CAR or expressed separately.

[0159] 8. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate immune responses if not inhibited. It has become evident that tumors co-opt certain immune checkpoint pathways as a primary mechanism of immune resistance, particularly to T cells specific for tumor antigens. Checkpoint inhibitors (CIs) are antagonists that can reduce the expression of checkpoint genes or gene products or decrease the activity of checkpoint molecules, blocking inhibitory checkpoints and restoring immune system function. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has transformed the oncology landscape, and these agents have led to long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. One aspect of the present application provides a therapeutic approach to overcome CI resistance by including genomically engineered functional derivative cells as provided herein in a combination therapy with CI. In some embodiments, checkpoint inhibitors are used in combination with a population of effector cells as described herein, by their simultaneous or sequential administration to a subject. In some other embodiments, the checkpoint inhibitor is expressed by the effector cells by genetically engineering the iPSCs with an exogenous polynucleotide sequence encoding the checkpoint inhibitor or a fragment or variant thereof and directing differentiation of the engineered iPSCs. Some embodiments of the combination therapy with effector cells described herein include at least one checkpoint inhibitor to target at least one checkpoint molecule, and the derivative cells have a genotype listed in Table 1.

[0160] In some embodiments, the exogenous polynucleotide sequence encoding the checkpoint inhibitor or a fragment thereof is co-expressed with the CAR, either in a separate construct or in a bicistronic construct. In some further embodiments, the sequence encoding the checkpoint inhibitor or a fragment thereof can be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence, exemplified as CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences for the checkpoint inhibitor and the CAR are in a single open reading frame (ORF). When the checkpoint inhibitor is delivered and expressed and secreted as a payload by derived effector cells that can infiltrate the tumor microenvironment (TME), it counteracts inhibitory checkpoint molecules upon binding to the TME, activating modalities such as CARs or activating receptors to activate effector cells. In one embodiment of the combination therapy, the derived effector cells are NK lineage cells. In another embodiment of the combination therapy, the derived effector cells are T lineage cells.

[0161] Suitable checkpoint inhibitors for combination therapy with derived effector cells as provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A 2AThese include, but are not limited to, antagonists of R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0162] In some embodiments, the antagonist that inhibits any of the above-mentioned checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a single variable novel antigen receptor (VNAR), a shark heavy chain antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single chain antigen binding fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single domain antigen binding fragments (sdAb, nanobodies), recombinant heavy chain only antibodies (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be more cost-effective to produce, easier to use, or more sensitive than whole antibodies. In some embodiments, the checkpoint inhibitor comprises at least one of atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR), IPH43 (anti-MICA), IPH33 (anti-TLR3), lilimumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and derivatives, functional equivalents, or biosimilars thereof.

[0163] In some embodiments, the antagonists that inhibit any of the above-mentioned checkpoint molecules are microRNA-based, as many miRNAs are found as regulators controlling the expression of immune checkpoints (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint antagonistic 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.

[0164] In some embodiments, a checkpoint inhibitor is co-expressed with the CAR and inhibits at least one of the following checkpoint molecules: PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2AR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR. In some embodiments, the checkpoint inhibitor co-expressed with the CAR in the derivative cells having the genotypes listed in Table 1 is selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and humanized or Fc-modified variants, fragments, and functional equivalents or biosimilars thereof. In some embodiments, the checkpoint inhibitor co-expressed with the CAR is atezolizumab, or humanized or Fc-modified variants, fragments, or functional equivalents or biosimilars thereof. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is nivolumab, or humanized or Fc-modified variants, fragments, or functional equivalents or biosimilars thereof. In some other embodiments, the checkpoint inhibitor co-expressed with the CAR is pembrolizumab, or a humanized, or Fc-engineered variant, fragment, or functional equivalent or biosimilar thereof.

[0165] In some other embodiments of the combination therapy comprising derived effector cells and at least one antibody that inhibits a checkpoint molecule provided herein, the antibody is not produced by or in the derived cells and is further administered prior to, simultaneously with, or after administration of the derived cells as provided herein. In some embodiments, the administration of one, two, three, or more checkpoint inhibitors in the combination therapy with the induced NK lineage cells or induced T lineage cells provided is simultaneous or sequential. In one embodiment of the combination therapy, the checkpoint inhibitor included in the treatment is one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of the combination therapy, the checkpoint inhibitor included in the treatment is atezolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments of the combination therapy, the checkpoint inhibitor included in the treatment is nivolumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof. In some embodiments of the combination therapy, the checkpoint inhibitor included in the treatment is pembrolizumab, or a humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0166] 9. Genetically engineered iPSC lines and derived cells provided herein In light of the above, the present application provides the following: - / - CD38 - / - and, optionally, CIITA - / -and one or more of an exogenous polynucleotide encoding an exogenous CD16, an exogenous polynucleotide encoding a cytokine signaling complex (IL), an exogenous polynucleotide encoding a CAR, an exogenous polynucleotide encoding an antibody, and an additional modality, wherein the derived cells are selected from the group consisting of iPSCs, iPS cell line cells, or derived cells thereof, comprising an exogenous polynucleotide encoding an exogenous CD16, an exogenous polynucleotide encoding a cytokine signaling complex (IL), an exogenous polynucleotide encoding a CAR, an exogenous polynucleotide encoding an antibody, and an additional modality, wherein the derived cells are selected from the group consisting of B2M - / - 、 CD38 - / - 、 (Optional CIITA - / - In some embodiments, the derived cells are hematopoietic lineage cells, including mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 + These include, but are not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutrophils, dendritic cells, and macrophages. In other embodiments, functional derivative hematopoietic cells include effector cells that have one or more functional attributes not present in the corresponding primary T, NK, NKT, and / or B cells.

[0167] In some embodiments, the derivative cells include NK or T lineage cells. - / - 、 CD38 - / - , and optionally CIITA - / -iPSC-derived NK or T lineage cells comprising one or more of a cytokine signaling complex (IL), exogenous CD16, and a CAR are useful to overcome or reduce tumor recurrence associated with tumor antigen escape observed with CAR-T alone treatment by combining the antibody with CAR-targeted therapy, where the antibody and CAR have specificity for different antigens of the tumor. Induced CAR-T cells expressing hnCD16 have acquired ADCC, providing an additional mechanism for tumor killing in addition to CAR targeting. In some embodiments, the derived cells include NK lineage cells. B2M - / - , CD38 - / - , and optionally CIITA - / - iPSC-derived NK cells containing one or more of a cytokine signaling complex (IL), exogenous CD16, and a CAR have enhanced cytotoxicity and are effective in recruiting bystander cells, including T cells, to infiltrate and kill tumor cells.

[0168] In some embodiments, when anti-CD38 antibodies are used to induce CD16-mediated enhanced ADCC, iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing effector cell elimination, i.e., reduction or depletion of CD38-expressing effector cells, thereby increasing the persistence and / or viability of iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or viability in vivo in the presence of an anti-CD38 therapeutic agent, which may be an anti-CD38 antibody. In some embodiments, the anti-CD38 antibody is daratumumab, isatuximab, or MOR202. In addition, because CD38 is upregulated on activated lymphocytes, such as T cells or B cells, CD38-specific antibodies can be used for lymphodepletion, thereby eliminating those activated lymphocytes, overcoming allorejection, and increasing the survival and persistence of CD38-negative effector cells without fratricide in the recipient of allogeneic effector cell therapy.

[0169] In some embodiments, the effector cells include T lineage cells, including B2M-negative and CD38-negative iPSC-derived T lineage cells, which have reduced cell depletion in the presence of anti-CD38 antibodies and acquired ADCC, providing an additional mechanism for T cell-mediated tumor killing. In some embodiments, the effector cells include NK lineage cells, including B2M-negative and CD38-negative iPSC-derived NK lineage cells, which have enhanced cytotoxicity and reduced NK cell fratricide in the presence of anti-CD38 antibodies.

[0170] Provided herein are iPSCs comprising B2M knockout, CD38 knockout, and optionally CIITA knockout, which can generate functional derived effector cells by directed differentiation. In some embodiments of effector cells comprising B2M negative / CD38 negative derived from engineered iPSCs, the cells are intact in HLA-II and still tolerate allorejection by activated recipient T cells, recipient B cells, and recipient NK cells. In some embodiments, iPSCs comprising B2M knockout and CD38 knockout and derived effector cells thereof further comprise CIITA knockout. In some embodiments, iPSCs comprising B2M knockout (and optionally CIITA knockout) and CD38 knockout and derived effector cells thereof comprise a CAR, which may or may not target CD38. In some embodiments, the CAR-expressing derived effector cells, which include B2M-negative, CD38-negative, and optionally CIITA-negative, further comprise exogenous CD16 and can be used with an anti-CD38 antibody to induce ADCC without causing effector cell elimination, thereby increasing the persistence and / or viability of iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or viability in vivo in combination therapy.

[0171] Further provided are iPSCs comprising B2M knockout, CD38 knockout, and optionally CIITA knockout, CAR, and one of the polynucleotides encoding at least one exogenous cytokine signaling complex (IL) that allows cytokine signaling that contributes to cell viability, persistence, and / or proliferation, and the iPSC line can produce functional derived effector cells with improved viability, persistence, proliferation, and effector function by hematopoietic differentiation. The exogenously introduced cytokine signaling complex includes any one, two, or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21 signaling. In some embodiments, introduced partial or complete peptides of cytokines and / or their respective receptors for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or temporary. In some embodiments, the transient / transient expression of the cell surface cytokine / cytokine receptor is via a retrovirus, Sendai virus, adenovirus, episome, minicircle, or RNA, including mRNA. - / - CD38 - / - (and optionally CIITA - / - ) Exogenous cell surface cytokines and / or receptors contained in iPSCs or derived cells thereof that contain IL7 enable IL7 signaling. In some embodiments, B2M - / - CD38 - / - (and optionally CIITA - / - ) Exogenous cell surface cytokines and / or receptors contained in iPSCs or derived cells thereof that contain IL10 enable IL10 signaling. In some embodiments, B2M - / - CD38 - / - (and optionally CIITA - / -) Exogenous cell surface cytokines and / or receptors contained in iPSCs or their derived cells that contain IL15 enable IL15 signaling. - / - CD38 - / - (and optionally CIITA - / - In some embodiments of the iPSCs comprising IL15, IL15 expression is via a construct described herein. - / - CD38 - / - (and optionally CIITA - / - The iPSCs and derived cells thereof that contain B2M ILs can autonomously maintain or improve cell proliferation, growth, proliferation, and / or effector function without contact with additionally supplied soluble cytokines in vitro or in vivo. In some embodiments, B2M - / - CD38 - / - The IL-containing iPSCs and their derived effector cells are HLA-II intact and have synergistically increased persistence and / or survival in the presence of activated recipient T cells, B cells, and NK cells. When anti-CD38 antibodies are used with the derived effector cells in combination therapy, the cells have synergistically increased persistence, survival, and effector function.

[0172] Also provided are iPSCs comprising a B2M knockout, a CD38 knockout, and optionally one or more of CIITA knockout, IL, CAR, and hnCD16, which are capable of directed differentiation to produce functional derived hematopoietic cells without the need for HLA-G and / or HLA-E expression to overcome alloreactive NK cells. In some embodiments, the derived hematopoietic cells include mesodermal cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 +These include, but are not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, bone marrow cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. iPSCs and their derived effector cells can be used with anti-CD38 antibodies to induce ADCC without effector cell elimination or allorejection by activated recipient T cells, recipient B cells, and recipient NK cells, thereby increasing the persistence and / or survival of iPSCs and their effector cells. In some embodiments, the effector cells have increased persistence and / or survival in vivo.

[0173] Also provided herein, as described above, are iPSCs or iPSC-derived cells, which further comprise a truncated fusion protein of IL15 and IL15Rα, which fusion protein does not comprise an intracellular domain. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 8, 9, or 10. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the truncated IL15 / IL15Rα fusion protein lacking the intracellular domain comprises the amino acid sequence of SEQ ID NO: 10. In yet other embodiments, the iPSCs or iPSC-derived cells comprising a truncated IL15 / IL15Rα fusion protein lacking the intracellular domain (IL15Δ) further comprise one or more of B2M knockout, CIITA knockout, CD38 knockout, hnCD16, CAR, and an exogenous cytokine signaling complex, and the iPSCs are capable of directed differentiation to produce functional derived hematopoietic cells, including mesoderm cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34+ These include, but are not limited to, hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, or derived effector cells that have one or more functional attributes not present in the corresponding primary T, NK, NKT, and / or B cells.

[0174] Thus, the present application provides iPSCs and their functional derived hematopoietic cells comprising any one of the following genotypes in Table 1. As provided in Table 1, unless specified as IL15Δ, "IL" represents any one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which particular cytokine signaling complex expression is selected. Furthermore, when the iPSCs and their functional derived hematopoietic cells have a genotype that includes both a CAR and an IL, the CAR and the IL can be included in a bicistronic expression cassette that includes a 2A sequence. In contrast, in some other embodiments, the CAR and the IL are in separate expression cassettes included in the iPSCs and their functional derived hematopoietic cells. In one particular embodiment, it is IL15 that is included in the iPSCs and their functional derived effector cells that express both a CAR and an IL, and the IL15 construct is included in an expression cassette together with the CAR or separately from the CAR.

[0175] [Table 11-1]

[0176] [Table 11-2]

[0177] 7. Additional Modifications In some embodiments, the genetic modification modalities include one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or survival rate of iPSCs or their derived cells. In some embodiments, the genetically modified iPSCs and their derived cells comprise the genotypes listed in Table 1. In some embodiments, iPSCs and derived effector cells thereof comprising any one of the genotypes in Table 1 contain a deletion or disruption of at least one of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or a deletion or disruption of at least one of HLA-E, HLA-G, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A, B, C, D ... 2A The method may additionally include the introduction of at least one of a surface triggering receptor for coupling to an R, a TCR, an Fc receptor, an antibody or functional variant or fragment thereof, a checkpoint inhibitor, and a bispecific, multispecific or universal engager.

[0178] II. Methods for targeted genome editing at selected loci in iPSCs Genome editing, or genome editing, or gene editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and / or replaced in the genome of a target cell. Targeted genome editing (interchangeable with "targeted genome editing" or "targeted gene editing") allows insertion, deletion, and replacement at preselected sites in the genome. When endogenous sequences are deleted at the insertion site during targeted editing, endogenous genes containing the affected sequences can be knocked out or knocked down by sequence deletion. Thus, targeted editing can also be used to precisely disrupt endogenous gene expression. The term "targeted integration," as also used herein, refers to a process that involves the insertion of one or more exogenous sequences, with or without the deletion of endogenous sequences at the insertion site. In contrast, randomly integrated genes are susceptible to position effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomeric regions are particularly prone to transgene silencing. The newly integrated genes may affect the surrounding endogenous genes and chromatin, altering cell behavior or promoting cell transformation. Therefore, inserting foreign DNA into preselected loci, such as safe harbor loci or genomic safe harbor (GSH), is important for safety, efficiency, copy number control, and reliable gene response regulation.

[0179] Targeted editing can be achieved by either a nuclease-independent approach or a nuclease-dependent approach, in which homologous recombination is guided by homologous sequences flanking the exogenous polynucleotide to be inserted via the host cell's enzymatic machinery.

[0180] Alternatively, targeted editing can be achieved at higher frequencies by specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing exploits DNA repair mechanisms including non-homologous end joining (NHEJ) that occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in random insertion or deletion (indels) of a small number of endogenous nucleotides. In contrast, in the presence of a donor vector containing exogenous genetic material flanked by a pair of homology arms, exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in "targeted integration". In some situations, the targeted integration site is intended to be within the coding region of a selected gene, and thus targeted integration can disrupt gene expression and result in simultaneous knock-in and knock-out (KI / KO) in one single editing step.

[0181] One or more transgenes can be inserted into selected positions in the locus of interest (GOI) to achieve simultaneous gene knockout. Suitable loci for simultaneous knock-in and knockout (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. Each site-specific targeting homology arm for site-selective insertion allows the transgene to be expressed either under the endogenous promoter of the site or under the exogenous promoter included in the construct. When two or more transgenes are inserted into the selected position of the CD38 locus, a linker sequence, such as a 2A linker or an IRES, is placed between any two transgenes. The 2A linker encodes, for example, a self-cleaving peptide (referred to as "F2A", "E2A", "P2A" and "T2A", respectively) derived from FMDV, ERAV, PTV-I or TaV, allowing separate proteins to be produced from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. In various embodiments, the exogenous promoter can be CAG or other constitutive, inducible, time-specific, tissue-specific or cell type-specific promoter, including but not limited to CMV, EF1α, PGK and UBC.

[0182] Available endonucleases that can introduce specific targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems. In addition, the DICE (dual integrase cassette exchange) system, which utilizes phiC31 and Bxb1 integrases, is also a promising tool for targeted integration.

[0183] ZFNs are targeted nucleases that contain a nuclease fused to a zinc finger DNA binding domain. "Zinc finger DNA binding domain" or "zinc finger DNA binding domain, 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 about 30 amino acids within the 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, the C2H2 zinc finger, the C3H zinc finger, and the C4 zinc finger. A "designed" zinc finger domain is a domain that does not occur in nature, whose design / construction results primarily from rational criteria, such as the application of substitution rules and computerized algorithms to process information from databases that store information on existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081, 6,453,242 and 6,534,261, and also WO 98 / 53058, 98 / 53059, 98 / 53060, 02 / 016536 and 03 / 016496, the complete disclosures of which are incorporated herein by reference. A "selected" zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trapping or hybrid selection. ZFNs are described in detail in U.S. Patent Nos. 7,888,121 and 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of ZFN in the art is the fusion of FokI nuclease with a zinc finger DNA binding domain.

[0184] TALENs are targeted nucleases that contain a nuclease fused to a TAL effector DNA binding domain. "Transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain", or "TALE DNA binding domain" refers to the polypeptide domain of a TAL effector protein that is involved in binding the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via the transactivation domain. The specificity of the TAL effector DNA binding domain depends on the effector variable number of imperfect 34 amino acid repeats that contain polymorphisms at selected repeat positions called repeat variable-diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application No. 2011 / 0145940, which is incorporated herein by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of a FokI nuclease to a TAL effector DNA binding domain.

[0185] Another example of a targeted nuclease for use in the methods of the present invention is a targeted Spo11 nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA binding domain having specificity for a DNA sequence of interest, such as a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc.

[0186] Further examples of targeted nucleases suitable for embodiments of the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used individually or in combination.

[0187] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases, CRISPR-associated nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr, restriction endonucleases, meganucleases, homing endonucleases, and the like.

[0188] Using Cas9 as an example, CRISPR / Cas9 requires two major components: (1) the Cas9 endonuclease and (2) the crRNA-tracrRNA complex. When co-expressed, the two components form a complex and are recruited to a target DNA sequence containing a PAM and a PAM-proximal seeding region. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target a selected sequence. These two components can then be delivered into mammalian cells via transfection or transduction.

[0189] DICE-mediated insertion uses a pair of recombinases, e.g., phiC31 and Bxb1, to provide unidirectional integration of exogenous DNA that is tightly restricted by each enzyme's own small attB and attP recognition sites. These targeted att sites do not naturally occur in mammalian genomes and must first be introduced into the genome at the desired integration site. See, e.g., U.S. Patent Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0190] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct into a cell to allow site-specific homologous recombination by a cellular host enzyme machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a ZFN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to allow ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell, and introducing a TALEN expression cassette comprising a DNA binding domain specific to a desired integration site into the cell to allow TALEN-mediated insertion. In another embodiment, a method of targeted integration in a cell includes introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell a gRNA comprising a Cas9 expression cassette and a guide sequence specific for a desired integration site to allow insertion via Cas9. In yet another embodiment, a method of targeted integration in a cell includes introducing into the cell a construct comprising one or more att sites for a pair of DICE recombinases at a desired integration site, introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing an expression cassette for DICE recombinase to allow targeted integration via DICE.

[0191] Potential sites for targeted integration include, but are not limited to, safe harbor loci, or genomic safe harbors (GSH), which are intragenic or extragenic regions of the human genome that, in theory, can accommodate predictable expression of the newly integrated DNA without adverse effects on the host cell or organism. A useful safe harbor should allow sufficient transgene expression to produce the desired levels of vector-encoded protein or non-coding RNA. A safe harbor should also not predispose cells to malignant transformation or alter cellular function. For an integration site to be a potential safe harbor locus, it should ideally meet criteria including but not limited to: absence of disruption of regulatory elements or genes as judged by sequence annotation, an intergenic region within a gene-dense region or a convergent position between two genes transcribed in opposite directions, distances that minimize the possibility of long-range interactions between vector-encoded transcriptional activators and promoters of adjacent genes, particularly cancer-associated and microRNA genes, and apparently ubiquitous transcriptional activity as reflected by widespread spatial and temporal expressed sequence tag (EST) expression patterns that indicate ubiquitous transcriptional activity. This latter attribute is particularly important in stem cells, where chromatin remodeling during differentiation typically results in the silencing of some loci and the potential activation of others. Within a region favorable for exogenous insertion, the precise locus selected for insertion should be devoid of repetitive elements and conserved sequences, allowing easy design of primers for amplification of homology arms.

[0192] Suitable sites for human genome editing, or specifically targeted integration, include, but are not limited to, the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the human orthologue of the mouse ROSA26 locus. In addition, the human orthologue of the mouse H11 locus may also be a suitable site for insertion using the compositions and methods of targeted integration disclosed herein. Furthermore, the collagen and HTRP loci may also be used as safe harbors for targeted integration. However, validation of each selected site has been shown to be necessary, especially in stem cells, for certain integration events, and optimization of the insertion strategy, including promoter selection, exogenous gene sequence and positioning, and construct design, is often required.

[0193] For targeted indels, the editing site is often contained in the endogenous gene whose expression and / or function is intended to be disrupted. In some embodiments, the endogenous gene containing the targeted indel is associated with immune response regulation and modification. In some other embodiments, the endogenous gene containing the targeted indel is associated with targeting modalities, receptors, signaling molecules, transcription factors, potential drug targets, immune response regulation and modification, or proteins that inhibit the engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of stem and / or progenitor cells and cells derived therefrom.

[0194] Thus, one aspect of the invention provides a method of targeted integration at selected loci that include a genomic safe harbor, or pre-selected loci that are known or proven to be safe and well-regulated for continuous or transient gene expression, such as the TRAC and TRBC loci, as provided herein. In one embodiment, the genomic safe harbor for the method of targeted integration includes one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR (TRAC or TRBC), or RUNX1, or other loci that meet the criteria of a genomic safe harbor. In one embodiment, a method of targeted integration into a cell comprising introducing into the cell a construct comprising one or more exogenous polynucleotides, and introducing into the cell a construct comprising a pair of homology arms specific to a desired integration site and one or more exogenous sequences to enable site-specific homologous recombination by the cellular host enzymatic machinery, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR, or RUNX1, or other locus that meets the criteria of the genomic safe harbor. Further integration sites include endogenous loci intended for disruption, e.g., reduction or knockout, including B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0195] In another embodiment, a method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a ZFN expression cassette comprising a DNA binding domain specific for a desired integration site to allow for ZFN-mediated insertion, the desired integration site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method of targeted integration in a cell includes introducing a construct comprising one or more exogenous polynucleotides into the cell and introducing a TALEN expression cassette comprising a DNA binding domain specific for a desired integration site into the cell to allow TALEN-mediated insertion, the desired integration site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.In another embodiment, a method of targeted integration in a cell comprises introducing into the cell a construct comprising one or more exogenous polynucleotides and introducing into the cell a Cas9 expression cassette specific for a desired integration site and a gRNA comprising a guide sequence to allow for Cas9-mediated insertion, the desired integration site comprising AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinase into a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides into the cell, and introducing an expression cassette for DICE recombinase to enable DICE-mediated targeted integration, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0196] Further, as provided herein, the above-mentioned methods for targeted integration with safe harbors are used to insert polynucleotides of interest, such as polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, and proteins that promote stem cell and / or progenitor cell engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival. In some other embodiments, the constructs comprising one or more exogenous polynucleotides further comprise one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the invention is a suicide gene encoding a safety switch protein. Suitable suicide gene systems for induced cell death include, but are not limited to, caspase 9 (or caspase 3 or 7) and AP1903, thymidine kinase (TK) and ganciclovir (GCV), cytosine deaminase (CD) and 5-fluorocytosine (5-FC). In addition, some suicide gene systems are cell type specific, for example, genetic modification of T lymphocytes with the B cell molecule CD20 can eliminate them upon administration of the mAb rituximab. Furthermore, modified EGFR containing an epitope recognized by cetuximab can be used to deplete genetically engineered cells when the cells are exposed to cetuximab. Thus, one aspect of the present invention provides a method for targeted integration of one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0197] In some embodiments, one or more exogenous polynucleotides integrated by the methods described herein are driven by an operably linked exogenous promoter included in the construct for targeted integration. The promoter may be inducible or constitutive, and may be time-specific, tissue-specific, or cell-type specific. Constitutive promoters suitable for the methods of the present invention include, but are not limited to, cytomegalovirus (CMV), elongation factor 1 alpha (EF1 alpha), 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 integrated by the methods described herein may be driven by endogenous promoters in the host genome at the integration site. In one embodiment, the methods described herein are used for targeted integration of one or more exogenous polynucleotides at the AAVS1 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the methods described herein are used for targeted integration at the ROSA26 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the methods described herein are used for targeted integration at the H11 locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous H11 promoter. In another embodiment, the methods described herein are used for targeted integration at the collagen locus in the genome of a cell. In one embodiment, at least one integrated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method described herein is used for targeted integration at the HTRP locus in the genome of a cell.In one embodiment, at least one integrated polynucleotide is driven by endogenous HTRP promoter.Theoretically, only correct insertion at the desired position will allow gene expression of exogenous gene driven by endogenous promoter.

[0199] In some embodiments, one or more exogenous polynucleotides included in the construct for the method of targeted integration are driven by one promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to increase the physical separation between the moieties and maximize access to the enzymatic machinery. The linker peptide of the linker sequence may be composed of amino acids selected to make the physical separation between the moieties (exogenous polynucleotides and / or proteins or peptides encoded therefrom) more flexible or more rigid, depending on the function involved. The linker sequence may be cleavable by a protease or may be chemically cleavable to yield separate moieties. 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 one that is naturally produced by the host or is exogenously introduced. Alternatively, the cleavage site in the linker may be one that can be cleaved upon exposure to a selected chemical, such as cyanogen bromide, hydroxylamine, or low pH. The optional linker sequence may serve a purpose other than providing a cleavage site. The linker sequence should allow for effective positioning of a moiety relative to another adjacent moiety in order for the moiety to function properly. The linker may also be a simple amino acid sequence of sufficient length to prevent any steric hindrance between the moieties. In addition, the linker sequence may provide for post-translational modifications including, but not limited to, phosphorylation sites, biotinylation sites, sulfation sites, gamma-carboxylation sites, and the like. In some embodiments, the linker sequence is flexible so as not to hold the biologically active peptide in a single undesired conformation. The linker may be comprised primarily of amino acids with small side chains, such as glycine, alanine, and serine, to provide flexibility. In some embodiments, about 80-90 percent or more of the linker sequence includes glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, a G4S linker peptide separates the terminal processing and endonuclease domains of the fusion protein. In other embodiments, a 2A linker sequence allows two separate proteins to be produced from a single translation. Suitable linker sequences can be easily identified empirically. In addition, suitable sizes and sequences of linker sequences 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 site (IRES). In some embodiments, any two consecutive linker sequences are different.

[0200] The method of introducing the construct containing the exogenous polynucleotide for targeted integration into the cell can be achieved using methods of gene transfer into cells known per se. In one embodiment, the construct comprises the backbone of a viral vector, such as an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a Sendai viral vector. In some embodiments, a plasmid vector is used to deliver and / or express the exogenous polynucleotide into the target cell (e.g., pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.). In some other embodiments, an episomal vector is used to deliver the exogenous polynucleotide into the target cell. In some embodiments, recombinant adeno-associated viruses (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV do not integrate into the host genome. In addition, episomal rAAV vectors mediate a much higher rate of homologous tropism gene targeting compared to traditional targeting plasmid transfection. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the genome of iPSCs. In some embodiments, the genomically modified iPSCs and their derived cells obtained using the methods and compositions described herein comprise at least one genotype listed in Table 1.

[0201] III. Methods for Obtaining and Maintaining Genome-Engineered iPSCs In various embodiments, the invention provides methods for obtaining and maintaining genomically engineered iPSCs that contain one or more targeted edits at one or more desired sites, where the one or more targeted edits remain intact and functional at each selected edit site in the expanded genomically engineered iPSCs or iPSC-derived non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions (i.e., targeted integrations and / or indels at selected sites) into the genome of the iPSCs and cells derived therefrom. Many advantages of obtaining genomically engineered derivative cells derived through editing and differentiation of iPSCs provided herein compared to direct manipulation of patient-derived peripheral blood-derived primary effector cells include an unlimited source of engineered effector cells, no need for repeated manipulation of effector cells, especially when multiple engineered modalities are involved, the resulting effector cells have extended telomeres and are younger due to less attrition, and the effector cell population is homogenous with respect to editing sites, copy number, and lack of allelic variation, random mutations, and expression variegation, primarily due to the ability to perform clonal selection in the engineered iPSCs provided herein.

[0202] In certain embodiments, genomically 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 a cell culture medium as shown in Table 2 as Fate Maintenance Medium (FMM), in which the iPSCs retain the targeted edits and functional modifications at the selected sites. The components of the medium may be present in the medium in amounts within the optimal ranges shown in Table 2. iPSCs cultured in FMM have been shown to continue to maintain their undifferentiated profile and basal or naive profile; provide genomic stability without the need for culture washing or selection, and readily give rise to all three somatic lineages, i.e., in vitro differentiation via embryoid bodies or monolayers (without formation of embryoid bodies), and in vivo differentiation by teratoma formation. See, e.g., WO 2015 / 134652, the disclosure of which is incorporated herein by reference.

[0203] [Table 12]

[0204] In some embodiments, genomically engineered iPSCs containing one or more targeted integrations and / or indels are maintained, passaged, or expanded in medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and free or essentially free of a TGFβ receptor / ALK5 inhibitor, wherein the iPSCs retain intact and functional targeted edits at the selected sites.

[0205] Another aspect of the invention provides a method of generating genomically engineered iPSCs through targeted editing of iPSCs or by first generating genomically engineered non-pluripotent cells by targeted editing and then reprogramming the selected / isolated genomically engineered non-pluripotent cells to obtain iPSCs containing the same targeted edit as the non-pluripotent cells. A further aspect of the invention provides genomically engineered non-pluripotent cells undergoing simultaneous reprogramming by introducing targeted integrations and / or targeted indels into the cells, the contacted non-pluripotent cells being under conditions sufficient for reprogramming, the reprogramming conditions comprising contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genomic engineering and reprogramming, the targeted integrations and / or targeted indels can be introduced into the non-pluripotent cells prior to initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally one or more small molecules, or essentially simultaneously.

[0206] In some embodiments, for simultaneous genomic engineering and reprogramming of non-pluripotent cells, targeted integrations and / or indels may also be introduced into non-pluripotent cells after the multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and vectors carrying the constructs are introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181 and CD30.

[0207] In some embodiments, reprogramming is initiated by contacting the non-pluripotent cells with at least one reprogramming factor, and optionally a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 2). In some embodiments, the genomically engineered iPSCs produced by any of the methods described above are further maintained and expanded using a mixture comprising a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 2).

[0208] In some embodiments of the method of generating genomically engineered iPSCs, the method comprises introducing one or more targeted integrations and / or indels into the iPSCs to genomically engineer the iPSCs to obtain a genomically engineered iPSC having at least one genotype listed in Table 1. Alternatively, the method of generating genomically engineered iPSCs comprises (a) introducing one or more targeted edits into a non-pluripotent cell to obtain a genomically engineered non-pluripotent cell comprising a targeted integration and / or indel at a selected site, and (b) contacting the genomically engineered non-pluripotent cell with one or more reprogramming factors, and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor, to obtain a genomically engineered iPSC comprising a targeted integration and / or indel at a selected site. Alternatively, a method of generating genomically engineered iPSCs includes: (a) contacting a non-pluripotent cell with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cell; (b) introducing one or more targeted integrations and / or indels into the reprogrammed non-pluripotent cell for genome engineering; and (c) obtaining a clonal genomically engineered iPSC that contains the targeted integrations and / or indels at the selected site. Any of the above methods may further include single cell sorting of the genomically engineered iPSCs to obtain clonal iPSCs, and / or screening for off-target editing and abnormal karyotypes in the genomically engineered iPSCs. Through clonal expansion of the genomically engineered iPSCs, a master cell bank is generated to include single cell sorted and expanded clonally engineered iPSCs having at least one phenotype as provided herein. The master cell bank is subsequently cryopreserved, providing a platform for further iPSC manipulation and a renewable source for manufacturing off-the-shelf engineered homogenous cell therapy products that are compositionally defined and uniform and can be mass-produced at significant scale in a cost-effective manner.

[0209] The reprogramming factor is 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 WO 2015 / 134652 and WO 2017 / 066634, the disclosures of which are incorporated herein by reference. The one or more reprogramming factors may be in the form of a polypeptide. The reprogramming factor may also be in the form of a polynucleotide encoding the reprogramming factor, and thus may be introduced into the non-pluripotent cell by vectors such as retrovirus, Sendai virus, adenovirus, episomes, plasmids, and minicircles. In certain embodiments, the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, the one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by plasmid combination. For example, see International Publication No. WO2019 / 075057(A1), the disclosure of which is incorporated herein by reference.

[0210] In some embodiments, non-pluripotent cells are transfected with multiple constructs comprising 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 targeting modalities, receptors, signaling molecules, transcription factors, pharmacologic active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, and / or survival of iPSCs or derived cells. In some embodiments, the exogenous polynucleotides encode RNAs, including but not limited to siRNAs, shRNAs, miRNAs, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue-specific or cell type-specific promoters. Thus, the polynucleotides are expressible under conditions that activate the promoter, for example, in the presence of an inducer, or in a specific differentiated cell type. In some embodiments, the polynucleotides are expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, the 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 transfected simultaneously or sequentially into non-pluripotent cells. Non-pluripotent cells subjected to targeted integration of multiple constructs can be contacted with one or more reprogramming factors simultaneously to initiate reprogramming simultaneously with genome manipulation, thereby obtaining genomically engineered iPSCs comprising multiple targeted integrations in the same pool of cells. Thus, this robust method allows for the derivation of clonal genomically engineered hiPSCs with multiple modalities integrated at one or more selected target sites by simultaneous reprogramming and manipulation strategies. In some embodiments, the genomically modified iPSCs and derived cells thereof obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0211] IV. Methods for obtaining genetically engineered effector cells by differentiating genomically engineered iPSCs Further aspects of the invention provide methods of in vivo differentiation of genomically engineered iPSCs by teratoma formation, where differentiated cells derived in vivo from genomically engineered iPSCs retain intact and functional targeted edits, including targeted integrations and / or indels, at desired sites. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma formation contain one or more inducible suicide genes integrated at one or more desired sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta2 microglobulin, CD38, GAPDH, TCR, or RUNX1, or other loci that meet the criteria of the genomic safe harbor. In some other embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma formation contain polynucleotides encoding targeting modalities or polynucleotides encoding proteins that promote trafficking, homing, viability, self-renewal, persistence, and / or survival of stem and / or progenitor cells. In some embodiments, differentiated cells derived in vivo from genomically engineered iPSCs via teratoma formation containing one or more inducible suicide genes further comprise one or more indels in endogenous genes associated with regulating and mediating immune responses. In some embodiments, the indels are comprised in one or more endogenous checkpoint genes. In some embodiments, the indels are comprised in one or more endogenous T cell receptor genes. In some embodiments, the indels are comprised in one or more endogenous MHC class I suppressor genes. In some embodiments, the indels are comprised in one or more endogenous genes associated with the major histocompatibility complex.In some embodiments, the indels are contained in one or more endogenous genes, including, but not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. In one embodiment, the genomically engineered iPSCs comprising one or more exogenous polynucleotides at selected sites further comprise a targeted edit in the gene encoding B2M (beta-2-microglobulin).

[0212] In certain embodiments, genomically engineered iPSCs containing one or more genetic modifications provided herein are used to induce hematopoietic cell lineages or other specific cell types in vitro, and the induced non-pluripotent cells retain the functional genetic modification comprising the targeted edit at the selected site. In one embodiment, the genomically engineered iPSC-derived cells are mesoderm cells with definitive hemogenic endothelial (HE) potential, definitive HE, CD34 + Cells derived from genomically engineered iPSCs, including but not limited to hematopoietic cells, hematopoietic stem progenitor cells, hematopoietic multipotent progenitors (MPPs), T cell precursors, NK cell precursors, myeloid cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, retain the functional genetic modification including the targeted edit at the desired site.

[0213] Applicable differentiation methods and compositions for obtaining iPSC-derived hematopoietic cell lineages include, for example, those set forth in WO 2017 / 078807, the disclosure of which is incorporated herein by reference. See WO 2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, methods and compositions for generating hematopoietic cell lineages through definitive hemogenic endothelium (HE) derived from pluripotent stem cells, including iPSCs, in a culture platform that is scalable and does not require monolayer EB formation under serum-free, feeder-free, and / or stroma-free conditions. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated and transdifferentiated cells, and cells of various lineages that have transitioned directly to a hematopoietic fate without passing through a pluripotent intermediate. Similarly, cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells and all intervening hematopoietic cell lineages.

[0214] A method for differentiating and expanding cells of hematopoietic lineage from pluripotent stem cells in monolayer culture includes contacting the pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, mesodermal cells derived from pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then subjected to contact with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with definitive hemogenic endothelial (HE) potential without forming embryoid bodies from the pluripotent stem cells. Subsequent contact with bFGF, and optionally with a ROCK inhibitor, and / or a WNT pathway activator, differentiates the mesodermal cells with definitive HE potential into definitive HE cells, which are also expanded during differentiation.

[0215] The methods for obtaining cells of hematopoietic lineage provided herein are superior to EB-mediated pluripotent stem cell differentiation because EB formation results in modest to minimal cell proliferation, does not allow for monolayer culture, which is important for many applications requiring uniform proliferation, and uniform differentiation of cells within a population, making it laborious and less efficient.

[0216] The provided monolayer differentiation platform promotes differentiation into definitive hemogenic endothelium resulting in the induction of hematopoietic stem cells and differentiated progeny such as T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale expansion, allowing the delivery of therapeutically relevant numbers of pluripotent stem cell-derived hematopoietic cells for a variety of therapeutic applications. Furthermore, monolayer culture using the methods provided herein results in functional hematopoietic lineage cells capable of a full range of in vitro differentiation, in vivo modification, and in vivo long-term hematopoietic self-renewal, reconstitution, and engraftment. As provided, iPSC-derived hematopoietic lineage cells include, but are not limited to, definitive hemogenic endothelium, hematopoietic multipotent progenitor cells, hematopoietic stem cells and progenitors, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0217] Thus, in various embodiments, a method for directing differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage comprises: (i) contacting the pluripotent stem cells with a composition comprising a BMP activator, and optionally bFGF, to initiate differentiation and proliferation of mesodermal cells from the pluripotent stem cells; and (ii) contacting the mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, to initiate differentiation and proliferation of mesodermal cells with definitive HE potential from the mesodermal cells, wherein the composition optionally comprises a TGFβ receptor activator. and (iii) contacting mesodermal cells having definitive HE potential with a composition comprising a ROCK inhibitor, bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator, to initiate differentiation and proliferation of definitive hemogenic endothelium from pluripotent stem cell-derived mesodermal cells having definitive hemogenic endothelial potential, wherein the composition optionally does not comprise a TGFβ receptor / ALK inhibitor.

[0218] In some embodiments, the method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not including a TGFβ receptor / ALK inhibitor, and seeding and expanding the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs comprising one or more genetic imprints, and the one or more genetic imprints comprised in the iPSC are retained in hematopoietic cells differentiated therefrom. In some embodiments of the method for directing differentiation of pluripotent stem cells into cells of a hematopoietic lineage, the differentiation of the pluripotent stem cells into cells of a hematopoietic lineage is in a monolayer culture form without the generation of embryoid bodies.

[0219] In some embodiments of the above-described methods, the obtained pluripotent stem cell-derived definitive hemogenic endothelial cells are CD34 + In some embodiments, the obtained definitive hemogenic endothelial cells are CD34 + CD43 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CD43 - CXCR4 - CD73 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CXCR4 - CD73 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CD43 - CD93 - In some embodiments, the definitive hemogenic endothelial cells are CD34 + CD93 - It is.

[0220] In some embodiments of the above-described method, the method further comprises: (i) contacting the pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, and optionally a BMP activator, to initiate differentiation of the definitive hemogenic endothelium into a pre-T cell precursor, and optionally (ii) contacting the pre-T cell precursor with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not including one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to initiate differentiation of the pre-T cell precursor into a T cell precursor or a T cell. In some embodiments of the method, the pluripotent stem cell-derived T cell precursor is CD34 + CD45 + CD7 + In some embodiments of this method, the pluripotent stem cell-derived T cell precursors are CD45 + CD7 + It is.

[0221] In some further embodiments of the above-described methods for directing differentiation of pluripotent stem cells into cells of a hematopoietic lineage, the method further comprises: (i) contacting the definitive hemogenic endothelium derived from the 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, IL3, IL7, and IL15, and optionally a BMP activator, to initiate differentiation of the definitive hemogenic endothelium into pre-NK cell precursors, and optionally (ii) contacting the pre-NK cell precursors derived from the pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and a medium free of one or more of VEGF, bFGF, TPO, a BMP activator, and a ROCK inhibitor, to initiate differentiation of the pre-NK cell precursors into NK cell precursors or NK cells. In some embodiments, the pluripotent stem cell-derived NK precursors are CD3 - CD45 + CD56 + CD7+ In some embodiments, the pluripotent stem cell-derived NK cells are CD3 - CD45 + CD56 + and, if necessary, NKp46 + , CD57 + and CD16 + The present invention is further defined by:

[0222] Thus, using the above differentiation methods, one or more of the following populations of iPSC-derived hematopoietic cells can be obtained: (i) CD34 iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2, using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; + In some embodiments, the medium is: HE cells (iCD34), (ii) definitive hemogenic endothelium (iHE), using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2, (iii) definitive HSC, 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 precursors (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 precursors (ipro-NK), using one or more culture media selected from iNK-A2 and iNK-B2, and / or (viii) NK cells (iNK), and iNK-B2. a. iCD34-C comprises a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator, and does not comprise a TGFβ receptor / ALK inhibitor. b. iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. c. iTC-A2 comprises a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, and IL7, and optionally a BMP activator. d. iTC-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. e. iNK-A2 comprises a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator. f. iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0223] In some embodiments, the genomically engineered iPSC-derived cells resulting from the above methods comprise one or more inducible suicide genes integrated at one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR alpha or beta constant regions, NKG2A, NKG2D, CD25, CD38, CD44, CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or other loci that meet the criteria for the genomic safe harbor. In some other embodiments, the genomically engineered iPSC-derived cells comprise polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, potential drug targets, or proteins that promote stem and / or progenitor cell trafficking, homing, viability, self-renewal, persistence, and / or survival. In some embodiments, the genomically engineered iPSC-derived cells comprising one or more suicide genes further comprise one or more indels in one or more endogenous genes associated with immune response regulation and mediation, including, but not limited to, checkpoint genes, endogenous T cell receptor genes, and MHC class I suppressor genes. In one embodiment, the genomically engineered iPSC-derived cells comprising one or more suicide genes further comprise an indel in the B2M gene, where B2M is knocked out.

[0224] In addition, applicable dedifferentiation methods and compositions for obtaining second fate genomically engineered hematopoietic cells from first fate genomically engineered hematopoietic cells include, for example, those shown in WO 2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided therein allow for partial reprogramming of starting non-pluripotent cells into non-pluripotent intermediate cells by limiting expression of the endogenous Nanog gene during reprogramming, and subjecting the non-pluripotent intermediate cells to conditions for differentiating the intermediate cells into a desired cell type. In some embodiments, the genomically modified iPSCs and their derived cells obtained using the methods and compositions herein comprise at least one genotype listed in Table 1.

[0225] V. Therapeutic Uses of Derived Immune Cells with Functional Modalities Differentiated from Genetically Engineered iPSCs The invention provides, in some embodiments, compositions comprising an isolated population or subpopulation of functionally enhanced derived immune cells differentiated from genomically engineered iPSCs using the disclosed methods and compositions. In some embodiments, the iPSCs comprise one or more targeted gene edits that can be retained in iPSC-derived effector 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 effector cells comprises iPSC-derived CD34 +In one embodiment, the isolated population or subpopulation of engineered effector cells comprises iPSC-derived HSC cells. In one embodiment, the isolated population or subpopulation of engineered effector cells comprises iPSC-derived pro-T cells or T cells. In one embodiment, the isolated population or subpopulation of engineered effector cells comprises iPSC-derived pro-NK cells or NK cells. In one embodiment, the isolated population or subpopulation of engineered effector cells comprises iPSC-derived immunomodulatory cells or myeloid-derived suppressor cells (MDSCs). In some embodiments, the iPSC-derived engineered effector cells are further conditioned ex vivo for improved therapeutic potential. In one embodiment, the isolated population or subpopulation of engineered effector cells derived from iPSCs comprises an increased number or proportion of naive T cells, stem cell memory T cells, and / or central memory T cells. In one embodiment, the isolated population or subpopulation of engineered effector cells derived from iPSCs comprises an increased number or proportion of type I NKT cells. In another embodiment, the isolated population or subpopulation of engineered effector cells derived from iPSCs comprises an increased number or proportion of adaptive NK cells. In some embodiments, the isolated population or subpopulation of engineered effector cells derived from iPSCs comprises an increased number or proportion of adaptive NK cells. + The isolated population or subpopulation of cells, HSC cells, T cells, NK cells, or myeloid-derived suppressor cells is allogeneic. In some other embodiments, the isolated population or subpopulation of cells, HSC cells, T cells, NK cells, or myeloid-derived suppressor cells is allogeneic. + The isolated population or subpopulation of cells, HSC cells, T cells, NK cells, or MDSCs is autologous.

[0226] In some embodiments, the iPSCs for differentiation contain genetic imprints selected to convey desired therapeutic attributes in derived effector cells, provided that cellular developmental biology is not disrupted during differentiation and that the genetic imprints are retained and functional in differentiated hematopoietic cells derived from the iPSCs.

[0227] In some embodiments, the genetic imprint of the pluripotent stem cells comprises (i) one or more genetic modification modalities obtained through genomic insertion, deletion or substitution in the genome of the pluripotent cells during or after reprogramming of non-pluripotent cells to iPSCs, or (ii) one or more retainable therapeutic properties of source-specific immune cells that are donor, disease or therapeutic response specific, where the pluripotent cells are reprogrammed from source-specific immune cells and the iPSCs retain the source therapeutic property, which is also contained in the iPSC-derived hematopoietic lineage cells.

[0228] In some embodiments, the genetic modification modalities include one or more of safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharma- ceutically active proteins and peptides, drug target candidates, or proteins that promote engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or survival of iPSCs or derived cells thereof. In some embodiments, the genetically modified iPSCs and derived cells thereof comprise a genotype listed in Table 1. In some other embodiments, the genetically modified iPSCs and derived cells thereof comprise a genotype listed in Table 1, including (1) a deletion or disruption of one or more of NLRC5, PD1, LAG3, and TIM3, and (2) a deletion or disruption of one or more of HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A Further included are additional genetic modification modalities including the introduction of surface triggering receptors for coupling with R, CAR, TCR, Fc receptors, or bispecific or multispecific engagers or universal engagers.

[0229] In yet some other embodiments, the iPSC-derived hematopoietic lineage cells comprise therapeutic attributes of source-specific immune cells in relation to a combination of at least two of the following: (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) resistance to the tumor microenvironment, (iv) recruitment of bystander immune cells and immune modulation, (iv) improved on-target specificity with reduced extra-tumor effects, and (v) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0230] In some embodiments, the iPSC-derived hematopoietic cells comprise a genotype listed in Table 1, and the cells express at least one cytokine and / or its receptor, including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, or any modified protein thereof, and express at least a CAR. In some embodiments, the engineered expression of the cytokine and CAR is NK cell specific. In some other embodiments, the engineered expression of the cytokine and CAR is T cell specific. In some embodiments, the iPSC-derived hematopoietic effector cells are antigen specific. In some embodiments, the antigen-specific derived effector cells target liquid tumors. In some embodiments, the antigen-specific derived effector cells target solid tumors. In some embodiments, the antigen-specific iPSC-derived hematopoietic effector cells are capable of rescuing tumor antigen escape.

[0231] Specifically, the present application provides methods of reducing or preventing allorejection of allogeneic effector cells by recipient activated immune cells in adoptive cell therapy, the methods comprising administering a combination therapy, the combination therapy comprising derived effector cells as described herein and an anti-CD38 therapeutic agent. In various embodiments, the derived effector cells are B2M - / - CD38 - / - (and optionally CIITA - / -), further comprising one or more of exogenous CD16, IL, CAR, antibodies, and any other modalities as shown in Table 1. In various embodiments, the anti-CD38 therapeutic of the combination therapy is an anti-CD38 antibody or fragment thereof. In some embodiments, the anti-CD38 antibody is daratumumab, isatuximab, or MOR202. In some embodiments, the anti-CD38 therapeutic is administered simultaneously with, prior to, or after administration of the derived effector cells. Thus, in some embodiments, the antibody is used in combination with a population of effector cells as described herein, by simultaneous or sequential administration to the subject. In other embodiments, such antibodies or fragments thereof can be expressed by the effector cells by genetically engineering the iPSCs with an exogenous polynucleotide sequence encoding the antibody or fragment thereof, and directing differentiation of the engineered iPSCs, as described herein. In some embodiments of the method, the allogeneic effector cells are iPSC-derived hematopoietic cells. In some embodiments of this method, the allogeneic effector cells are iPSC-derived T cells, NK cells, or NKT cells.

[0232] In further embodiments of the method of reducing or preventing allorejection of allogeneic effector cells by recipient activated immune cells in adoptive cell therapy, the method further comprises administering an antibody specific for the same or different upregulated surface protein as targeted by the CAR, and / or one or more additional therapeutic agents. In some embodiments of the method, the antibody comprises at least one of anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, anti-CD38 antibody, anti-CD25 antibody, anti-CD69 antibody, anti-CD71 antibody, anti-CD44 antibody, or any of their humanized or Fc-modified variants or fragments, functional equivalents, and biosimilars. In some embodiments of the therapeutic agent used in the methods, the therapeutic agent comprises a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a supplement thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

[0233] A variety of diseases can be ameliorated by introducing the derived effector cells of the invention into a subject suitable for adoptive cell therapy. In some embodiments, the iPSC-derived hematopoietic cells provided herein are for allogeneic adoptive cell therapy. Additionally, the invention provides, in some embodiments, therapeutic uses of the above-described therapeutic compositions and / or combination therapies by introducing the compositions into a subject suitable for adoptive cell therapy, the subject having an autoimmune disorder, hematological malignancy, solid tumor, or infection associated with HIV, RSV, EBV, CMV, adenovirus, or BK polyomavirus.

[0234] Examples of hematological malignancies include acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphomas, non-Hodgkin's lymphomas, and the like. Examples of solid cancers include, but are not limited to, cancers of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testis, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, and esophagus. Examples of various autoimmune diseases include alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes mellitus (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigoid / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary Examples of viral infections include, but are not limited to, biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's disease). Examples of viral infections include, but are not limited to, HIV- (human immunodeficiency virus), HSV- (herpes simplex virus), KSHV- (Kaposi's sarcoma-associated herpes virus), RSV- (respiratory syncytial virus), EBV- (Epstein-Barr virus), CMV- (cytomegalovirus), VZV (varicella zoster virus), adenovirus-, lentivirus-, and BK polyomavirus-associated diseases.

[0235] Treatment using the derived hematopoietic lineage cells of the embodiments disclosed herein can be performed upon presentation of symptoms or to prevent relapse. The terms "treating", "treatment" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of completely or partially preventing the disease, and / or therapeutic, in terms of partially or completely curing the disease and / or adverse effects resulting from the disease. As used herein, "treatment" encompasses any intervention of a disease in a subject, including: preventing the disease from arising in a subject susceptible to the disease but not yet diagnosed as having it, inhibiting the disease, i.e. arresting its development, or alleviating the disease, i.e. reversing the disease. Therapeutic agents or compositions can be administered before, during, or after the onset of the disease or injury. Treatment of ongoing diseases, where treatment stabilizes or reduces undesirable clinical symptoms in the patient, is also of particular interest. In certain embodiments, the subject in need of treatment has a disease, condition, and / or injury in which at least one associated symptom can be inhibited, ameliorated, and / or ameliorated by cell therapy. Certain embodiments contemplate that subjects in need of cell therapy include, but are not limited to, bone marrow or stem cell transplant candidates, subjects who have undergone chemotherapy or radiation therapy, subjects having or at risk of having a hyperproliferative disorder or cancer, e.g., a hyperproliferative disorder or cancer of the hematopoietic system, subjects having or at risk of developing a tumor, e.g., a solid tumor, subjects having or at risk of having a viral infection or a disease associated with a viral infection.

[0236] When assessing responsiveness to a treatment comprising the derived hematopoietic lineage cells of the embodiments disclosed herein, response can be measured by at least one of clinical benefit rate, survival to death, pathologic complete response, semi-quantitative measurement of pathologic response, clinical complete response, clinical partial response, clinical stable disease, recurrence-free survival, metastasis-free survival, disease-free survival, circulating tumor cell reduction, circulating marker response, and RECIST (Response Evaluation Criteria In Solid Tumors) criteria.

[0237] The therapeutic compositions comprising the iPSC-derived hematopoietic lineage cells disclosed herein can be administered to a subject before, during, and / or after other treatments. Thus, methods of combination therapy can include administration or preparation of iPSC-derived effector cells before, during, and / or after the use of additional therapeutic agents. As described above, the one or more additional therapeutic agents include peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, dsRNA (double-stranded RNA), mononuclear blood cells, feeder cells, feeder cell components or their supplements, vectors comprising one or more polynucleic acids of interest, antibodies, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). Administration of the iPSC-derived immune cells can be separated in time by hours, days, or weeks from administration of the additional therapeutic agent. Additionally or alternatively, administration can be combined with other bioactive agents or modalities, such as, but not limited to, anti-tumor agents, non-pharmacologic therapies such as surgery, etc.

[0238] In some embodiments of the combined cell therapy, the therapeutic combination comprises iPSC-derived hematopoietic lineage cells provided herein and an additional therapeutic agent that is an antibody or fragment thereof. 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 capacity. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents for administered iPSC-derived hematopoietic lineage cells include anti-CD20 (e.g., rituximab, veltuzumab, ofatumumab, ublituximab, ocaratu- zumab, obinutuzumab), anti-HER2 (e.g., trastuzumab, pertuzumab), anti-CD52 (e.g., alemtuzumab), anti-EGFR (e.g., cetuximab), anti-GD2 (e.g., dinutuximab), anti-PDL1 (e.g., avelumab), anti-CD38 (e.g., daratumumab, isatuximab, MOR202), anti-CD123 (e.g., 7G3, CSL362), anti-SLAMF7 (elotuzumab), anti-CD25 (e.g., daclizumab, basiliximab, M-A251, 2A3, BC69, 24204, 22722 or 24212), anti-CD69 (e.g., MAB23591, FN50, 298614, or AF2359), anti-CD71 (e.g., CY1G4 or DF1513), anti-CD44 (e.g., bivatuzumab, RG7356, or G44-26) and humanized or Fc-modified variants or fragments thereof or functional equivalents or biosimilars thereof.

[0239] In some embodiments, the additional therapeutic agent comprises one or more checkpoint inhibitors. A checkpoint refers to a cellular molecule, often a cell surface molecule, that can suppress or downregulate an immune response if not inhibited. A checkpoint inhibitor is an antagonist that can reduce the gene expression or gene product of a checkpoint or reduce the activity of a checkpoint molecule. Suitable checkpoint inhibitors for combination therapy with derived effector cells, including NK cells or T cells, are provided above.

[0240] Some embodiments of combination therapies comprising derived effector cells provided further comprise at least one inhibitor targeting a checkpoint molecule. Some other embodiments of combination therapies with derived effector cells provided comprise two, three, or more inhibitors such that two, three, or more checkpoint molecules are targeted. In some embodiments, the effector cells for combination therapies described herein are derived NK cells as provided. In some embodiments, the effector cells for combination therapies described herein are derived T cells. In some embodiments, the derived NK cells or T cells for combination therapies are functionally enhanced as provided herein. In some embodiments, two, three, or more checkpoint inhibitors can be administered in combination therapy simultaneously, before, or after administration of derived effector cells. In some embodiments, two or more checkpoint inhibitors are administered simultaneously or one at a time (sequentially).

[0241] In some embodiments, the antagonist that inhibits any of the above-mentioned checkpoint molecules is an antibody. In some embodiments, the checkpoint inhibitory antibody can be a murine antibody, a human antibody, a humanized antibody, a camelid Ig, a single variable novel antigen receptor (VNAR), a shark heavy chain antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single chain antigen binding fragment (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single domain antigen binding fragments (sdAb, nanobodies), recombinant heavy chain only antibodies (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be more cost-effective to produce, easier to use, or more sensitive than whole antibodies. In some embodiments, the one or two or three or more checkpoint inhibitors comprise at least one of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof.

[0242] Combination therapy including derived effector cells and one or more check inhibitors is being investigated for the treatment of cutaneous T-cell lymphoma (NHL), non-Hodgkin's lymphoma (NHL), mycosis fungoides, Pagetoid reticulosis, Sézary syndrome, granulomatous lax skin, lymphomatoid papulosis, pityriasis lichenoides chronica, pityriasis lichenoides acutea, and CD30 + Cutaneous T-cell lymphoma, secondary cutaneous CD30 +Large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, Hodgkin lymphoma (HL), Head and neck tumors: squamous cell carcinoma, rhabdomyosarcoma, Lewis lung carcinoma (LLC), non-small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, gastric cancer, prostatic small cell neuroendocrine carcinoma The present invention is applicable to the treatment of liquid and solid cancers, including but not limited to, glioblastoma, hepatoma, oral squamous cell carcinoma, pancreatic cancer, papillary thyroid cancer, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal carcinoma.

[0243] In some embodiments, other than the derived effector cells as provided herein, the combination for therapeutic use includes one or more additional therapeutic agents, including chemotherapeutic agents or radioactive moieties. Chemotherapeutic agents refer to cytotoxic antitumor agents, i.e., chemical agents that are found to preferentially kill tumor cells, or disrupt the cell cycle of rapidly proliferating cells, or eradicate stem cancer cells, and are used therapeutically to prevent or reduce the proliferation of neoplastic cells. Chemotherapeutic agents may also be referred to as antitumor or cytotoxic drugs or agents, and are well known in the art.

[0244] In some embodiments, chemotherapeutic agents include anthracyclines, alkylating agents, alkyl sulfonates, aziridines, ethylenimines, methylmelamine, 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, mechlorethamine, mephalin, chlorambucil, heparinmethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine), antimetabolites (methotrexate, fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids (vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide, etoposide orthoquinone, and teniposide), antibiotics (daunorubicin, doxorubicin, mitoxantrone, bisantrene, actinomycin D, plicamycin, puromycin, and gramicidin D), paclitaxel, colchicine, cytochalasin B, emetine, maytansine, and amsacrine.Additional agents include amine glutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calcitonin, capecitabine, celecoxib, and cerebrospinal fluid. Cimab, cladribine, cloflavine, cytarabine, dacarbazine, denileukin diftitox, diethylstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine, 5-fluorouracil, fludarabine, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hydroxyurea, ibritumomab, Idarubicin, ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole, leucovorin, leuprolide, levamisole, mechlorethamine, megestrol, melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pemetrexed, pegadromase, These include pegaspargase, pentostatin, pipobroman, plicamycin, porifeprosan, porfimer, procarbazine, quinacrine, rituximab, sargramostim, streptozocin, tamoxifen, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitable agents are those approved for human use, including those approved as chemotherapeutic or radiotherapeutic agents and known in the art.Such agents can be found in either many standard physician or oncologist reference works (e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, NY, 1995) or on the National Cancer Institute website (fda.gov / cder / cancer / druglistfrarne.htm), both of which are updated from time to time.

[0245] Immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide, stimulate both NK cells and T cells. As provided herein, IMiDs can be used in conjunction with iPSC-derived therapeutic immune cells for cancer treatment.

[0246] In addition to the isolated population of iPSC-derived hematopoietic lineage cells contained in the therapeutic composition, a composition suitable for administration to a patient can further include one or more pharma- ceutically acceptable carriers (additives) and / or diluents (e.g., a pharma- ceutically acceptable vehicle, e.g., cell culture medium), or other pharma- ceutically acceptable components. Pharmaceutically acceptable carriers and / or diluents will be determined, in part, by the particular composition being administered, as well as by the particular method used to administer the therapeutic composition. Thus, there are a wide variety of suitable formulations of the therapeutic compositions of the present embodiments (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, the disclosure of which is incorporated herein by reference in its entirety). th (see ed. 1985).

[0247] In one embodiment, the therapeutic composition comprises pluripotent cell-derived T cells generated by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived NK cells generated by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises pluripotent cell-derived CD34 cells generated by the methods and compositions disclosed herein. +In one embodiment, the therapeutic composition comprises a pluripotent cell-derived HSC generated by the methods and compositions disclosed herein. In one embodiment, the therapeutic composition comprises a pluripotent cell-derived MDSC generated by the methods and compositions disclosed herein. The therapeutic composition comprising a population of iPSC-derived hematopoietic lineage cells disclosed herein can be administered individually or in combination with other suitable compounds by intravenous, intraperitoneal, enteral, or tracheal administration methods to affect a desired therapeutic goal.

[0248] These pharma- ceutically acceptable carriers and / or diluents can be present in an amount sufficient to maintain the pH of the therapeutic composition at about 3 to about 10. Thus, the buffer can be as much as about 5% on a weight-to-weight basis of the total composition. Electrolytes such as, but not limited to, sodium chloride and potassium chloride can also be included in the therapeutic composition. In one aspect, the pH of the therapeutic composition ranges from about 4 to about 10. Alternatively, the pH of the therapeutic composition ranges from 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 in one of the above pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the therapeutic composition has a pH in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

[0249] The present invention also provides, in part, the use of pharma- ceutically acceptable cell culture media in certain compositions and / or cultures of the embodiments of the invention. Such compositions are suitable for administration to a human subject. Generally speaking, any medium that supports the maintenance, growth, and / or health of iPSC-derived effector cells according to the embodiments of the invention is suitable for use as a pharmaceutical cell culture medium. In certain embodiments, the pharma- ceutical acceptable cell culture medium is a serum-free and / or feeder-free medium. In various embodiments, the serum-free medium is free of animal components and may optionally be protein-free. Optionally, the medium may include recombinant proteins that are biopharmaceutical acceptable. Animal component-free medium refers to a medium in which components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, protein-free medium is defined as being substantially free of protein. Those skilled in the art will appreciate that the examples of media described above are illustrative and in no way limit the formulation of media suitable for use in the present invention, and there are many suitable media known and available to those skilled in the art.

[0250] iPSC-derived hematopoietic lineage cells are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, pro-T cells, pro-NK cells, CD34 + The iPSC-derived hematopoietic lineage cells can comprise about 95% to about 100% T cells, NK cells, pro-T cells, pro-NK cells, CD34 cells, HSCs, B cells, myeloid-derived suppressor cells (MDSCs), regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells. In some embodiments, the iPSC-derived hematopoietic lineage cells can comprise about 95% to about 100% T cells, NK cells, pro-T cells, pro-NK cells, CD34 + In some embodiments, the present invention provides a method for treating a subject in need of cell therapy that comprises approximately 95% T cells, NK cells, pro-T cells, pro-NK cells, CD34 HE cells, or myeloid-derived suppressor cells (MDSCs). + Therapeutic compositions comprising purified T cells or NK cells, such as compositions comprising HE cells, or isolated populations of myeloid-derived suppressor cells (MDSCs), are provided.

[0251] In one embodiment, the combination cell therapy comprises an anti-CD38 therapeutic protein or peptide and a population of NK cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, the derived NK cells comprise B2M negative and CD38 negative. In another embodiment, the combination cell therapy comprises an anti-CD38 antibody therapeutic protein or peptide and a population of T cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, the derived T cells comprise B2M negative and CD38 negative. In some embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202 and a population of NK or T cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, the derived NK or T cells comprise B2M negative, CD38 negative, and CIITA negative. In still some other embodiments, the combination cell therapy comprises daratumumab and a population of NK cells or T cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, the derived NK cells or T cells comprising one or more of B2M negative, CIITA negative, CD38 negative, and exogenous CD16 and CAR. In yet some additional embodiments, the combination cell therapy comprises daratumumab, isatuximab, or MOR202, and a population of NK cells or T cells derived from genomically engineered iPSCs comprising a genotype listed in Table 1, the derived NK cells or T cells comprising B2M negative, CD38 negative, and CIITA negative, and exogenous CD16, CAR, and one or more exogenous cytokine signaling complexes.

[0252] As will be appreciated by those skilled in the art, both autologous and allogeneic hematopoietic lineage cells derived from iPSCs based on the methods and compositions herein can be used in cell therapy as described above. In the case of autologous transplantation, the isolated population of derived hematopoietic lineage cells is fully or partially HLA-matched with the patient. In another embodiment, the derived hematopoietic lineage cells are HLA-mismatched with the subject, and the derived hematopoietic lineage cells are NK cells or T cells, including HLA-I deficiency and, optionally, HLA-II deficiency.

[0253] 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 5 × 10 9 In some embodiments, the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1 x 10 per dose. 5 cells ~ approx. 1 x 10 6 Cells, per dose, approximately 0.5 x 10 6 cells ~ approx. 1 x 10 7 Cells, per dose, approximately 0.5 x 10 7 cells ~ approx. 1 x 10 8 Cells, per dose, approximately 0.5 x 10 8 cells ~ approx. 1 x 10 9 Cells, per dose, approximately 1 x 10 9 Cells ~ approx. 5 x 10 9 Cells, per dose, approximately 0.5 x 10 9 cells ~ approx. 8 x 10 9 Cells, per dose, approximately 3 x 10 9 cells ~ approx. 3 x 10 10 cells, or any range in between. Generally, for a 60 kg patient / subject, 1 x 10 8 Cells / dose is 1.67 x 10 6 Converted to cells / kg.

[0254] In one embodiment, the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a portion of blood or a single umbilical cord, or at least 0.1 x 10 5 At least 0.5 x 10 cells / kg body weight 5 Cells / kg body weight, at least 1 x 10 5Cells / kg body weight, at least 5 × 10 5 Cells / kg body weight, at least 10 x 10 5 At least 0.75 x 10 cells / kg body weight 6 At least 1.25 x 10 cells / kg body weight 6 Cells / kg body weight, at least 1.5 x 10 6 Cells / kg body weight, at least 1.75 x 10 6 At least 2 x 10 cells / kg body weight 6 At least 2.5 x 10 cells / kg body weight 6 Cells / kg body weight, at least 3 x 10 6 Cells / kg body weight, at least 4 x 10 6 Cells / kg body weight, at least 5 × 10 6 Cells / kg body weight, at least 10 x 10 6 Cells / kg body weight, at least 15 x 10 6 Cells / kg body weight, at least 20 x 10 6 Cells / kg body weight, at least 25 x 10 6 Cells / kg body weight, at least 30 x 10 6 cells / kg body weight, 1×10 8 cells / kg body weight, 5×10 8 cells / kg body weight or 1 x 10 9 cells / kg body weight.

[0255] In one embodiment, a dose of derived hematopoietic lineage cells is delivered to the subject. In an exemplary embodiment, the effective amount of cells provided to the subject is at least 2×10 6 Cells / kg, at least 3 x 10 6 Cells / kg, at least 4 x 10 6 Cells / kg, at least 5 × 10 6 Cells / kg, at least 6 × 10 6 Cells / kg, at least 7 × 10 6 Cells / kg, at least 8 × 10 6 Cells / kg, at least 9 × 10 6 cells / kg, or at least 10 x 10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0256] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, or approximately 10 x 10 6 cells / kg, or more cells / kg, including all intervening cell doses.

[0257] In another exemplary embodiment, the effective amount of cells provided to a subject is about 2×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 4×10 6 cells / kg ~ approx. 10×10 6 cells / kg, approximately 5×10 6 cells / kg ~ approx. 10×10 6 cells / kg, 2×10 6 cells / kg~about 6×10 6 cells / kg, 2×10 6 cells / kg~about 7×10 6 cells / kg, 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 3×10 6 cells / kg~about 6×10 6 cells / kg, 3×10 6 cells / kg~about 7×10 6 cells / kg, 3×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 4×10 6 cells / kg~about 6×10 6 cells / kg, 4×10 6 cells / kg~about 7×10 6 cells / kg, 4×10 6 cells / kg ~ approx. 8×10 6 cells / kg, 5×10 6cells / kg~about 6×10 6 cells / kg, 5×10 6 cells / kg~about 7×10 6 cells / kg, 5×10 6 cells / kg ~ approx. 8×10 6 cells / kg or 6 x 10 6 cells / kg ~ approx. 8×10 6 Cells / kg and includes all intervening cell doses.

[0258] In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a single dose treatment. In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a multiple dose treatment. In some embodiments, the multiple dose treatment is one dose every day, every 3 days, every 7 days, every 10 days, every 15 days, every 20 days, every 25 days, every 30 days, every 35 days, every 40 days, every 45 days, every 50 days, or any number of doses in between.

[0259] The compositions comprising the population of derived hematopoietic lineage cells of the present invention may be sterile, suitable for administration to a human patient / subject, and may be administered immediately (i.e., without further treatment). A cell-based composition that is ready to be administered means that the composition does not require any further processing or manipulation before implantation or administration to a subject. In other embodiments, the present invention provides isolated populations of derived hematopoietic lineage cells that are expanded and / or conditioned prior to administration of one or more agents, including small chemical molecules. Compositions and methods for modulating immune cells, including iPSC-derived effector cells, are described in detail, for example, in WO 2017 / 127755, the relevant disclosures of which are incorporated herein by reference. For derived hematopoietic lineage cells engineered to express a recombinant TCR or CAR, the cells may be activated and expanded using methods described, for example, in U.S. Pat. No. 6,352,694.

[0260] In certain embodiments, the primary and costimulatory signals for derived hematopoietic lineage cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. If bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" formation) or to separate surfaces (i.e., in a "trans" formation). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal can be bound to the cell surface and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agent can be in a soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor, or an antibody or other binding agent that binds the agent, as disclosed in U.S. Patent Publication Nos. 2004 / 0101519 and 2006 / 0034810 (the disclosures of which are incorporated by reference) for artificial antigen presenting cells (aAPCs) contemplated for use in activating and expanding T lymphocytes in embodiments of the invention.

[0261] Some variation in dosage, frequency and protocol will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose, frequency and protocol for the individual subject. EXAMPLES

[0262] The following examples are offered by way of illustration and not by way of limitation.

[0263] Example 1 - Materials and Methods To effectively select suicide systems under the control of various promoters and test them in combination with different safe harbor locus integration strategies, Applicant's proprietary hiPSC platform, which allows single cell passaging and high-throughput 96-well plate-based flow cytometry sorting, was used to enable the derivation of clonal hiPSCs with single or multiple gene modulations.

[0264] Maintenance of hiPSCs in small cell cultures: hiPSCs were routinely passaged as single cells once the cultures reached 75%–90% confluency. For single cell dissociation, hiPSCs were washed once with PBS (Mediatech) and treated with Accutase (Millipore) for 3–5 min at 37°C, followed by pipetting to ensure single cell dissociation. The single cell suspension was then mixed with equal volumes of conventional medium, centrifuged at 225 × g for 4 min, resuspended in FMM, and seeded onto matrigel-coated surfaces. Passages were typically 1:6–1:8, transferred to tissue culture plates precoated with matrigel for 2–4 h at 37°C, and fed with FMM every 2–3 days. Cell cultures were maintained in a humidified incubator set at 37°C and 5% CO2.

[0265] Using human iPSC manipulation with ZFNs, CRISPR for targeted editing of the desired modality:ROSA26 targeted insertion as an example, for ZFN-mediated genome editing, 2 million iPSCs were transfected with a mixture of 2.5 μg ZFN-L (FTV893), 2.5 μg ZFN-R (FTV894) and 5 μg donor construct for AAVS1 targeted insertion. For CRISPR-mediated genome editing, 2 million iPSCs were transfected with a mixture of 5 μg ROSA26-gRNA / Cas9 (FTV922) and 5 μg donor construct for ROSA26 targeted insertion. Transfection was performed using a Neon transfection system (Life Technologies) using the parameters 1500V, 10 ms, 3 pulses. On the second or third day after transfection, flow cytometry was used to measure transfection efficiency if the plasmid contained an artificial promoter-driver GFP and / or RFP expression cassette. On the fourth day after transfection, puromycin was added to the medium at a concentration of 0.1 μg / mL for the first 7 days and at a concentration of 0.2 μg / mL after 7 days to select target cells. During puromycin selection, cells were passaged onto fresh Matrigel-coated wells on day 10. From day 16 of puromycin selection onwards, viable cells were selected for GFP + The percentage of iPS cells was analyzed by flow cytometry.

[0266] Bulk and clonal sorting of genome-edited iPSCs: iPSCs containing genome-targeted edits using ZFN or CRISPR-Cas9 express GFP after 20 days of puromycin selection. + SSEA4 + TRA181 +Bulk and clonal sorting of iPSCs. Single cell dissociated targeted iPSC pools were resuspended in chilled staining buffer containing Hanks Balanced Salt Solution (MediaTech), 4% fetal bovine serum (Invitrogen), 1x penicillin / streptomycin (Mediatech), and 10 mM Hepes (Mediatech), freshly made for optimal performance. Conjugated primary antibodies such as SSEA4-PE, TRA181-Alexa Fluor-647 (BD Biosciences) were added to the cell solution and incubated on ice for 15 min. All antibodies were used at 7 μL in 100 μL staining buffer per million cells. The solution was washed once with staining buffer, spun down at 225 g for 4 min, resuspended in staining buffer containing 10 μM thiazobib, and kept on ice for flow cytometric sorting. Flow cytometric sorting was performed on a FACS Aria II (BD Biosciences). For bulk sorting, GFP was used as the primary antibody. + SSEA4 + TRA181 +Cells were gated and sorted into 15 ml standard tubes filled with 7 ml of FMM. For clonal sorting, a 100 μM nozzle was used to eject sorted cells directly into a 96-well plate at a concentration of 3 events per well. Each well was pre-filled with 200 μL of FMM supplemented with 5 μg / mL fibronectin and 1× penicillin / streptomycin (Mediatech) and was previously coated overnight with 5× Matrigel. For 5× Matrigel pre-coating, one aliquot of Matrigel was added to 5 mL of DMEM / F12, incubated overnight at 4°C, properly resuspended, and finally added to the 96-well plate at 50 μL per well and incubated overnight at 37°C. The 5× Matrigel was aspirated immediately before adding medium to each well. Once sorting was completed, the 96-well plate was centrifuged at 225 g for 1-2 min before incubation. The plate was left undisturbed for 7 days. On day 7, 150 μL of medium was removed from each well and replaced with 100 μL of FMM. The wells were re-fed with an additional 100 μL of FMM on day 10 after sorting. Colony formation was detected as early as day 2, and most colonies grew between 7 and 10 days after sorting. For the first passage, wells were washed with PBS and dissociated with 30 μL of Accutase for approximately 10 min at 37 °C. The need for prolonged Accutase treatment reflects the compactness of colonies that did not play in culture for long periods. After the cells were observed to be dissociated, 200 μL of FMM was added to each well and pipetted several times to disrupt the colonies. The dissociated colonies were transferred to another well of a 96-well plate previously coated with 5× Matrigel and centrifuged at 225 g for 2 min before incubation. This one-to-one passage is performed to expand the initial colonies before they grow. Subsequent passaging was routinely performed with 3-5 min of Accutase treatment and expansion 1:4-1:8 at 75-90% confluency into large wells previously coated with 1x Matrigel in FMM. Each clonal cell line was analyzed for GFP fluorescence and TRA1-81 expression levels. Clonal populations were cryopreserved to serve as master cell banks, with nearly 100% GFP fluorescence and TRA1-81 expression levels. +and TRA1-81 + Clonal lines with .DELTA. were selected for further screening and analysis, including but not limited to off-target editing and / or karyotype of engineered iPSCs. Flow cytometry analysis was performed on a Guava EasyCyte 8 HT (Millipore) and analyzed using Flowjo (FlowJo, LLC).

[0267] Example 2 - HLA-I Deficiency and CD16 Matching in iPSC-Derived Effector Cells To achieve HLA complex modification, for example through B2M knockout, iPSC lines were transfected with a B2M-targeting gRNA pair for CRISPR-mediated editing. The B2M-edited iPSCs were then genetically engineered to knock out CD38 and insert an exogenous CD16, such as hnCD16, thereby knocking out the B2M - / - , CD38 - / - After iPSC clone selection, transgene copy number verification and karyotype verification, modified iPSCs were then generated that contained CD38 - / - Genome-edited HLA-I-deficient clonal iPSCs containing CD16 were differentiated according to the methods provided herein to express CD38 - / - HLA-I-deficient effector cells with CD16 (HLA-I null CD38 - / - CD16). B2M wild type and CD38 - / - iPSC-derived effector cells bearing CD16 also express HLA-I WT CD38 - / - CD16-bearing iPSCs were obtained and used as a control for the phenotypic and functional analysis of HLA-I deficient effector cells.

[0268] iNKs derived from either a B2M wild-type or B2M knockout iPSC background were differentiated and expanded. Phenotyping using flow cytometry analysis for NK cell markers showed that cells from both backgrounds shared a very similar surface profile for typical NK cell markers (Figure 1A).

[0269] iNKs derived from either the B2M wild-type iPSC background or the B2M knockout iPSC background further contain hnCD16 in addition to the CD38 knockout. Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of NK cell-mediated lysis via binding of CD16 to antibody-coated target cells. To assess ADCC function, hnCD16-expressing B2M iPSCs were cultured in the presence and absence of an anti-CD38 antibody, e.g., daratumumab. - / - iNK cells were co-cultured with the Nalm-6 leukemia cell line. Flow cytometric analysis of the cells at 48 hours of co-culture revealed that B2M - / - We show that the iNK line exhibits very similar levels of ADCC activity compared to the B2M WT iNK line (Figure 1B). Thus, HLA-I deficiency is a potent inhibitor of B2M - / - It does not adversely affect the exogenous CD16-mediated ADCC mechanism in CD16 effector cells.

[0270] Example 3 - HLA Deletion, CD38 Conditioning, and Allorejection Protection for Effector Cells In this assay, the iNK cells used were B2M - / - CIITA - / - CD38 - / -Defective in both HLA-I and HLA-II compared to WT iNK of the same specific donor background that contains hnCD16 and is therefore intact in both HLA-I and HLA-II (double knockout, "dKO" or "B2M / CIITA KO" in Figure 2A). All cell populations shown lacked CD38 expression. Primed and expanded allogeneic donor T cells (E) were co-cultured with dKO iNK cells and WT iNK cells, respectively (T), at a 1:1 E:T ratio (allo-T cells:iNK). As shown in Figure 2B, allogeneic T cells showed a reduced ability to attack these HLA-I deficient iNKs than against B2M WT iNK cells. Thus, HLA-I deficiency may be protective against allogeneic T cell reactivity and promote greater effector cell persistence in an allogeneic cell therapy setting.

[0271] Previously, it was found that expression of immunosuppressive proteins (e.g., HLA-E or HLA-G) on HLA-I-deficient effector cells could prevent the proliferation of allogeneic peripheral blood NK (pbNK) cells, thus reducing pbNK cell recognition and cytotoxicity against effector cells. Furthermore, modified versions of HLA-E or HLA-G to avoid cleavage could further enhance the persistence of HLA-I-deficient effector cells. To test the inhibitory effects of various ligands, pbNK cells from 18 donors were co-cultured with K562 target cells expressing the indicated inhibitory ligands, and the resulting fold changes in cytotoxicity of NK cell subsets expressing specific HLA receptors are shown in Figure 3. As shown, HLA-E surface-expressing effector cells can inactivate NK cells expressing the inhibitory receptor NKG2A, but HLA-E surface expression was also observed to activate NKG2C of pbNK cells, which leads to adverse effects including pbNK cell recognition and consequent killing of HLA-E expressing cells. The data indicate that a subset of NK cells is resistant to inhibitory pathways involving CD47 and HLA-E signaling. Thus, since the inhibitory receptors recognizing HLA-E and HLA-G are stochastically expressed, i.e., they are not expressed by all cells, it appears that HLA-E / G does not provide complete protection from primary NK cell-based recognition, and there are corresponding activating receptors recognizing HLA-E (and possibly HLA-G) that may cause accelerated rejection.

[0272] To avoid the leakage of HLA-E / G protection of HLA-I deficient cells against allogeneic primary NK, the present application provides an alternative or additional approach that provides a more complete protection to reduce recognition and cytotoxicity by pbNK against HLA-I deficient (e.g. B2M KO) or HLA-I and HLA-II deficient (e.g. B2M / CIITA dKO) effector cells. The approach, which does not require HLA-E / G modification, is via CD38 conditioning using anti-CD38 antibodies to eliminate pbNK cells activated by effector cells without exposing the effector cells to deleterious effects due to their lack of CD38 expression.

[0273] As shown in Figure 4, B2M WT CD38 - / - or B2M - / - CD38 - / - iNK cells were co-cultured with allogeneic pbNK cells isolated from healthy donors and primed overnight with IL15 at an E:T ratio of 1:5 or 1:1 (pbNK:iNK). - / - CD38 - / - The level of pbNK cytotoxicity against iNK cells was WT CD38 - / - This is consistent with the susceptibility of HLA-I-deficient cells to NK cell lysis. On the other hand, co-culture in the presence of daratumumab resulted in B2M cells that were amenable to CD38 conditioning with CD38-specific antibodies. - / - CD38 - / - CD38 conditioning thus provides a protective effect for HLA-I-deficient cells that would otherwise be subject to host primary NK cell recognition and cytotoxicity.

[0274] In another experiment, allogeneic pbNK cells from healthy donors were pretreated in duplicate for 48 hours with various concentrations of daratumumab, as shown in Figures 5A and 5B. After 48 hours, B2M WT CD38 - / - , B2M - / -CD38 - / - , or B2M - / - CIITA - / - CD38 - / - iNK cells were added to the appropriate wells at a ratio of 1:1 (pbNK:iNK) and cells were cultured for an additional 48 h. As shown in Figure 5B, pretreatment with daratumumab significantly reduced the number of pbNK in the co-cultures, shown normalized to wells without daratumumab, thereby resulting in an increase in the number of B2M KO and B2M / CIITA dKO, signifying protection from allorejection (Figure 5A).

[0275] In subsequent assays, allogeneic PBMCs (peripheral blood mononuclear cells, which contain T, NK and other immune cells) from different healthy donors were cultured alone or in vitro with CD38 - / - Co-culture with iNK cells revealed that the size of the PBMC subpopulation expressing CD38 when cultured alone (Figure 6A, day 9 of culture) was significantly greater than that of the allogeneic CD38 PBMC subpopulation expressing CD38 PBMCs. - / - PBMCs primed with iNK cells, and thus subject to allorecognition of iNK cells in coculture, have increased CD38 expression, leading to an increase in the size of the CD38-expressing alloreactive PBMC subpopulation (FIG. 6B, day 9 of coculture). This observation suggested that a CD38 conditioning step (e.g., anti-CD38 antibody infusion) during or after infusion of allogeneic effector cells could be effective in promoting greater effector cell persistence in adoptive cell therapy, provided that the effector cells are not challenged by the CD38 antagonist used in the CD38 conditioning step.

[0276] Further analysis included mixed lymphocyte reaction (MLR) and allogeneic PBMCs were compared with B2M - / - , CD38 - / -, IL15RF and hnCD16-bearing iPSC-derived iNK cells were co-cultured at a 5:1 ratio with or without daratumumab (i.e., 10 μg, 5 μg, 1 μg, 0.1 μg, 0.01 μg, and no daratumumab, Figures 7A and 7B). PBMC and iNK cell counts were collected on day 11 of co-culture under each daratumumab dose condition. As shown in Figure 7A, PBMC cell counts are dose-dependently decreased in the presence of daratumumab. Although HLA-I-deficient effector cells are protected against T cell-mediated alloreactivity (see above and Figure 2), pbNK cells in PBMCs show alloreactivity against these cells due to their HLA-I deficiency, resulting in lysis of HLA-I-deficient effector cells (see the "No Darat" column in Figures 7A and 7B). As a result, HLA deficiency alone may not be sufficient to extend the survival rate of allogeneic effector cells. In this assay, the sensitivity of iPSC-derived HLA-I-deficient cells to allogeneic PBMC reactivity is observed to be rescued in the presence of daratumumab, more clearly at dose levels higher than about 0.01 μg. Thus, a dose-dependent effect of daratumumab on both the elimination of alloreactive host cells and the improvement of survival rate of donor iNK cells was observed.

[0277] Healthy donor PBMCs were cultured with or without daratumumab (e.g., 10 μg / mL, 5 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, and no daratumumab) to generate HLA-I deficient iNK cells (B2M - / - , CD38 - / - MLR assays using donor PBMCs co-cultured with CD38 - / -, and compared to those obtained in coculture with iNK cells bearing IL15RF and hnCD16 (i.e., B2M WT iNK). Over the course of coculture (>15 days) at each dose level, there was an effect of daratumumab on both eliminating alloreactive host cells (PBMCs) and improving the viability of donor iNK cells with both HLA-I deficiency and CD38 knockout over time (Figures 8A and 8B), with iNK cell numbers increasing in a daratumumab dose-dependent manner. Absolute T cell counts in PBMCs in WT iNK cocultures (Figure 8C) show that daratumumab prevented T cell proliferation, and that B2M KO to iNK (Figure 8D) was sufficient to prevent T cell proliferation. Absolute pbNK cell counts in PBMCs in both WT iNK cocultures (Figure 8E) and B2M KO iNK cocultures (Figure 8F) show that daratumumab prevented pbNK cell proliferation as well. Without wishing to be bound by theory, the synergistic effect on the protection of effector iNK cells against alloreactive host cells may be due to multiple reasons, including, but not limited to, the following: (1) HLA-I deficiency of effector iNK cells reduces the elimination of effector iNK cells caused by host alloreactive T cells, (2) allogeneic effector cell priming upregulates CD38 expression of alloreactive cells in PBMCs, thereby increasing the susceptibility of alloreactive host cells to anti-CD38 antibodies, (3) enhanced ADCC by exogenous CD16 allows for anti-CD38 antibody targeting, and (4) CD38 expression of effector cells may increase the susceptibility of alloreactive host cells to anti-CD38 antibodies. - / - The phenotype avoids anti-CD38 antibody targeting, but the antibody specifically eliminates alloreactive host cells that express CD38.

[0278] Samples of the same co-cultures described above with respect to Figures 8A-8F were analyzed using the HLA-A2 + Expression of CD25, 4-1BB, and CD38 in PBMCs (iNKs are HLA-A2 negative) was analyzed by flow cytometry. The data show that B2M KO on iNKs significantly reduced the expression of CD38 in PBMCs. + CD25 + , CD38 + 41BB+ and CD25 + 41BB + The results show that daratumumab treatment was sufficient to reduce activation in cultures with WT iNK. + 41BB + PBMCs (Figure 9D). The cumulative data further suggests that the combination of B2M KO and daratumumab can potently inhibit allogeneic T and NK cell responses. Furthermore, the data suggest the possibility of additional or alternative strategies in which 4-1BB alloreactive cells can be selectively depleted by activation of 4-1BB-targeted alloprotective receptors (ADRs). Some exemplary ADRs are described, for example, in International Publication No. WO 2019 / 210081, which is incorporated herein by reference.

[0279] In a separate MLR assay, allogeneic PBMCs were cultured in B2M with or without daratumumab (10 μg, 1 μg, 0.1 μg, or no daratumumab). - / - , C.I.T.A. - / - , CD38 - / - , IL15RF and hnCD16-bearing iPSC-derived iNK cells (B2M / CIITA dKO). As shown in Figure 10A, co-culture with daratumumab dose-dependently protects iNK cells co-cultured with HLA-mismatched PBMCs. Absolute alloreactive NK cell counts (Figure 10B) show that daratumumab also dose-dependently prevented alloreactive NK cell proliferation when B2M / CIITA dKO iNK cells were co-cultured with HLA-mismatched PBMCs. B2M / CIITA dKO iNK cells with intact HLA-I and HLA-II were co-cultured with iPSC-derived iNK cells (B2M / CIITA dKO). As shown in Figure 10A, co-culture with daratumumab dose-dependently protects iNK cells co-cultured with HLA-mismatched PBMCs. Absolute alloreactive NK cell counts (Figure 10B) show that daratumumab also dose-dependently prevented alloreactive NK cell proliferation when B2M / CIITA dKO iNK cells were co-cultured with HLA-mismatched PBMCs. WT iNK cells (i.e., CD38 - / - Flow cytometry analysis of B2M / CIITA dKO iNK cells compared to iNK cells with CD4+, IL15RF, and hnCD16 (denoted "WT iNK") showed that B2M / CIITA dKO iNK cells up-regulated CD4+ and IL15RF+ hnCD16 when co-cultured with HLA-mismatched PBMCs. + and CD8 +We demonstrate that the combination of B2M / CIITA dKO and daratumumab does not stimulate T cell proliferation (FIG. 10C). Collectively, the data support that the combination of B2M / CIITA dKO and daratumumab can potently inhibit allogeneic T and NK cell responses.

[0280] Example 4 - Anti-CD38 antibodies extend survival and persistence of effector cells relative to primary / host cells A flow-based caspase 3 / 7 killing assay was performed to assess the degree of fratricide mediated via antibody-dependent cellular cytotoxicity (ADCC) by pbNK cells in combination with anti-CD38 antibodies against effector cells. In this assay, iPSC-derived CAR-T (CAR-iT) cells containing an exemplary CD19-CAR introduced into the TRAC locus and either CD38KO ("CD38 KO iT") or CD38 wild-type ("WT iT") were co-cultured with pbNK cells for approximately 3 hours in the presence of an anti-CD38 monoclonal antibody, daratumumab, or an anti-CD20 monoclonal antibody, rituximab, as a negative control. Both antibodies were serially diluted 1:3 from approximately 30 μg / mL to 0 μg / mL. CAR-iT cells were plated at approximately 1E5 cells / well and pbNK cells were added at a 3:1 ratio. iT cells and pbNK cells were distinguished by differential fluorescent labeling, and the specific cytotoxicity (cell death) of each cell type was assessed independently by flow cytometry using a reporter of caspase 3 / 7 activity.

[0281] pbNK cells express endogenous CD16 and CD38, and as a result, as shown in FIG. 11A, pbNK cells undergo anti-CD38-directed fratricide in the presence of anti-CD38 mAb dose-dependently CD16-mediated ADCC, whereas anti-CD20 mAb (negative control) has no effect on these cells. The iT cell-specific cytotoxicity shown in FIG. 11B demonstrated that WT iT cells, but not C38 KO iT cells, are susceptible to ADCC when combined with pbNK and anti-CD38 mAb. Although CD16-expressing pbNK cells recognize and kill WT iT cells coated with anti-CD38 mAb, CD38 KO iT cells are resistant to ADCC by pbNK cells combined with anti-CD38, because CD38-specific mAb cannot bind to CD38 KO iT cells and induce pbNK activation via CD16 cross-linking.

[0282] To test whether daratumumab can deplete pbNK cells in vivo, pbNK cells were injected into NSG mice or IL15 transgenic NSG mice (NSG-ILtg) in an MLR assay, and the persistence of injected pbNK cells was monitored over time in peripheral blood. Briefly, pbNK cells and iNK cells were injected alone or with one dose of daratumumab. Cell numbers were normalized to the group without daratumumab. As shown in Figure 12, pbNK cells persisted in the NSG-ILtg model (>22 days), but the addition of daratumumab reversed cell viability, which is consistent with the in vitro observations.

[0283] Another observation in this analysis is that the administration of anti-CD38 antibodies can be controlled, and thus, upon the occurrence of adverse effects from allogeneic effector cells, the effector cells can be tapered or eliminated in a controllable manner, so that they are eliminated by allorejection. In addition, anti-CD38 antibodies can also be used as a preconditioning strategy to eliminate alloreactive cells prior to the infusion of allogeneic effector cells, and thus can be separated in time from the infusion of allogeneic effector cells. These are some of the advantages not offered by incorporating HLA-E / G into HLA-I-deficient cells to overcome allorejection by peripheral T, NK and other alloreactive cells, providing flexibility in the treatment process and a mode of control of effector cell numbers in response to patient response during treatment.

[0284] The effect of CD38 conditioning on the viability and persistence of B2M KO iNK and B2M / CIITA dKO iNK cells in the presence of primary NK cells was also further evaluated in vivo. Subsequently, WT iNK cells, B2M KO iNK cells, and B2M / CIITA dKO iNK cells were co-injected into NSG-ILtg mice with and without daratumumab, and the persistence of iNK cells was evaluated and compared in peripheral blood, spleen, and bone marrow around day 14. When pbNK cells were injected alone, pbNK were detected in the circulation, but were significantly reduced in the presence of daratumumab (Figure 13A). As shown, when WT iNK cells, B2M KO iNK cells, and B2M / CIITA dKO iNK cells were injected alone without pbNK cells, each of the donor iNK cells persisted in the circulation at similar levels (Figure 13B). WT iNK cells co-injected with pbNK persisted in the presence and absence of daratumumab (Figure 13C). However, B2M KO iNK cells and B2M / CIITA dKO iNK cells co-injected with pbNK were rejected by pbNK in the absence of daratumumab, but were protected from pbNK allorejection in the presence of daratumumab (Figures 13D and 13E). Furthermore, in blood, spleen, and bone marrow, pbNK was significantly decreased in the presence of daratumumab, which resulted in the persistence of B2M KO and B2M / CIITA KO iNK (Figure 14A). In addition, daratumumab downregulated CD38 expression, which contributed to the protection of iNK from PBNK allorejection. + dKO cells and B2M / CIITA dKO cells, which were induced by pbNK cytotoxicity against HLA-I-deficient cells, were significantly depleted (Figures 14B and 14C). Thus, co-injection with anti-CD38 antibody conditioning treatment reversed the selective depletion of B2M KO and B2M / CIITA dKO cells by pbNK cytotoxicity against HLA-I-deficient cells, leading to improved survival.

[0285] Increased life span of B2M KO CD38KO hnCD16 IL15RF iNK cells ("B2M KO" and "B2M / CIITA dKO") versus B2M WT CD38KO hnCD16 IL15RF iNK cells (denoted as "WT") in the presence of anti-CD38 antibody, and CD38 expression from the respective tissue samples. + The associated clearance of subpopulations (peripheral NK cells, activated B cells and T cells) as provided herein demonstrates the ability of anti-CD38 antibodies in suppressing activated recipient immune cells by targeting their upregulated CD38 in recipients of B2M KO or B2M / CIITA dKO effector cells, thereby reducing allorejection to allogeneic effector cells (which are not the anti-CD38 antibody target).

[0286] The effect of CD38 conditioning on host immune reconstitution was further evaluated in human subjects undergoing adoptive cell therapy. As shown in Figure 15, lymphocyte absolute profiles (indicated by arrows along the x-axis) from four multiple myeloma patients (subjects A-D) treated with CD38KO hnCD16 iNK effector cells in combination with daratumumab show that the use of daratumumab attenuates lymphocyte recovery after lymphodepleting chemotherapy (LDC, subjects A-D), in contrast to a patient not receiving daratumumab (representative subject E), where lymphocyte recovery begins by D4 and continues until the end of the treatment cycle. Lymphocyte data from subject A, who received daratumumab before LDC and weekly thereafter, were further subjected to Uniform Manifold Approximation and Projection (UMAP) visualization. As shown in Figure 16, overlaid lymphocyte data files from each time point show clustering of different cell types by color. Individual UMAP visualization by time point shows endogenous CD4 +We demonstrate CD38 expression on T cells, B cells, and NK cells. As shown, daratumumab eliminated the majority of CD38-expressing lymphocytes by C1D-5, prior to LDC, and maintained suppression of CD38-expressing cells throughout the treatment cycle. Thus, in the presence of anti-CD38 antibodies, CD38 + The observed clearance of subpopulations (peripheral NK cells, activated B cells and T cells) further supports the ability of anti-CD38 antibodies to suppress activated recipient immune cells by targeting their upregulated CD38, thereby reducing allorejection to allogeneic effector cells in recipients of the effector cells provided herein and extending the therapeutic window of adoptive cell therapy.

[0287] Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are representative of exemplary embodiments and are not intended as limitations on the scope of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the disclosure disclosed herein without departing from the scope and spirit of the invention.

[0288] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0289] The disclosure illustratively described herein can be practiced without any element or elements, limitations, or limitations not specifically disclosed herein. Thus, for example, in each example herein, the terms "comprise", "consist essentially of" and "consist of" can be replaced with either of the other two terms. The terms and expressions used are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but rather, it is recognized that various modifications are possible within the scope of the disclosure as claimed. Thus, although the disclosure has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may occur to those skilled in the art, and such modifications and variations are within the scope of the invention as defined by the appended claims.

Claims

1. A cell or a population thereof, wherein (i) the cell is an induced pluripotent stem cell (iPSC), a cloned iPSC, an iPSC cell line cell, or a derivative cell obtained by differentiating the iPSC; (ii) the cell or population thereof comprises (a) HLA-I deficiency, (b) CD38 knockout, and optionally (c) an exogenous polynucleotide encoding CD16 or a variant thereof.

2. The cell further comprises (i) an exogenous polynucleotide encoding a cytokine signaling complex comprising a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its receptor; (ii) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (iii) HLA-II deficiency; and (iv) an exogenous polynucleotide encoding HLA-G, HLA-E, or a variant thereof, and has improved persistence in the presence of alloreactive host cells in adoptive cell therapy incorporating CD38 conditioning. The cell or population thereof according to claim 1.

3. The cell further comprises (i) at least one of the genotypes listed in Table 1; (ii) knockout of one or both of CD58 and CD54; (iii) disruption of at least one of B2M, CII TA, TAP1, TAP2, tapasin, NLR C5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT; (v) does not contain an exogenous polynucleotide encoding HLA-G, HLA-E, or a variant thereof; (iv) comprising the introduction of at least one of 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, AR, TCR, Fc receptor, antibody or a functional variant or fragment thereof, checkpoint inhibitor, engager, and surface trigger receptor for coupling with bispecific or multispecific or universal engager, and / or ２Ａ a method for producing a recombinant cell, a recombinant cell obtained by the method, a method for producing a pharmaceutical composition, a pharmaceutical composition obtained by the method, and a method for treating a disease using the pharmaceutical composition The HLA-I deficiency comprises disruption of at least one of B2M, TAP1, TAP2, and tapasin; and / or The HLA-II deficiency comprises disruption of at least one of CII TA, RFX5, RFXAP, and RFXANK. The cell or population thereof according to claim 1.

4. The derivative cell is (b) used as an allogeneic effector cell, and compared to its natural corresponding cell obtained from peripheral blood, cord blood, or any other donor tissue, has the following (a) Derived CD34 ＋ cells, derived hematopoietic stem progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell precursors, derived NK cell precursors, derived T cells, derived NKT cells, derived NK cells, or derived B cells, or (i) improved persistence and / or survival rate; (ii) increased resistance to activated recipient immune cells. ​ (iii) Increased cytotoxicity, (iv) Improved tumor infiltration, (v) Enhanced or acquired ADCC, (vi) Enhanced ability to migrate tumor-infiltrating bystander immune cells to the tumor site and / or activate or mobilize them, (vii) Enhanced ability to reduce tumor immunosuppression, (viii) Improved ability to rescue tumor antigen escape, and (ix) Reduced fratricide, A derivative NK cell or derivative T cell having at least one of the characteristics including

5. The CD16 or its variant is (a) High-affinity non-cleavable CD16 (hnCD16) or its variant, (b) F176V and S197P in the extracellular domain of CD16, (c) Complete or partial extracellular domain derived from CD64, (d) Non-natural (or non-CD16) transmembrane domain, (e) Non-natural (or non-CD16) intracellular domain, (f) Non-natural (or non-CD16) signaling domain, (g) Non-natural stimulatory domain, and (h) A cell or population thereof according to claim 1, comprising at least one of a transmembrane domain, a signaling domain, and a stimulatory domain that is not derived from CD16 and is derived from the same or a different polypeptide.

6. (a) The non-natural transmembrane domain is derived from CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a T cell receptor (TCR) polypeptide, (b) The non-natural stimulatory domain is derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or an NKG2D polypeptide, (c) The non-natural signaling domain is derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or an NKG2D polypeptide, or The cell or population thereof according to claim 5, wherein (d) the non-natural transmembrane domain is derived from NKG2D, the non-natural stimulatory domain is derived from 2B4, and the non-natural signaling domain is derived from CD3ζ. **Claim 7** The CAR is (i) specific for T cells or NK cells, (ii) a bispecific antigen-binding CAR, (iii) a switchable CAR, (iv) a dimerized CAR, (v) a split CAR, (vi) a multi-chain CAR, (vii) an inducible CAR, (viii) co-expressed with a cytokine signaling complex comprising a partial or complete peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, optionally in a separate construct or in a bicistronic construct, (ix) co-expressed with a checkpoint inhibitor, optionally in a separate construct or in a bicistronic construct, and / or (x) optionally (1) inserted into the TRAC or TRBC locus, and / or driven by the endogenous promoter of the TCR, and / or the TCR is knocked out by the CAR insertion, (2) inserted into a safe harbor locus, or (3) inserted into a locus intended for disruption, the cell or population thereof according to claim 2. **Claim 8** The CAR is (i) specific for CD19, BCMA, B7H3, MICA / B, or MR1, and / or (ii)ADGRE2, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigen of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2, 3, 4, EGFR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), mucin 1 (Muc-1), mucin 16 (Muc-16), mesothelin (MSLN), NKCS1, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, tumor fetal antigen (h5T4), PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and a cell or a population thereof according to claim 2, which is specific for any one of pathogen antigens.

9. The cytokine signal transduction complex is (a) a cell surface-expressed exogenous cytokine containing at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or their respective receptors and / or a partial or complete peptide of the receptor, or (b) (i) Co-expression of IL15 and IL15Rα with a self-cleaving peptide in between, (ii) A fusion protein of IL15 and IL15Rα, (iii) An IL15 / IL15Rα fusion protein (IL15Δ) in which the intracellular domain of IL15Rα is shortened, (iv) A fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) A fusion protein of IL15 and IL15Rβ, (vi) A fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either natural or modified, and (vii) A homodimer of IL15Rβ, of which at least one is, Any one of (i) to (vii) is optionally co-expressed with a CAR in a separate construct or in a bicistronic construct, Optionally, (c) The cell or population thereof according to claim 2, which is transiently expressed.

10. The cell is a derived NK cell or a derived T cell, the derived NK cell can mobilize and / or migrate T cells to the tumor site, and the derived NK cell or the derived T cell can reduce tumor immunosuppression in the presence of one or more checkpoint inhibitors. The cell or population thereof according to claim 1.

11. The one or more checkpoint inhibitors are antagonists against one or more checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A ２Ａ RR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR, the cell or population thereof according to claim 10.

12. The one or more checkpoint inhibitors are, (a) One or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (b) At least one of atezolizumab, nivolumab, and pembrolizumab, The cell or population thereof according to claim 10.

13. The cell is, (i) One or more exogenous polynucleotides integrated into one safe harbor locus or a locus intended for disruption, or (ii) Three or more exogenous polynucleotides integrated into different safe harbor loci or loci intended for disruption, The cell or population thereof according to claim 1.

14. The cell or population thereof according to claim 13, wherein the safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, TCR or RUNX1, or the locus intended for disruption comprises B2M, TAP1, TAP2, tapasin, NLRRC5, CII TA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL - B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

15. The CD38 conditioning is (i) via a CD38 antagonist comprising an anti - CD38 antibody or a CAR (CD38 - CAR) that specifically binds to CD38, (ii) via daratumumab, isatuximab, or MOR202, (iii) via daratumumab, (iv) comprising administering a CD38 antagonist to a subject in need of the adoptive cell therapy before, during, or after infusion of the cell or population thereof for the therapy, (v) comprising pre - loading the cell or population thereof with a CD38 antagonist in vitro and then infusing the pre - loaded cell or population thereof, (vi) eliminating or reducing the number of alloreactive host cells, (vii) delaying host immune reconstitution, and / or (viii) extending the survival rate and persistence of the cell or population thereof in the presence of alloreactive host cells of a subject in need of the adoptive cell therapy, the cell or population thereof according to claim 2.

16. The alloreactive host cells are (i) comprising primary T cells, primary B cells, and / or primary NK cells that are allogeneic to the cell or population thereof, (ii) sensitized to the CD38 conditioning by the cell or population thereof, and / or (iii) dose - dependently eliminated by CD38 conditioning via a CD38 antagonist, the cell or population thereof according to claim 2.

17. A composition comprising a CD38 antagonist and the cell or population thereof according to any one of claims 1 - 16.

18. The composition according to claim 17, further comprising one or more therapeutic agents.

19. The composition according to claim 18, wherein the one or more therapeutic agents comprise a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small interfering RNA, double-stranded RNA (dsRNA), a mononuclear cell, a feeder cell, a feeder cell component or a replenishing factor thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

20. (i) the checkpoint inhibitor is (a) One or more antagonists against checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A ２Ａ RR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or (ii) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide, the composition according to claim 19.

21. The antibody is (a) an anti-CD20 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody, an anti-CD123 antibody, an anti-GD2 antibody, an anti-PDL1 antibody, an anti-CD25 antibody, an anti-CD69 antibody, an anti-CD71 antibody, or an anti-CD44 antibody, or (b) one or more of rituximab, belzutifan, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daclizumab, basiliximab, M-A251, 2A3, BC69, 24204, 22722, 24212, MAB23591, FN50, 298614, AF2359, CY1G4, DF1513, veltuzumab, RG7356, G44-26, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof and functional equivalents and biosimilars thereof, the composition according to claim 19.

22. The CD38 antagonist is (i) an anti-CD38 antibody or a CD38-CAR, or (ii) daratumumab, isatuximab, or MOR202, or (iii) daratumumab, or (iv) provided to a subject in need of adoptive cell therapy before, during, or after injection of the cells or population thereof, the composition according to claim 17.

23. A composition according to any one of claims 17 to 22, for use in the manufacture of an adoptive cell therapy for the treatment of autoimmune disorders, hematological malignancies, solid tumors, cancers, or viral infections.

24. Use in the manufacture of a medicament for reducing or preventing the alloreactivity of host cells against allogeneic effector cells in said adoptive cell therapy provided to a subject in need thereof, of a cell or population thereof according to any one of claims 1 to 16, wherein said use comprises use in combination with CD38 conditioning.

25. The cell or population for use according to claim 24, wherein the host cells comprise alloreactive immune cells including primary T cells, primary B cells, and / or primary NK cells.

26. Said CD38 conditioning comprises (i) comprising the use of a CD38 antagonist, or (ii) preloading the allogeneic effector cells with a CD38 antagonist in vitro, and said CD38 conditioning (a) eliminates or reduces the number of alloreactive host cells, (b) extends the survival and persistence of the allogeneic effector cells to a controllable extent by a given dose of said CD38 antagonist, and / or (c) delays host immune reconstitution.

27. Said CD38 antagonist comprises (i) an anti-CD38 antibody or CD38-CAR, (ii) daratumumab, isatuximab, or MOR202, and / or (iii) comprises daratumumab.

28. The cell or population for use according to claim 26, wherein the alloreactive host cells comprise upregulated CD38 expression.

29. The cell or population for use according to claim 24, wherein the medicament is for use in combination with a therapeutic agent.

30. The cell or population for use according to claim 29, wherein the therapeutic agent comprises a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small molecule RNA, dsRNA (double-stranded RNA), a mononuclear cell, a feeder cell, a feeder cell component or a replenishing factor thereof, a vector comprising one or more target polynucleic acids, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

31. (i) the checkpoint inhibitor is (a) One or more antagonists against checkpoint molecules including PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A ２Ａ R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxp1, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirilumab, monalizumab, nivolumab, pembrolizumab, and derivatives or functional equivalents thereof, or (c) at least one of atezolizumab, nivolumab, and pembrolizumab, or (ii) the therapeutic agent comprises one or more of venetoclax, azacitidine, and pomalidomide, the cell or population for use according to claim 30.

32. The cell or population for use according to claim 24, wherein the medicament is a medicament for use that does not require or minimally requires lymphodepletion using a combination of cyclophosphamide and fludarabine (Cy / Flu).

Images