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WO2025029810A1 - Squelette de ciblage de tumeur solide favorisant la différenciation et la fonction de cellules effectrices - Google Patents

Squelette de ciblage de tumeur solide favorisant la différenciation et la fonction de cellules effectrices Download PDF

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Publication number
WO2025029810A1
WO2025029810A1 PCT/US2024/040194 US2024040194W WO2025029810A1 WO 2025029810 A1 WO2025029810 A1 WO 2025029810A1 US 2024040194 W US2024040194 W US 2024040194W WO 2025029810 A1 WO2025029810 A1 WO 2025029810A1
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cell
receptor
cells
antigen
seq
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Inventor
Eigen Peralta
Bi-Huei YANG
Tom Tong LEE
Bahram Valamehr
Pei-Fang Tsai
Hui-yi CHU
Martin HOSKING
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Fate Therapeutics Inc
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Fate Therapeutics Inc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
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    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2824—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD58
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N2510/00—Genetically modified cells

Definitions

  • the one or several genetic modifications include one or more of DNA insertion, deletion, and substitution, and which modifications are retained and remain functional in subsequently derived cells after differentiation, expansion, passaging and/or transplantation.
  • the present disclosure provides a cell or population thereof, wherein (i) the cell is (a) an immune cell; (b) an induced pluripotent cell (iPSC); or (c) a derivative effector cell obtained from differentiating the iPSC; (ii) the cell comprises a construct comprising at least one of: (a) a polynucleotide encoding an allo-immune defense receptor (ADR); and (b) a polynucleotide encoding IL2, IL18, or a IL7RF; (iii) the construct is inserted into a T cell receptor (TCR) locus, thereby knocking out the TCR; and (iv) the construct is expressed under control of an endogenous TCR promoter.
  • ADR allo-immune defense receptor
  • TCR T cell receptor
  • the cell or population thereof further comprises a construct at a first integration site (site 1), wherein: (i) the site 1 construct comprises two or more of: (a) a polynucleotide encoding a TGF ⁇ signaling redirector receptor (TGF ⁇ -SRR) comprising a partial or full peptide of the extracellular domain (ECD) of transforming growth factor beta receptor (TGF ⁇ R); (b) a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof; and (c) a polynucleotide encoding an exogenous CD16 or a variant thereof; and (ii) the site 1 construct comprises an exogenous promoter that regulates expression of the polynucleotides in the site 1 construct.
  • site 1 construct comprises two or more of: (a) a polynucleotide encoding a TGF ⁇ signaling redirector receptor (TGF ⁇ -SRR) comprising a partial or full peptide of the
  • the cell or population thereof further comprises a construct at a second integration site (site 2), wherein: (i) the site 2 construct comprises a polynucleotide encoding a first chimeric antigen receptor (CAR1); (ii) the site 2 construct optionally further comprises one or more of: (a) a polynucleotide encoding a second chimeric antigen receptor (CAR2) that is different from CAR1 in antigen specificity; (b) a polynucleotide encoding a T cell enhancer (TCE); and (c) a polynucleotide encoding a cytokine; (iii) the site 2 differs from the site 1; and (iv) the site 2 construct comprises an exogenous promoter that regulates expression of the polynucleotides in the site 2 construct.
  • CAR1 chimeric antigen receptor
  • the site 2 construct optionally further comprises one or more of: (a) a polynucleotide encoding
  • the site 2 comprises one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, TCR constant region, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR ⁇ or ⁇ constant region (TRAC or TRBC), NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD54, CD56, CD58, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, ASB2, or TIGIT.
  • the ADR is specific to 41BB.
  • the ADR comprises a 41BB-specific ligand operably linked to a signaling domain promoting effector cell activation, and wherein ADR comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NOs: 66 - 69.
  • the ADR comprises a signaling domain, and wherein the signaling domain comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 60.
  • the present disclosure provides a cell or population thereof, wherein: (i) the cell is (a) an immune cell; (b) an induced pluripotent cell (iPSC); or (c) a derivative effector cell obtained from differentiating the iPSC; and (ii) the cell comprises a polynucleotide encoding a IL2, a IL18, or a IL7/IL7R fusion protein; wherein the polynucleotide is inserted into a TCR locus, thereby knocking out a TCR gene; and wherein the construct is expressed under an endogenous TCR promoter.
  • the checkpoint inhibitor comprises: (a) one or more antagonists to checkpoint molecules comprising 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, lirimumab, monalizumab, nivolumab, pe
  • the engager comprises: (i) a bispecific T cell engager (BiTE); (ii) a bispecific killer cell engager (BiKE); or (iii) a tri-specific Attorney Docket No.: FATE-173/01WO killer cell engager (TriKE); or wherein the engage comprises (a) a first binding domain recognizing an extracellular portion of CD3, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variants thereof of the cell or a by-stander immune effector cell; and (b) a second binding domain specific to an antigen comprising any one of: B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3
  • the present disclosure provides a master cell bank (MCB) comprising the iPSC according to any of the various aspects or embodiments herein.
  • MBC master cell bank
  • the present disclosure provides a therapeutic use of a composition disclosure herein.
  • the therapeutic use comprises introducing the composition to a subject in need of an adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematological malignancy, a solid tumor, cancer, or a virus infection.
  • the present disclosure provides a method of improving T cell differentiation. In some embodiments, the method comprises regulating ADR expression under an endogenous promoter that is temporally regulated during T cell differentiation.
  • the endogenous promoter is activated upon commitment to lymphoid lineage, and wherein the endogenous promoter activity is further heightened upon commitment to T cell lineage.
  • the endogenous promoter comprises a promoter of at least one of TRAC, TIM-3, ASB2, and TIGIT.
  • the ADR is co-expressed with a cytokine comprising IL2, IL18, or IL7RF.
  • the derivative effector cell is an immune effector cell
  • the method comprises: (a) obtaining a genetically engineered iPSC, wherein the iPSC comprises edits comprising: (i) a polynucleotide encoding a TCR promoter-driven ADR and/or a cytokine, (ii) a polynucleotide encoding a TCE, (iii) a polynucleotide encoding a CAR, (iv) a polynucleotide encoding a TGF ⁇ - SRR, (v) a polynucleotide encoding a C-X-C-motif chemokine receptor or a variant thereof, (vi) a polynucleotide encoding an exogenous CD16 or variant thereof, and (vii) a CD38 knockout; (b) differentiating the genetically engineered iPSC to a derivative CD34 + cell; and (c) different
  • obtaining the genetically engineered iPSC comprises: (i) generating a first pre-MCB (master cell bank) iPSC by introducing to an iPSC a construct comprising polynucleotides encoding a TGF ⁇ -SRR, a C-X-C-motif chemokine Attorney Docket No.: FATE-173/01WO receptor or a variant thereof, and a CD16 variant to a first integration site (site 1); (ii) generating a second pre-MCB iPSC by introducing to the first pre-MCB iPSC a construct comprising a polynucleotide encoding an allo-immune defense receptor (ADR) and a polynucleotide encoding one of IL2, IL18 and a IL7RF at a TCR locus; and (iii) generating a MCB iPSC by introducing to the second pre-MCB iPSC a construct comprising
  • the site 2 differs from the site 1, and wherein the site 1 and site 2 comprise any two of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, TCR constant region, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD54, CD56, CD58, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, ASB2, and TIGIT loci.
  • the site 2 comprise CD38, CD54, CD56, CD58, TIM3, TIGIT, H11 and PH12; wherein integration at any of CD38, CD54, CD56, CD58, TIM3, ASB2, or TIGIT knocks out an endogenous gene.
  • the TCR constant region is a constant region of TCR alpha (TRAC) or TCR beta (TRBC); wherein endogenous gene at TRAC or TRBC is knocked out; and wherein the construct is under control of TRAC or TRBC endogenous promoter.
  • the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3;
  • the TGF ⁇ -SRR comprises a TGF ⁇ R2-IL2R ⁇ , a TGF ⁇ R2-IL12R ⁇ , a TGF ⁇ R2-IL18R ⁇ , or a TGF ⁇ R2-trIL12R ⁇ redirector receptor;
  • the CD16 variant is a high affinity non-cleavable CD16 (hnCD16);
  • the ADR is specific to 4-1BB; or
  • the TCE is TCF1 or CD27.
  • the method further comprises genetically engineering the first pre-MCB iPSC, the second pre-MCB iPSC, or the MCB iPSC by one or more of: (a) introducing HLA-I deficiency, and/or HLA-II deficiency; (b) deleting or disrupting one or more of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD44, CD54, CD56, CD58, CD69, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, ASB2, and TIGIT; or (c) introducing at least one of HLA-G, HLA-E, 4-1BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, chimeric
  • the genetic engineering comprises targeted editing.
  • the targeted editing is carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods.
  • the present disclosure provides a method of treating a subject in need of an adoptive cell therapy, wherein the method comprises infusing the subject with effector cells, wherein the effector cells comprise the derivative cell or population thereof according to any of the various aspect or embodiments herein.
  • the effector cells Attorney Docket No.: FATE-173/01WO comprise a CAR specific to an antigen expressed on a cancer cell, wherein the antigen comprises at least one of B7H3, BCMA, CD19, CD20, CD22, CD38, CD52, CD79b, CD123, EGFR, EGP2/EpCAM, GD2, GPRC5D, HER2, KLK2, MICA/B, MSLN, VEGF-R2, PSMA and PDL1.
  • the method further comprises administering one or more therapeutic agents to the subject, wherein the one or more therapeutic agents comprise: (i) a cytokine, an antibody, an engager, a checkpoint inhibitor, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD); (ii) an anti-CD38 antibody comprising daratumumab, isatuximab, or MOR202; (iii) an engager comprising a BiTE (bi-specific T cell engager) or a TriKE (tri-specific Killer cell engager); (iv) a checkpoint inhibitor comprising atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, or pembrolizumab; and/or (v) a chemotherapeutic agent comprising cyclophospham
  • FIG.3 provides illustrative results showing that effector cells comprising a selected TCE and/or cytokine signaling in a configuration as indicated have T cell signatures marked by TRAC-CAR and intracellular CD3 (icCD3) expression.
  • FIGS.4A-4B show illustrative results for tumor volume for individual mice plotted against days post tumor implant for each effector cell group as indicated, and show that certain edits and/or configurations resulted in enhanced TGI compared to the CAR-iT cell control group.
  • FIG.8 shows illustrative results for progressive and titrated expression of ADR under endogenous TRAC promoter regulation during the iT cell differentiation process.
  • FIG.9 shows illustrative results for appropriate lymphoid commitment in the process of T cell differentiation from iPSC comprising TRAC regulated ADR.
  • FIGS.10 A-10B provide illustrative results showing that the TRAC regulated ADR expression in CAR iT cells enables resistance to allogenic immune cells in vivo.
  • FIGS.11A-11B provide illustrative results showing that CD58 knockout does not impact ADR function, and synergizes with ADR to resist allogeneic rejection.
  • FIG.12 provides illustrative results showing that TRAC regulated IL2 expression improves the anti-tumor efficacy and intratumoral persistence of CAR iT cells in vivo.
  • FIGS.13A-13B provide illustrative results showing that IL2 production regulated temporally by TRAC following CAR activation is much lower than that of primary CAR T cells.
  • FIGS.14A-14D provide illustrative results showing that engineering of IL2 into alternative T cell loci enables higher IL2 expression after CAR activation, but only TRAC regulated IL2 expression enhances the persistence and sustained in vivo efficacy of CAR iT cells.
  • FIGS.15A-15C provide schematics and illustrative results showing that TRAC regulated IL2 supports hnCD16 and ADR mediated efficacy in effector cells.
  • FIGS.16A-16B provide illustrative results showing that iT cells with constitutively regulated CAR maintain cytolytic activity over multiple rounds of tumor challenge in vitro without expected exhaustion.
  • FIG.17 provides illustrative results showing that effector cells having a constitutive CAR outperform those having TRAC regulated CAR in a subcutaneous in vivo model of solid tumor.
  • FIGS.18A-18B provide illustrative results showing that constitutive expression of TCF1 reinforces T cell lineage commitment in engineered iPSC differentiation.
  • FIGS.19A-19B provide schematics and illustrative results showing that iT cells comprising an ADR_2 backbone (TRAC-ADR-IL2, CD38KO_ TGF ⁇ -SRR-hnCD16-CXCR2), and constitutively expressed CAR-TCF1 from the CD58 locus demonstrated improved in vivo antitumor activity in comparison control cells without TCF1.
  • ADR_2 backbone TRAC-ADR-IL2, CD38KO_ TGF ⁇ -SRR-hnCD16-CXCR2
  • FIGS.20A-20C provide illustrative results showing that constitutively expressed TCF1 improves ADR_2 iT cells in both allogenic defense and antitumor function.
  • FIG.21 provides illustrative results showing the workflow and strategic design for generating a Master Cell Bank (MCB) having at least three targeted integration sites hosting multiplex configured edits via multiple MCB intermediaries (pre-MCBs).
  • FIGS.22A-22C provide illustrative flow cytometry analyses confirming CD58 targeted CAR and TCF1 insertion in various configurations.
  • 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.
  • an element means one element or more than one element.
  • the use of the alternative e.g., “or” should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the term “and/or” should be understood to mean either one, or both of the alternatives.
  • the term “about” or “approximately” refers to a quantity, 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 quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “substantially free of” and “essentially free of” are used interchangeably, and when used to describe a composition, such as a cell population or culture media, refer to a composition that is free of a specified substance or its source thereof, such as, 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance or its source thereof, or is undetectable as measured by conventional means.
  • the term “free of” or “essentially free of” a certain ingredient or substance in a composition also means that no such ingredient or substance is (1) included in the composition at any concentration, or (2) included in the composition at a functionally inert, low concentration.
  • ex vivo procedures involve living cells or tissues taken from an organism and cultured in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours or longer, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be “in vitro,” though in certain embodiments, this term can be used interchangeably with ex vivo. [00066] The term “in vivo” refers generally to activities that take place inside an organism.
  • reprogramming or “dedifferentiation” or “increasing cell potency” or “increasing developmental potency” refer to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state.
  • a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state.
  • a reprogrammed cell is one that is in a less differentiated state than the same cell in a non- reprogrammed state.
  • embryonic stem cell refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. They do not contribute to the extra-embryonic membranes or the placenta (i.e., are not totipotent).
  • multipotent stem cell refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (i.e., ectoderm, Attorney Docket No.: FATE-173/01WO mesoderm and endoderm), but not all three. Thus, a multipotent cell can also be termed a “partially differentiated cell.” Multipotent cells are known in the art, and examples of multipotent cells include adult stem cells, such as for example, hematopoietic stem cells and neural stem cells. “Multipotent” indicates that a cell may form many types of cells in a given lineage, but not cells of other lineages.
  • pluripotency Two types have previously been described: the “primed” or “metastable” state of pluripotency akin to the epiblast stem cells (EpiSC) of the late blastocyst, and the “na ⁇ ve” or “ground” state of pluripotency akin to the inner cell mass of the early/preimplantation blastocyst.
  • EpiSC epiblast stem cells
  • HE definitive hemogenic endothelium
  • iHE plural stem cell-derived definitive hemogenic endothelium
  • Hematopoietic stem and progenitor cells are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T cells, B cells, Attorney Docket No.: FATE-173/01WO NK cells).
  • T cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells).
  • T cell can also refer to a genetically engineered T cell, such as a T cell modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • a T cell or T cell like effector cell can also be differentiated from a stem cell or progenitor cell (“a derived T cell” or “a derived T cell like effector cell”, or collectively, “a derivative T lineage cell”).
  • a derived T cell like effector cell may have a T cell lineage in some respects, but at the same time has one or more functional features that are not present in a primary T cell.
  • a T cell, a T cell like effector cell, a derived T cell, a derived T cell like effector cell, or a derivative T lineage cell are collectively termed as “a T lineage cell”.
  • CD4 + T cells refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune response. They are characterized by secretion profiles following stimulation, which may include secretion of cytokines such as IFN-gamma, TNF- alpha, IL2, IL4 and IL10.
  • cytokines such as IFN-gamma, TNF- alpha, IL2, IL4 and IL10.
  • CD4 are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes/macrophages.
  • CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility Attorney Docket No.: FATE-173/01WO complex) class II-restricted immune responses. On T-lymphocytes they define the helper/inducer subset.
  • CD8 + T cells refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells.
  • CD8 are differentiation antigens found on thymocytes and on cytotoxic and suppressor T-lymphocytes.
  • CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.
  • NK cell or “Natural Killer cell” refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3).
  • An NK cell can be any NK cell, such as a cultured NK cell, e.g., a primary NK cell, or an NK cell from a cultured or expanded NK cell or a cell-line NK cell, e.g., NK-92, or an NK cell obtained from a mammal that is healthy or with a disease condition.
  • adaptive NK cell and “memory NK cell” are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56 + , expressing at least one of NKG2C and CD57, and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceR ⁇ , and EAT-2.
  • isolated subpopulations of CD56 + NK cells comprise expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-1.
  • CD56 + can be dim or bright expression.
  • effector cell includes, and in some contexts is interchangeable with, immune cells, “differentiated immune cells,” and primary or differentiated cells that are edited and/or modulated to carry out a specific activity in response to stimulation and/or activation.
  • Non-limiting examples of effector cells include primary- sourced or iPSC-derived T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.
  • the term “isolated” or the like refers to a cell, or a population of cells, which has been separated from its original environment, i.e., the environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist.
  • the term includes a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample.
  • the term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated form a cell culture or cell suspension. Therefore, an “isolated cell” is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
  • Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions and cells cultured in a medium that is non-naturally occurring.
  • Isolated cells may be obtained by separating the desired cells, or populations thereof, from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.
  • purify or the like refers to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.
  • a “construct” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo.
  • a “vector,” as used herein refers to any nucleic acid construct capable of directing the delivery or transfer of a foreign genetic material to target cells, where it can be replicated and/or expressed.
  • vector comprises the construct to be delivered.
  • a vector can be a linear or a circular molecule.
  • a vector can be integrating or non-integrating.
  • the major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes.
  • Viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors, and the like.
  • a “gene of interest” or “a polynucleotide sequence of interest” is a DNA sequence that is transcribed into RNA and in some instances translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences.
  • a gene or polynucleotide of interest can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences.
  • a gene of interest may encode an miRNA, an shRNA, a native polypeptide (i.e., a polypeptide found in nature) or fragment thereof; a variant polypeptide (i.e., a mutant of the native polypeptide having less than 100% sequence identity with the native polypeptide) or fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.
  • sequence of a polynucleotide is composed of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds.
  • a polypeptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids of a polypeptide.
  • the terms refer to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are 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.
  • the polypeptides Attorney Docket No.: FATE-173/01WO include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or a combination thereof.
  • subunit refers to each separate polypeptide chain of a protein complex, where each separate polypeptide chain can form a stable folded structure by itself.
  • CD3 complex is composed of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ subunits, which form the CD3 ⁇ /CD3 ⁇ , CD3 ⁇ /CD3 ⁇ , and CD3 ⁇ /CD3 ⁇ dimers.
  • CD3 ⁇ /CD3 ⁇ , CD3 ⁇ /CD3 ⁇ , and CD3 ⁇ /CD3 ⁇ dimers are composed of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ subunits, which form the CD3 ⁇ /CD3 ⁇ , CD3 ⁇ /CD3 ⁇ , and CD3 ⁇ /CD3 ⁇ dimers.
  • domains contiguous portions of the polypeptide chain frequently fold into compact, local, semi-independent units that are called “domains”.
  • Many protein domains may further comprise independent “structural subunits”, also called subdomains, contributing to a common function of the domain.
  • subdomain refers to a protein domain inside of a larger domain, for example, a binding domain within an ectodomain of a cell surface receptor; or a stimulatory domain or a signaling domain of an endodomain of a cell surface receptor.
  • “Operably-linked” or “operatively linked,” interchangeable with “operably connected” or “operatively connected,” refers to the association of nucleic acid sequences on a single nucleic acid fragment (or amino acids in a polypeptide with multiple domains) so that the function of one is affected by the other.
  • Fusion proteins or “chimeric proteins”, as used herein, are proteins created through genetic engineering to join two or more partial or whole polynucleotide coding sequences encoding separate proteins, and the expression of these joined polynucleotides results in a single peptide or multiple polypeptides with functional properties derived from each of the original proteins or fragments thereof. Between two neighboring polypeptides of different sources in the fusion protein, a linker (or spacer) peptide can be added.
  • the term “genetic imprint” refers to genetic or epigenetic information that contributes to preferential therapeutic attributes in a source cell or an iPSC, and is retainable in the source cell derived iPSCs, and/or the iPSC-derived hematopoietic lineage cells.
  • a source cell is a non-pluripotent cell that may be used for generating iPSCs through reprogramming, and the source cell derived iPSCs may be further differentiated to Attorney Docket No.: FATE-173/01WO specific cell types including any hematopoietic lineage cells.
  • the genetic imprint contributing to preferential therapeutic attributes may include any context- specific genetic or epigenetic modifications which manifest a retainable phenotype, i.e., a preferential therapeutic attribute, that is passed on to derivative cells of the selected source cell, irrespective of the underlying molecular events being identified or not.
  • Donor-, disease-, or treatment response- specific source cells may comprise genetic imprints that are retainable in iPSCs and derived hematopoietic lineage cells, which genetic imprints include but are not limited to, prearranged monospecific TCR, for example, from a viral specific T cell or invariant natural killer T (iNKT) cell; trackable and desirable genetic polymorphisms, for example, homozygous for a point mutation that encodes for the high-affinity CD16 receptor in selected donors; and predetermined HLA requirements, i.e., selected HLA-matched donor cells exhibiting a haplotype with increased population.
  • prearranged monospecific TCR for example, from a viral specific T cell or invariant natural killer T (iNKT) cell
  • iNKT invariant natural killer T
  • predetermined HLA requirements i.e., selected HLA-matched donor cells exhibiting a haplotype with increased population.
  • preferential therapeutic attributes include improved engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity of a derived cell.
  • a preferential therapeutic attribute may also relate to antigen targeting receptor expression; HLA presentation or lack thereof; resistance to tumor microenvironment; induction of bystander immune cells and immune modulations; improved on-target specificity with reduced off-tumor effect; and/or resistance to treatment such as chemotherapy.
  • derivative cells having one or more therapeutic attributes are obtained from differentiating an iPSC that has genetic imprint(s) conferring a preferential therapeutic attribute incorporated thereto, such derivative cells are also called “synthetic cells”.
  • a synthetic cell possesses one or more non-native cell functions when compared to its closest counterpart primary cell, whether the synthetic cell is differentiated from engineered pluripotent cells or obtained by engineering a primary cell from natural/native sources, such as peripheral blood, umbilical cord blood, or other donor tissues.
  • natural/native sources such as peripheral blood, umbilical cord blood, or other donor tissues.
  • synthetic effector cells, or synthetic NK cells or synthetic T cells are cells differentiated from a genomically modified iPSC, as compared to their primary counterpart Attorney Docket No.: FATE-173/01WO obtained from natural/native sources such as peripheral blood, umbilical cord blood, or other donor tissues.
  • the synthetic cell possesses one or more non-native cell functions when compared to its closest counterpart primary cell.
  • an NK cell with an “enhanced therapeutic property” refers to a therapeutic property of a cell that is enhanced as compared to a typical immune cell of the same general cell type.
  • an NK cell with an “enhanced therapeutic property” will possess an enhanced, improved, and/or augmented therapeutic property as compared to a typical, unmodified, and/or naturally occurring NK cell.
  • Therapeutic properties of an immune cell may include, but are not limited to, cell engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity.
  • an immune cell are also manifested by antigen targeting receptor expression; HLA presentation or lack thereof; resistance to tumor microenvironment; induction of bystander immune cells and immune modulations; improved on-target specificity with reduced off-tumor effect; and/or resistance to treatment such as chemotherapy.
  • the term “engager” refers to a molecule, e.g., a fusion polypeptide, which is capable of forming a link between an immune cell (e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, a neutrophil), and a tumor cell; and activating the immune cell.
  • engagers include, but are not limited to, bi-specific T cell engagers (BiTEs), bi-specific killer cell engagers (BiKEs), tri-specific killer cell engagers (TriKEs), or multi-specific killer cell engagers, or universal engagers compatible with multiple immune cell types.
  • the term “surface triggering receptor” refers to a receptor capable of triggering or initiating an immune response, e.g., a cytotoxic response. Surface triggering receptors may be engineered, and may be expressed on effector cells, e.g., a T cell, a NK cell, a NKT cell, a B cell, a macrophage, or a neutrophil.
  • the surface triggering receptor facilitates bi- or multi- specific antibody engagement between the effector cells and a specific target cell (e.g., a tumor cell) independent of the effector cells’ natural receptors and cell types.
  • a specific target cell e.g., a tumor cell
  • universal it is meant that the surface triggering receptor can be expressed in, and activate, any effector cells irrespective of the cell type, and all effector cells expressing the universal receptor can be coupled or linked to the engagers recognizable by the surface triggering receptor, regardless of the engager’s tumor binding specificities.
  • engagers having the same tumor targeting specificity are used to couple with the universal surface triggering receptor.
  • engagers having different tumor targeting specificity are used to couple with the Attorney Docket No.: FATE-173/01WO universal surface triggering receptor.
  • a surface triggering receptor generally comprises a co-stimulatory domain for effector cell activation and an anti-epitope that is specific to the epitope of an engager.
  • a bi- specific engager is specific to the anti-epitope of a surface triggering receptor on one end, and is specific to a tumor antigen on the other end.
  • the term “safety switch protein” refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of a cell therapy.
  • the safety switch protein expression is conditionally controlled to address safety concerns for transplanted engineered cells that have permanently incorporated the gene encoding the safety switch protein into its genome. This conditional regulation could be variable and might include control through a small molecule-mediated post-translational activation and tissue- specific and/or temporal transcriptional regulation.
  • the safety switch protein could mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation and/or antibody-mediated depletion.
  • the safety switch protein is activated by an exogenous molecule, e.g., a prodrug, that when activated, triggers apoptosis and/or cell death of a therapeutic cell.
  • a prodrug 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.
  • suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof.
  • a prodrug that is administered in the event of an adverse event is activated by the suicide-gene product and kills the transduced cell.
  • the term “pharmaceutically active proteins or peptides” refers to proteins or peptides that are capable of achieving a biological and/or pharmaceutical effect on an organism.
  • a pharmaceutically active protein has healing, curative or palliative properties against a disease and may be administered to ameliorate, relieve, alleviate, reverse or lessen the severity of a disease.
  • a pharmaceutically active protein also has prophylactic properties and is used to prevent the onset of a disease or to lessen the severity of such disease or pathological condition when it does emerge.
  • “Pharmaceutically active proteins” include an entire protein or peptide or pharmaceutically active fragments thereof. The term also includes pharmaceutically active analogs of the protein or peptide or analogs of fragments of the protein or peptide.
  • pharmaceutically active protein also refers to a plurality of proteins or peptides that act cooperatively or synergistically to provide a therapeutic benefit.
  • pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth suppressing proteins, antibodies or fragments thereof, growth factors, and/or cytokines.
  • signal molecule refers to any molecule that modulates, participates in, inhibits, activates, reduces, or increases, cellular signal transduction.
  • Signal transduction refers to the transmission of a molecular signal in the form of chemical modification by recruitment of protein complexes along a pathway that ultimately triggers a biochemical event in the cell.
  • Examples of signal transduction pathways are 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.
  • targeting modality refers to a molecule, e.g., a polypeptide, that is genetically incorporated into a cell to promote antigen and/or epitope specificity that includes, but is not limited to, i) antigen specificity as it relates to a unique chimeric antigen receptor (CAR) or T cell receptor (TCR), ii) engager specificity as it relates to monoclonal antibodies or bispecific engagers, iii) targeting of transformed cells, iv) targeting of cancer stem cells, and v) other targeting strategies in the absence of a specific antigen or surface molecule.
  • CAR unique chimeric antigen receptor
  • TCR T cell receptor
  • engager specificity as it relates to monoclonal antibodies or bispecific engagers
  • targeting of transformed cells iv) targeting of cancer stem cells
  • other targeting strategies in the absence of a specific antigen or surface molecule.
  • the term “specific” or “specificity” can be used to refer to the ability of a molecule, e.g., a receptor or an engager, to selectively bind to a target molecule, in contrast to non-specific or non-selective binding.
  • the term “adoptive cell therapy” as used herein refers to a cell-based immunotherapy that relates to the transfusion of autologous or allogeneic lymphocytes, whether the immune cells are isolated from a human donor, or effector cells obtained from in vitro differentiation of a pluripotent cell; whether they are genetically modified or not; or whether they are primary donor cells or cells that have been passaged, expanded, or immortalized, ex vivo, after isolation from a donor.
  • radiation refers to the emission or transmission of energy in the form of waves or particles.
  • Exemplary forms of radiation include, but are not limited to, electromagnetic radiation (e.g., radio waves, microwaves, infrared, visible light, ultraviolet, x- rays, and gamma radiation), particle radiation (e.g., alpha radiation, beta radiation, proton radiation and neutron radiation), and acoustic radiation (e.g., ultrasound, sound and seismic waves).
  • electromagnetic radiation e.g., radio waves, microwaves, infrared, visible light, ultraviolet, x- rays, and gamma radiation
  • particle radiation e.g., alpha radiation, beta radiation, proton radiation and neutron radiation
  • acoustic radiation e.g., ultrasound, sound and seismic waves.
  • the amount of radiation is measured as a Gray (Gy), which is defined as the absorption of one joule of radiation energy per kilogram of matter.
  • Gray Gray
  • the amount of radiation applied varies depending on the type and stage of cancer being treated.
  • the typical dose for a solid epithelial tumor ranges from 60 to 80 Gy, while lymphomas are typically treated with 20 to 40 Gy.
  • Preventive (adjuvant) doses are Attorney Docket No.: FATE-173/01WO typically around 45–60 Gy in 1.8–2 Gy fractions (for, e.g., breast, head, and neck cancers).
  • radiation may be used as a sensitizing agent as disclosed herein.
  • “radiation therapy” or “radiotherapy” are used interchangeably to refer to a type of cancer treatment that involves use of radiation to damage cells by destroying the genetic material that controls how cells grow and divide.
  • radiation therapy often refers to external beam radiation therapy, wherein high-energy beams (e.g., x-rays, gamma rays, photons, protons, neutrons, ions, and any other forms of energy applicable to such treatments) are produced by a machine outside of the subject being treated, and are aimed at a precise point on the subject’s body.
  • high-energy beams e.g., x-rays, gamma rays, photons, protons, neutrons, ions, and any other forms of energy applicable to such treatments
  • radiation therapy also includes brachytherapy, wherein seeds, ribbons, or capsules that contain or are otherwise linked to a radiation source are placed inside the subject’s body in or near a tumor or cancer cell.
  • brachytherapy low-dose rate implants, high-dose rate implants, and permanent implants.
  • radiation therapy is systemic radiation therapy, wherein radioactive drugs (e.g., radiopharmaceuticals or radionuclides, including radiopeptides) are given to the subject orally or intravenously and collect within the subject’s body at the tumor or area where cancers cells are located.
  • radioactive drugs e.g., radiopharmaceuticals or radionuclides, including radiopeptides
  • radiopharmaceuticals incorporate a radioactive compound linked to a targeting molecule (such as an antibody) that specifically binds to a tumor antigen.
  • radioactive compounds useful in radiopharmaceuticals include, but are not limited to calcium-47, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gallium-68, hydrogen- 3, indium-111, iodine-123, iodine-125, iodine-131, iorn-59, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, radium-223, rubidium-82, samarium-153, selenium-75, sodium-22, sodium-24, strontium-89, technetium-99m, thallium-201, xenon-133, and yttrium-90.
  • lymphodepletion and “lympho-conditioning” are used interchangeably to refer to the destruction of lymphocytes and T cells, typically prior to immunotherapy.
  • the purpose of lympho-conditioning prior to the administration of an 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.
  • lympho-conditioning is typically accomplished by administering one or more chemotherapeutic agents to the subject prior to a first dose of the adoptive cell therapy.
  • lympho-conditioning precedes the first dose of the adoptive Attorney Docket No.: FATE-173/01WO cell therapy by a few hours to a few days.
  • chemotherapeutic agents useful for lympho-conditioning include, but are not limited to, cyclophosphamide (CY), fludarabine (FLU), and those described below.
  • CY cyclophosphamide
  • FLU fludarabine
  • a sufficient lymphodepletion through anti-CD38 mAb could provide an alternative conditioning process for the present iNK cell therapy, without or with minimal need of a CY/FLU-based lympho-conditioning procedure, as further described herein.
  • homing or “trafficking” refers to active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., tumor), or organ).
  • a “homing molecule” refers to a molecule that directs cells to a target site.
  • a homing molecule functions to recognize and/or initiate interaction of a cell to a target site.
  • a homing molecule is a chemokine receptor.
  • chemokine receptor refers to a cell surface molecule that binds to a chemokine.
  • a chemokine receptor can comprise a naturally occurring or recombinant chemokine receptor or a variant thereof.
  • chemokine receptors include, but are not limited to, a CXC chemokine receptor (for example, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (for example, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), a CX3C chemokine receptor (for example, CX3CR1), an XC chemokine receptor (for example, XCR1), or a variant thereof.
  • a “therapeutically sufficient amount”, as used herein, includes within its meaning a non-toxic, but sufficient and/or effective amount of a particular therapeutic agent and/or pharmaceutical composition to which it is referring to provide a desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient’s general health, the patient’s age and the stage and severity of the condition being treated. In particular embodiments, a “therapeutically sufficient amount” is sufficient and/or effective to ameliorate, reduce, and/or improve at least one symptom associated with a disease or condition of the subject being treated. [000117] Differentiation of pluripotent stem cells requires a change in the culture system, such as changing the stimuli agents in the culture medium or the physical state of the cells.
  • EBs embryoid bodies
  • Embryoid bodies are three- dimensional clusters that have been shown to mimic embryo development as they give rise to numerous lineages within their three-dimensional area.
  • simple EBs for example, aggregated pluripotent stem cells elicited to differentiate
  • EB formation is initiated Attorney Docket No.: FATE-173/01WO by bringing pluripotent stem cells into close proximity with one another in three-dimensional multilayered clusters of cells.
  • pluripotent stem cell aggregates require further differentiation cues, as aggregates maintained in pluripotent culture maintenance medium do not form proper EBs. As such, the pluripotent stem cell aggregates need to be transferred to differentiation medium that provides eliciting cues towards the lineage of choice.
  • EB-based culture of pluripotent stem cells typically results in generation of differentiated cell populations (i.e., ectoderm, mesoderm and endoderm germ layers) with modest proliferation within the EB cell cluster.
  • EBs Although proven to facilitate cell differentiation, EBs, however, give rise to heterogeneous cells in variable differentiation states because of the inconsistent exposure of the cells in the three-dimensional structure to the differentiation cues within the environment. In addition, EBs are laborious to create and maintain. Moreover, cell differentiation through EB formation is accompanied with modest cell expansion, which also contributes to low differentiation efficiency. [000118] In comparison, “aggregate formation,” as distinct from “EB formation,” can be used to expand the populations of pluripotent stem cell derived cells. For example, during aggregate-based pluripotent stem cell expansion, culture media are selected to maintain proliferation and pluripotency.
  • Cell proliferation generally increases the size of the aggregates, forming larger aggregates, which can be mechanically or enzymatically dissociated into smaller aggregates to maintain cell proliferation within the culture and increase numbers of cells.
  • cells cultured within aggregates in maintenance culture media maintain markers of pluripotency.
  • the pluripotent stem cell aggregates require further differentiation cues to induce differentiation.
  • “monolayer differentiation” is a term referring to a differentiation method distinct from differentiation through three-dimensional multilayered clusters of cells, i.e., “EB formation.”
  • Monolayer differentiation avoids the need for EB formation to initiate differentiation.
  • a “dissociated cell” or “single dissociated cell” refers to a cell that has been substantially separated or purified away from other cells or from a surface (e.g., a culture plate surface).
  • a surface e.g., a culture plate surface.
  • cells can be dissociated from an animal or tissue by mechanical or enzymatic methods.
  • cells that aggregate in vitro can be Attorney Docket No.: FATE-173/01WO enzymatically or mechanically dissociated from each other, such as by dissociation into a suspension of clusters, single cells or a mixture of single cells and clusters.
  • adherent cells can be dissociated from a culture plate or other surface. Dissociation thus can involve breaking cell interactions with extracellular matrix (ECM) and substrates (e.g., culture surfaces), or breaking the ECM between cells.
  • ECM extracellular matrix
  • a “master cell bank” or “MCB” refers to a clonal master engineered iPSC line, which is a clonal population of iPSCs that have been engineered to comprise one or more therapeutic attributes, have been characterized, tested, qualified, and expanded, and have been shown to reliably serve as the starting cellular material for the production of cell-based therapeutics through directed differentiation in manufacturing settings.
  • an MCB is maintained, stored, and/or cryopreserved in multiple vessels to prevent genetic variation 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 processes.
  • feeder cells are terms describing cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimulation, growth factors and nutrients for the support of the second cell type.
  • the feeder cells are optionally from a different species as the cells they are supporting.
  • certain types of human cells, including stem cells can be supported by primary cultures of mouse embryonic fibroblasts, or immortalized mouse embryonic fibroblasts.
  • peripheral blood derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells.
  • the feeder cells may typically be inactivated when being co-cultured with other cells by irradiation or treatment with an anti-mitotic agent such as mitomycin to prevent them from outgrowing the cells they are supporting.
  • Feeder cells may include endothelial cells, stromal cells (for example, epithelial cells or fibroblasts), and leukemic cells.
  • one specific feeder cell type may be a human feeder, such as a human skin fibroblast.
  • Another feeder cell type may be mouse embryonic fibroblasts (MEF).
  • various feeder cells can be used in part to maintain pluripotency, direct differentiation towards a certain lineage, enhance proliferation capacity and promote maturation to a specialized cell type, such as an effector cell.
  • a “feeder-free” (FF) environment refers to an environment such as a culture condition, cell culture or culture media which is essentially free of feeder or stromal cells, and/or which has not been pre-conditioned by the cultivation of feeder cells.
  • Pre- conditioned” medium refers to a medium harvested after feeder cells have been cultivated within the medium for a period of time, such as for at least one day. Pre-conditioned medium contains many mediator substances, including growth factors and cytokines secreted by the feeder cells Attorney Docket No.: FATE-173/01WO cultivated in the medium.
  • a feeder-free environment is free of both feeder or stromal cells and is also not pre-conditioned by the cultivation of feeder cells.
  • HLA deficient including HLA class I deficient, HLA class II deficient, or both, refers to cells that either lack, or no longer maintain, or have a reduced level of surface expression of a complete MHC complex comprising an HLA class I protein heterodimer and/or an HLA class II heterodimer, such that the diminished or reduced level is less than the level naturally detectable by other cells or by synthetic methods.
  • Modified HLA deficient iPSC refers to an HLA deficient iPSC that is further modified by introducing genes expressing proteins related, but not limited to improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, costimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cellular cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptor (CAR), T cell receptor (TCR), CD16 Fc Receptor, BCL11b, NOTCH, RUNX1, IL15, 4-1BB, DAP10, DAP12, CD24, CD3 ⁇ , 4-1BBL, CD47, CD113, and PDL1.
  • non-classical HLA class I proteins e.g., HLA-E and HLA-G
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the cells that are “modified HLA deficient” also include cells other than iPSCs.
  • the term “ligand” refers to a substance that forms a complex with a target molecule to produce a signal by binding to a site on the target.
  • the ligand may be a natural or artificial Attorney Docket No.: FATE-173/01WO substance capable of specific binding to the target.
  • the ligand may be in the form of a protein, a peptide, an antibody, an antibody complex, a conjugate, a nucleic acid, a lipid, a polysaccharide, a monosaccharide, a small molecule, a nanoparticle, an ion, a neurotransmitter, or any other molecular entity capable of specific binding to a target.
  • the target to which the ligand binds may be a protein, a nucleic acid, an antigen, a receptor, a protein complex, or a cell.
  • a ligand that binds to and alters the function of the target and triggers a signaling response is called “agonistic” or “an agonist”.
  • a ligand that binds to a target and blocks or reduces a signaling response is “antagonistic” or “an antagonist.”
  • antibody is used herein in the broadest sense and refers generally to an immune-response generating molecule that contains at least one binding site that specifically binds to a target, wherein the target may be an antigen, or a receptor that is capable of interacting with certain antibodies.
  • an NK cell can be activated by the binding of an antibody or the Fc region of an antibody to its Fc-gamma receptors (Fc ⁇ R), thereby triggering the ADCC (antibody-dependent cellular cytotoxicity) mediated effector cell activation.
  • Fc ⁇ R Fc-gamma receptors
  • antibody includes, but is not limited to, native antibodies and variants thereof, fragments of native antibodies and variants thereof, peptibodies and variants thereof, and antibody mimetics that mimic the structure and/or function of an antibody or a specified fragment or portion thereof, including single chain antibodies and fragments thereof.
  • An antibody may be a murine antibody, a human antibody, a humanized antibody, a camel IgG, a single variable new antigen receptor (VNAR), a shark heavy-chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single-domain antibody (dAb), an anti-idiotype antibody, a bi-specific-, multi-specific- or multimeric- antibody, or antibody fragment thereof.
  • Anti-idiotype antibodies are specific for binding to an idiotope of another antibody, wherein the idiotope is an antigenic determinant of an antibody.
  • a bi-specific antibody may be a BiTE (bi-specific T cell engager) or a BiKE (bi-specific killer cell engager), and a multi-specific antibody may be a TriKE (tri-specific Killer cell engager).
  • antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fabc, pFc, Fd, single chain fragment variable (scFv), tandem scFv (scFv)2, single chain Fab (scFab), disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb), camelid heavy-chain IgG and Nanobody® fragments, recombinant heavy- chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the antibody.
  • Fc receptors are classified based on the type of antibody that they recognize. For example, those that bind the most common class of antibody, IgG, are called Attorney Docket No.: FATE-173/01WO Fc-gamma receptors (Fc ⁇ R), those that bind IgA are called Fc-alpha receptors (Fc ⁇ R) and those that bind IgE are called Fc-epsilon receptors (Fc ⁇ R).
  • the classes of FcRs 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 include several members, Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32), Fc ⁇ RIIB (CD32), Fc ⁇ RIIIA (CD16a), and Fc ⁇ RIIIB (CD16b), which differ in their antibody affinities due to their different molecular structures.
  • “Chimeric Receptor” is a general term used to describe an engineered, artificial, or a hybrid receptor protein molecule that is made to comprise two or more portions of amino acid sequences that are originated from at least two different proteins. The chimeric receptor proteins have been engineered to give a cell the ability to initiate signal transduction and carry out downstream function upon binding of an agonistic ligand to the receptor.
  • 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.
  • CARs chimeric antigen receptors
  • CFRs chimeric fusion receptors
  • CCFcRs chimeric Fc receptors
  • CFcR Chimeric Fc Receptor
  • one or more stimulatory domains can be introduced to the intracellular portion of the engineered Fc receptor to enhance cell activation, expansion and function upon triggering of the receptor.
  • the chimeric Fc receptor binds to an Fc fragment, or the Fc region of an antibody, or the Fc region comprised in an engager or a binding molecule and activates the cell function with or without bringing the targeted cell close in vicinity.
  • a Fc ⁇ receptor can be engineered to comprise selected transmembrane, stimulatory, and/or signaling domains in the intracellular region that respond to the binding of IgG at the extracellular domain, thereby generating a CFcR.
  • a CFcR is produced by engineering CD16, a Fc ⁇ receptor, by replacing its transmembrane domain and/or intracellular domain.
  • the extracellular domain of CD64 or the high-affinity variants of CD16 can be incorporated.
  • the proteolytic cleavage site comprising a serine at position 197 is eliminated or is replaced such at the extracellular domain of the receptor is non-cleavable, i.e., not subject to shedding, thereby obtaining a hnCD16-based CFcR.
  • CD16 a Fc ⁇ R receptor
  • CD16b has been identified to have two isoforms, Fc receptors Fc ⁇ RIIIa (CD16a) and Fc ⁇ RIIIb (CD16b).
  • CD16a is a transmembrane protein expressed by NK Attorney Docket No.: FATE-173/01WO cells, which binds monomeric IgG attached to target cells to activate NK cells and facilitate antibody-dependent cell-mediated cytotoxicity (ADCC).
  • “High affinity CD16,” “non-cleavable CD16,” or “high affinity non-cleavable CD16” (abbreviated as hnCD16), as used herein, refers to a natural or non-natural variant of CD16.
  • the wildtype CD16 has low affinity and is subject to ectodomain shedding, a proteolytic cleavage process that regulates the cells surface density of various cell surface molecules on leukocytes upon NK cell activation.
  • F176V and F158V are exemplary CD16 polymorphic variants having high affinity.
  • a CD16 variant having the cleavage site (position 195-198) in the membrane-proximal region (position 189-212) altered or eliminated is not subject to shedding.
  • the cleavage site and the membrane-proximal region are described in detail in WO2015/148926, the complete disclosure of which is incorporated herein by reference.
  • the CD16 S197P variant is an engineered non-cleavable version of CD16.
  • a CD16 variant comprising both F158V and S197P has high affinity and is non-cleavable.
  • Another exemplary high affinity and non-cleavable CD16 (hnCD16) variant is an engineered CD16 comprising an ectodomain originated from one or more of the 3 exons of the CD64 ectodomain.
  • cells comprising a set of engineered components that collectively complement (and in some cases synergize with) one another to enhance the activity of an effector cell, in the context of treating a tumor in general, and for a solid tumor microenvironment in particular.
  • the selected set of engineered components are referred to herein as a “backbone;” for its compatibility with any tumor antigen binding molecule to be expressed in the effector cell, including but not limited to, a CAR, an antibody, a bispecific antibody, and a TCR.
  • a backbone does not require any particular physical relationship between the individual components of the set, or their location within the cell; although certain association and/or arrangements (e.g., order in a co-expression construct of two or more of the individual components) may be optimized for higher expression level or ease of processing, among other considerations in a manufacturing setting.
  • a backbone may comprise integration of two expression cassettes, each at a different location in the genome of the cell.
  • the backbone comprises a plurality of genomic modifications, such as the insertion of one or more polynucleotides and/or modification to knockout one or more genes. Modifications may be made simultaneously or sequentially.
  • Non-limiting examples of effector cell function that may be increased by the modifications of the backbone include one or more of improving cell growth, proliferation, expansion, and/or effector function autonomously without contacting additionally supplied soluble cytokines in vitro or in vivo, as well as enhanced homing, trafficking, depletion or reduction of alloreactive host immune cells, and retention at tumor sites, in which the tumor cells could be sensitized to synergize with the functional features provided to the effector cells.
  • a solid tumor targeting backbone of the present Attorney Docket No.: FATE-173/01WO disclosure can be particularly beneficial in the context of an iPSC comprising the backbone, such as by providing a master cell bank providing a source of starting cells that can be modified by the simple addition of a tumor antigen binding molecule for an indication intended to be treated, and then being used as a source for differentiating enhanced effector cells with therapeutic properties for one or more intended tumor indications.
  • iPSC-derived cells are functionally improved and suitable for adoptive cell therapies following a combination of selective modalities being introduced to the cells at the level of iPSC through genomic engineering. It was previously unclear whether altered iPSCs comprising one or more provided genetic edits still have the capacity to enter cell development, and/or to mature and generate functional differentiated cells while retaining modified activities and/or properties.
  • Unanticipated failures during directed cell differentiation from iPSCs have been attributed to aspects including, but not limited to, development stage specific gene expression or lack thereof, requirements for HLA complex presentation, protein shedding of introduced surface expressing modalities, and the need for reconfiguration of differentiation protocols enabling phenotypic and/or functional change in the cell.
  • the present application has shown that the one or more selected genomic modifications as provided herein does not negatively impact iPSC differentiation potency, and the functional effector cells derived from the engineered iPSC have enhanced and/or acquired therapeutic properties attributable to the individual or combined genomic modifications retained in the effector cells following the iPSC differentiation.
  • genomic modifications and combinations thereof as may be described in the context of iPSC and iPSC-derived effector cells are applicable to primary sourced cells, including primary immune cells such as T, NK, or immunregulatory cells, whether cultured or expanded, the modification of which results in engineered immune cells useful for adoptive cell therapy.
  • primary immune cells such as T, NK, or immunregulatory cells, whether cultured or expanded, the modification of which results in engineered immune cells useful for adoptive cell therapy.
  • CAR-T cells Unlike liquid tumors where uniformly-expressed antigens are accessible and can be effectively targeted, tumor access, lack of tumor-exclusive antigen targets, and antigen heterogeneity are significant barriers to the successful development of CAR-T cells Attorney Docket No.: FATE-173/01WO in solid tumors. In addition, inherent genetic engineering variability seen with patient- and donor- derived immune cells limits the wide application of CAR-T cell therapy.
  • the present application provides genomic engineering aspects in the form of a solid tumor targeting backbone, as well as other genetic modalities, to improve on-target specificity with reduced off- tumor effect in the off-the-shelf adoptive cell therapy setting using effector cells derived from engineered iPSCs, to evade allorejection, as well as to overcome suppressive tumor microenvironment, a heightened challenge especially with solid tumors.
  • C-X-C Motif Chemokine Receptor Overexpression [000136] Chemokines are a family of homogeneous serum proteins of about 7 to about 16 kDa originally characterized by their ability to induce leukocyte migration.
  • chemokines have four characteristic cysteines (Cys) and are classified into C-X-C (or alpha, CXC), C-C (or beta), C (or gamma), and CX3C (or delta) chemokine classes, according to motifs displayed by the first two cysteines. Subfamilies of C-X-C (or alpha, CXC) are further classified, according to the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine, into two groups: ELR-CXC chemokines and non-ELR-CXC chemokines.
  • Cys characteristic cysteines
  • CXC chemokine receptor 2 also known as CD128, interleukin 8 receptor beta (IL8R ⁇ ), or L8 receptor type B
  • CD128, interleukin 8 receptor beta (IL8R ⁇ ), or L8 receptor type B is a chemokine receptor mostly expressed by neutrophils, mast cells, monocytes, and macrophages.
  • CD56 dim NK cells express CXCR2, however its expression can be downregulated upon NK cell activation.
  • T cells typically do not express CXCR2.
  • iPSCs and iPSC-derived T cells do not express CXCR2 without transducing exogenous polynucleotides encoding CXCR2 as disclosed in this application.
  • the chemokine IL8 (also known as CXCL8) is secreted by mononuclear macrophages, neutrophils, eosinophils, T lymphocytes, epithelial cells, and fibroblasts, and functions as a chemotactic factor by guiding the neutrophils to the site of infection.
  • CXCL8 is also secreted by tumor cells and promotes tumor migration, invasion, angiogenesis and metastasis.
  • CXCL8 is one of the ligands to multiple CXC chemokine receptors including CXCR1 and CXCR2.
  • CXC chemokine receptor 3 also known as G Protein-coupled Receptor 9 (GPR9) and CD183, is a G Protein-coupled receptor that binds to the chemokines CXCL9, CXCL10, and CXCL11.
  • CXCR3 is expressed primarily in activated T-helper type 1 (Th1) lymphocytes, but is also present in natural killer cells, macrophages, dendritic cells, and B lymphocyte subsets.
  • the present application provides effector cells or iPSCs genetically engineered to comprise, among other editing as contemplated and described herein, a solid tumor targeting backbone comprising, among other genetic modalities, a C-X-C motif chemokine receptor.
  • the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3, or variants thereof.
  • a non-limiting example of the amino acid sequence of human CXCR2 is one registered as UniProtKB No: P25025.
  • the CXCR2 comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 1. In some embodiments, the CXCR2 comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 1. In some embodiments, the CXCR2 comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 1. In some embodiments, the CXCR2 comprises the amino acid sequence of SEQ ID NO: 1.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm recognized in the art.
  • CXCR2 UniProtKB No: P25025
  • a non-limiting example of the amino acid sequence of human CXCR3 is one registered as UniProtKB No: P49682.
  • the CXCR3 comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 2.
  • the CXCR3 comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 2.
  • the CXCR3 comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 2.
  • the CXCR3 comprises the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide encoding the C-X-C motif chemokine receptor or variant thereof is inserted in a selected locus of a primary-sourced effector cell or an iPSC for deriving functional effector cells comprising the same genetic editing through directed differentiation.
  • the selected locus for insertion of the C-X-C motif chemokine receptor comprises a safe harbor locus, a gene locus intended to be disrupted or knocked out, a gene locus that provides an endogenous promoter that provides spacial and/or temporal control of the exogenous gene expression.
  • the selected locus for C-X-C motif chemokine receptor insertion comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.
  • the selected locus for C-X-C motif chemokine receptor insertion is the TCR locus.
  • the selected locus for C-X-C motif chemokine receptor insertion is the CD38 locus.
  • the C-X-C motif chemokine receptor is co-expressed with one or more exogenous polynucleotides encoding a polypeptide of interest through separate expression constructs, or a single bi- or tri- cistronic expression cassete.
  • the single bi- or tri- cistronic expression cassete comprising the C-X-C motif chemokine receptor and one or more exogenous polynucleotides encoding a polypeptide of interest comprises a 2A sequence, such that the C-X-C motif chemokine receptor and the additional polynucleotide(s) are in a single open reading frame (ORF).
  • ORF open reading frame
  • Self-cleaving peptides are found in members of the Picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV) and porcine tescho virus- 1 (PTV-I) (Donnelly, ML, et al, J. Gen. Virol, 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732 (2001)), and cardioviruses such as Theilovirus (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis viruses.
  • aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV) and porcine tescho virus- 1 (PTV
  • the 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are Attorney Docket No.: FATE-173/01WO sometimes also referred to as “F2A”, “E2A”, “P2A”, and “T2A”, respectively.
  • the exogenous polynucleotides that could be co-expressed with the C-X-C motif chemokine receptor encode one or more polypeptides comprising a CAR, a CD16 or a variant thereof, a cytokine, a cytokine receptor, a cytokine signaling complex, a chimeric fusion receptor, a chimeric Fc receptor, an engager, a checkpoint inhibitor, an Fc receptor, or an antibody or functional variant or fragment thereof.
  • the exogenous polynucleotides that are co-expressed with the C-X-C motif chemokine receptor in a bi-cistronic cassette do not encode a CAR.
  • At least one exogenous polynucleotide that is co-expressed in a bi-cistronic cassette with the C-X-C motif chemokine receptor encodes an exogenous CD16.
  • the primary-sourced or derived effector cells comprising the C-X-C motif chemokine receptor or variant thereof are T lineage cells.
  • the primary-sourced or derived effector cells comprising the C-X-C motif chemokine receptor or a variant thereof are NK lineage cells.
  • a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one modification or phenotype as provided herein, including but not limited to, a C-X-C motif chemokine receptor or a variant thereof, wherein the cell bank provides clonal engineered iPSCs for additional engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derivative NK and T cells, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective manner.
  • TGF ⁇ Transforming growth factor beta
  • CAF cancer associated fibroblast
  • TGF ⁇ exists in its latent form in the tumor microenvironment, and is known to suppress T cell effector function, in part, through Smad- mediated downregulation of the target genes granzyme, perforin, and interferon. Furthermore, the detection of a TGF ⁇ gene expression signature correlates with T cell exclusion from tumors and resistance to immunotherapy.
  • TGF ⁇ R transforming growth factor beta receptor
  • a “signaling (or signal) redirector receptor” or “SRR” redirects the signaling of an Attorney Docket No.: FATE-173/01WO extracellular domain from a first receptor (e.g., a TGF ⁇ receptor) through an intracellular domain from a different receptor (e.g., a cytokine receptor) by joining the extracellular domain of the first receptor and intracellular domains of the different receptor.
  • a first receptor e.g., a TGF ⁇ receptor
  • a different receptor e.g., a cytokine receptor
  • iPSCs and derivative cells therefrom comprise a polynucleotide encoding a TGF ⁇ redirector receptor (TGF ⁇ -SRR), which comprises a partial or full peptide of an extracellular domain (ECD) of TGF ⁇ R.
  • TGF ⁇ redirector receptor comprises: (i) an extracellular domain, or a fragment thereof, of transforming growth factor beta receptor (TGF ⁇ R); and (ii) an intracellular domain (ICD), or a fragment thereof, of a cytokine receptor comprising IL2R, IL12R, IL18R, IL21R, or any combination thereof.
  • the TGF ⁇ redirector receptor comprising the ECD and ICD as described above further comprises a transmembrane domain (TM).
  • the transmembrane (TM) domain of the TGF ⁇ redirector receptor can: (i) originate from the same molecule providing the intracellular domain, (ii) originate from the same molecule providing the extracellular domain, or (iii) may be modified or replaced with a transmembrane domain of any other membrane bound proteins.
  • the cytokine receptor providing an intracellular domain or a fragment thereof of the TGF ⁇ redirector receptor comprises at least one of an IL2R (e.g., IL2R ⁇ ), IL4R, IL6R, IL7R, IL9R, IL10R, IL11R, IL12R (e.g., IL12R ⁇ ), IL15R, IL18R (e.g. IL18R ⁇ ), and IL21R (e.g., IL21R ⁇ ).
  • an IL2R e.g., IL2R ⁇
  • IL4R IL6R
  • IL7R IL7R
  • IL9R IL10R
  • IL11R IL12R
  • IL15R IL18R
  • IL21R e.g., IL21R ⁇
  • the extracellular domain (ECD) of TGF ⁇ R comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 3. In some embodiments, the extracellular domain (ECD) of TGF ⁇ R comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 3. In some embodiments, the extracellular domain (ECD) of TGF ⁇ R comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 3. In some embodiments, the extracellular domain (ECD) of TGF ⁇ R comprises the amino acid sequence of SEQ ID NO: 3.
  • the intracellular domain (ICD) of IL2R ⁇ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 4. In some embodiments, the intracellular domain (ICD) of IL2R ⁇ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 4. In some embodiments, the intracellular domain (ICD) of IL2R ⁇ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 4. In some embodiments, the intracellular domain (ICD) of IL2R ⁇ comprises the amino acid sequence of Attorney Docket No.: FATE-173/01WO SEQ ID NO: 4.
  • the intracellular domain (ICD) of IL12R ⁇ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 5. In some embodiments, the intracellular domain (ICD) of IL12R ⁇ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 5. In some embodiments, the intracellular domain (ICD) of IL12R ⁇ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 5. In some embodiments, the intracellular domain (ICD) of IL12R ⁇ comprises the amino acid sequence of SEQ ID NO: 5.
  • a fragment of the intracellular domain of IL12R ⁇ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 6. In some embodiments, a fragment of the intracellular domain of IL12R ⁇ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 6. In some embodiments, a fragment of the intracellular domain of IL12R ⁇ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 6. In some embodiments, a fragment of the intracellular domain of IL12R ⁇ comprises the amino acid sequence of SEQ ID NO: 6.
  • the intracellular domain (ICD) of IL18R ⁇ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 7. In some embodiments, the intracellular domain (ICD) of IL18R ⁇ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 7. In some embodiments, the intracellular domain (ICD) of IL18R ⁇ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 7. In some embodiments, the intracellular domain (ICD) of IL18R ⁇ comprises the amino acid sequence of SEQ ID NO: 7.
  • the intracellular domain (ICD) of IL21R ⁇ comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL21R ⁇ comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL21R ⁇ comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 8. In some embodiments, the intracellular domain (ICD) of IL21R ⁇ comprises the amino acid sequence of SEQ ID NO: 8.
  • the signaling receptor comprises an extracellular domain or a fragment thereof of Attorney Docket No.: FATE-173/01WO TGF ⁇ R and an intracellular domain or a fragment thereof of the cytokine receptor IL12R ⁇ , thereby forming a TGF ⁇ R2-IL12R ⁇ signaling redirector receptor.
  • the signaling receptor comprises an extracellular domain or a fragment thereof of TGF ⁇ R and an intracellular domain or a fragment thereof of the cytokine receptor IL18R ⁇ , thereby forming a TGF ⁇ R2-IL18R ⁇ signaling redirector receptor.
  • the signaling receptor comprises an extracellular domain or a fragment thereof of TGF ⁇ R and an intracellular domain or a fragment thereof of the cytokine receptor IL21R, thereby forming a TGF ⁇ R2-IL21R signaling redirector receptor.
  • TGF ⁇ R2-IL12R ⁇ signaling redirector receptor comprises an amino acid sequence having sequence identity of at least 80%, 85%, 90%, 95%, or 97%, 98%, or 99% to a sequence represented by SEQ ID NO: 9 (termed specifically as TGF ⁇ R2-trIL12R ⁇ throughout the application).
  • TGF ⁇ R2-IL12R ⁇ signaling redirector receptor comprises an amino acid sequence having sequence identity of at least 90% to SEQ ID NO: 9.
  • TGF ⁇ R2-IL12R ⁇ signaling redirector receptor comprises an amino acid sequence having sequence identity of at least 95% to SEQ ID NO: 9. In some embodiments, TGF ⁇ R2-IL12R ⁇ signaling redirector receptor comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the transmembrane domain (TM) sequence represented by SEQ ID NO: 10 that is comprised within SEQ ID NO: 9 may vary in sequence or in length, or may even be replaced with a transmembrane domain of another transmembrane protein.
  • TGF ⁇ R2-TM-trIL12R ⁇ any of the TGF ⁇ -SRRs provided herein may be introduced to iPSCs using one or more of the construct designs described above, and to their derivative cells upon iPSC differentiation.
  • iPSC induced pluripotent cell
  • a Attorney Docket No.: FATE-173/01WO clonal iPSC, a clonal iPS cell line, or iPSC-derived cells comprising at least one engineered modality as disclosed herein are provided.
  • a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least a TGF ⁇ -SRR as described in this section, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, which are well-defined and uniform in composition, and can be mass produced at a significant scale in a cost-effective manner.
  • the present invention provides immune cells, iPSCs, and iPSC derived cells comprising a solid tumor targeting backbone comprising a polynucleotide encoding a TGF ⁇ redirector receptor (“TGF ⁇ -SRR” in Table 4), among other genetic modalities, wherein the cells, such as derivative T and NK cells, are useful for overcoming or reducing tumor microenvironment suppression associated with a tumor, and particularly, a solid tumor.
  • TGF ⁇ -SRR TGF ⁇ redirector receptor
  • the iPSC and derivative cells thereof comprise a solid tumor targeting backbone comprising two or more of: a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, a polynucleotide encoding a TGF ⁇ redirector receptor, and and/or one or more additional genomic edits as described herein, without adversely impacting the differentiation potential of the iPSC and function of the derived effector cells, such as derivative T and NK cells.
  • a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least an exogenously introduced polynucleotide encoding a TGF ⁇ redirector receptor, and optionally a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, which are well-defined and uniform in composition, and can be mass produced at a significant scale in a cost-effective manner. 3.
  • CD16 knock-in [000153] CD16 has been identified as two isoforms, Fc receptors Fc ⁇ RIIIa (CD16a; NM_000569.6) and Fc ⁇ RIIIb (CD16b; NM_000570.4).
  • CD16a is a transmembrane protein expressed by NK cells, which binds monomeric IgG attached to target cells to activate NK cells and facilitate antibody-dependent cell-mediated cytotoxicity (ADCC).
  • CD16b is exclusively expressed by human neutrophils.
  • “High affinity CD16,” “non-cleavable CD16,” or “high affinity non-cleavable CD16” (abbreviated as hnCD16), as used herein, refers to various CD16 variants.
  • the wildtype CD16 has low affinity and is subject to ectodomain shedding, a proteolytic cleavage process that regulates cell surface density of various cell surface molecules on Attorney Docket No.: FATE-173/01WO leukocytes upon NK cell activation.
  • F176V also called F158V in some publications
  • S197P variant is an example of genetically engineered non-cleavable version of CD16.
  • An engineered CD16 variant comprising both F176V and S197P has high affinity and is non-cleavable, which was described in greater detail in WO2015/148926, the complete disclosure of which is incorporated herein by reference.
  • a chimeric CD16 receptor with the ectodomain of CD16 essentially replaced with at least a portion of CD64 ectodomain can also achieve the desired high affinity and non-cleavable features of a CD16 receptor capable of carrying out ADCC.
  • the replacement ectodomain of a chimeric CD16 comprises one or more of EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoform or polymorphic variant).
  • various embodiments of an exogenous CD16 introduced to a cell include functional CD16 variants and chimeric receptors thereof.
  • the functional CD16 variant is a high-affinity non-cleavable CD16 receptor (hnCD16).
  • An hnCD16 in some embodiments, comprises both F176V and S197P; and in some embodiments, comprises F176V and with the cleavage region eliminated.
  • an hnCD16 comprises a sequence having identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in-between, when compared to any of the exemplary sequences, SEQ ID NOs.11, 12 and 13, and each comprises at least a portion of CD64 ectodomain.
  • the hnCD16 comprises an amino acid sequence of at least 90% identity to any of SEQ ID NOs.11-13, and optionally one or more of F176V, S197P, and at least a portion of CD64 ectodomain. In some embodiments, the hnCD16 comprises an amino acid sequence of at least 95% identity to any of SEQ ID NOs.11-13, and optionally one or more of F176V, S197P, and at least a portion of CD64 ectodomain. In some embodiments, the hnCD16 comprises the amino acid sequence of SEQ ID NO 11. In some embodiments, the hnCD16 comprises the amino acid sequence of SEQ ID NO 12.
  • the hnCD16 comprises the amino acid sequence of SEQ ID NO 13.
  • SEQ ID NO: 11 MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSY RITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRN GKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTL SCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQV LGLQLPTPVWFHYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (340 a.a.
  • CD64 domain-based construction; CD16TM; CD16ICD) Attorney Docket No.: FATE-173/01WO SEQ ID NO: 12 MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSY RITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRN GKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTL SCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQV LGLFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (336 a.a.
  • effector cells or iPSCs genetically engineered to comprise a solid tumor targeting backbone that comprises, among other editing as contemplated and described herein, an exogenous CD16 or a variant thereof, wherein the effector cells are cells from primary sources or derived from iPSC differentiation, or wherein the genetically engineered iPSCs are capable of differentiating into derived effector cells comprising the exogenous CD16 or a variant thereof introduced to the iPSCs.
  • the exogenous CD16 is a high-affinity non-cleavable CD16 receptor (hnCD16).
  • the exogenous CD16 comprises at least a portion of the CD64 ectodomain.
  • the exogenous CD16 is in a form of a CD16-based chimeric Fc receptor (CFcR) that comprises a transmembrane domain, a stimulatory domain and/or a signaling domain that is not derived from CD16.
  • CcR CD16-based chimeric Fc receptor
  • the primary-sourced or derived effector cells comprising the exogenous CD16 or variant thereof are NK lineage cells.
  • the primary- sourced or derived effector cells comprising the exogenous CD16 or variant thereof are T lineage cells.
  • the exogenous CD16 or functional variants thereof comprised in iPSC or effector cells has high affinity in binding to a ligand that triggers downstream signaling upon such binding.
  • ligands binding to the exogenous CD16 or functional variants thereof include not only ADCC antibodies or fragments thereof, but also to bi-, tri-, or multi- specific engagers or binders that recognize the CD16 or CD64 extracellular binding domains of the exognous CD16. Examples of bi-, tri-, or multi- specific engagers or binders are further described below in this application.
  • a derivative effector cell comprising a solid tumor targeting Attorney Docket No.: FATE-173/01WO backbone, or a cell population thereof, preloaded with one or more pre-selected ADCC antibodies through an exogenous CD16 expressed on the derivative effector cell, in an amount sufficient for therapeutic use in a treatment of a condition, a disease, or an infection as further detailed in this application, wherein the exogenous CD16 comprises an extracellular binding domain of CD64, or of a CD16 having F176V and S197P.
  • an exogenous CD16 comprises a CD16-, or variants thereof, based CFcR.
  • a chimeric Fc receptor (CFcR) is produced to comprise 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 the intracellular-domain.
  • non-native used herein means that the transmembrane, stimulatory or signaling domain are derived from a different receptor other than the receptor which provides the extracellular domain.
  • the various embodiments of the CD16-based chimeric Fc receptor as described above are capable of binding, with high affinity, to the Fc region of an antibody or fragment thereof; or to a bi-, tri-, or multi- specific engager or binder.
  • the stimulatory and/or signaling domains of the chimeric receptor Upon binding, the stimulatory and/or signaling domains of the chimeric receptor enable the activation and cytokine secretion of the effector cells, and the killing of the tumor cells targeted by the antibody, or the bi-, tri-, or multi- specific engager or binder having a tumor antigen binding component as well as the Fc region.
  • the CFcR could contribute to effector cells’ killing ability while increasing the effector cells’ proliferation and/or expansion potential.
  • the present disclosure also provides a derivative NK or T cell comprising a solid tumor targeting backbone, or a cell population thereof, preloaded with one or more pre-selected ADCC antibodies in an amount sufficient for therapeutic use in a treatment of a condition, a disease, or an infection as further detailed in this application.
  • the preloaded CD38 antibody is daratumumab.
  • the derived NK or T cells comprise a solid tumor targeting backbone comprising a TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X-C-motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, among other edits as provided herein.
  • said derived NK or T cells are preloaded with one or more of an anti-HER2 antibody (e.g., trastuzumab, Attorney Docket No.: FATE-173/01WO pertuzumab), an anti-EGFR antibody (e.g., cetuximab), or an anti-PDL1 antibody (e.g., avelumab).
  • an anti-HER2 antibody e.g., trastuzumab, Attorney Docket No.: FATE-173/01WO pertuzumab
  • an anti-EGFR antibody e.g., cetuximab
  • an anti-PDL1 antibody e.g., avelumab
  • the cell or population thereof comprising the solid tumor targeting backbone, and optionally a CAR, and an exogenous CD16 or a variant thereof (“CD16 exo ” in Table 4)
  • CD16 exo may further comprise one or more additional engineered modalities described herein, and/or as shown in Table 4.
  • a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one phenotype as provided herein, including but not limited to, a solid tumor targeting backbone comprising, among other genetic modalities, an exogenous CD16 or a variant thereof, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derivative NK and T cells, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective manner. 4.
  • Allo-Immune Defense Receptor (ADR) expression [000161] Unwanted activation of T- and NK- cells often promotes allo-immune reactions leading to development of graft-versus-host disease (GvHD). Although some steps may be taken to reduce the reactivity of allogeneic cells in the recipient individual, such cells would still be targeted by the immune system of the recipient (primarily T- and NK-cells), which would recognize them as foreign leading to rejection and limiting therapeutic benefit.
  • GvHD graft-versus-host disease
  • the present application provides a solid tumor targeting backbone comprising an allo-immune defense receptor (ADR), among other components.
  • ADR allo-immune defense receptor
  • Another aspect of the application provides immune cells, iPSCs, and iPSC-derived effector cells that are genetically engineered to comprise, among other editing as contemplated and described herein, a 4-1BB or CD38 specific allo-immune defense receptor (ADR) for effector cell potentiation as well as selective depletion of alloreactive host NK cells and T cells with upregulated 4-1BB and/or CD38 expression, the latter of which include pathogenic T cells, and regulatory T cells, while sparing resting cells in the recipient.
  • ADR allo-immune defense receptor
  • the ADR comprises an extracellular domain that targets 4-1BB upregulated on Attorney Docket No.: FATE-173/01WO host T or NK cells when they are activated, and a signaling domain promoting effector cell activation.
  • the 41BB-ADR extracellular domain may comprise any suitable ligand for 4-1BB, including 4-1BBL, an antibody (or functional fragment thereof) that targets 4-1BB, a fusion of Fc with 4-1BBL, or functional derivatives or fragments thereof.
  • the 41BB-ADR extracellular domain comprises 4-1BBL, or a fragment thereof effective to bind 4-1BB.
  • the 41BB-ADR extracellular domain comprises an amino acid sequence with at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 14. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence with at least about 90% sequence identity to SEQ ID NO: 14. In some embodiments, the 41BB-ADR extracellular domain comprises an amino acid sequence with at least about 95% sequence identity to SEQ ID NO: 14. In some embodiments, the 41BB-ADR extracellular domain comprises the amino acid sequence of SEQ ID NO: 14.
  • the CD38-ADR comprises an extracellular domain comprising a CD38 binding domain or fragments thereof.
  • the CD38 binding domain or fragment thereof is from an anti-CD38 antibody.
  • the anti-CD38 antibody comprises a murine antibody, a human antibody, a humanized antibody, a camel Ig, a single variable new antigen receptor (VNAR), a shark heavy- chain-only antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or an antibody fragment thereof.
  • VNAR single variable new antigen receptor
  • Ig NAR shark heavy- chain-only antibody
  • Non-limiting examples of antibody binding domain or fragments thereof 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 fragments (sdAb, Nanobody), recombinant heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody.
  • the CD38 binding domain or fragments thereof comprised in the CD38-ADR comprises a variable region of the heavy chain and/or a variable region of the light chain represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, and/or at least about 80% identity to SEQ ID NOs: 15 and 16 respectively, SEQ ID NOs: 17 and 18 respectively, SEQ ID NOs: 19 and 20 Attorney Docket No.: FATE-173/01WO respectively, SEQ ID NOs: 21 and 22 respectively, SEQ ID NOs: 23 and 24 respectively, SEQ ID NOs: 25 and 26 respectively, SEQ ID NOs: 27 and 28 respectively, SEQ ID NOs: 29 and 30 respectively, SEQ ID NOs: 31 and 32 respectively, SEQ ID NOs: 33 and 34 respectively, SEQ ID NOs: 35 and 36 respectively, SEQ ID NOs: 37 and 38 respectively, SEQ ID NOs: 39 and 40 respectively, SEQ ID NOs: 41 and
  • CD38-ADR extracellular domain comprises an amino acid sequence with at least about 90% sequence identity to the VH and/or VL sequence of any of pairs 1-23 in Table 1A. In some embodiments, the CD38-ADR extracellular domain comprises an amino acid sequence with at least about 95% sequence identity to the VH and/or VL sequence of any of pairs 1-23 in Table 1A. In some embodiments, the CD38-ADR extracellular domain comprises the amino acid sequence of the VH and/or VL sequence of any of pairs 1-23 in Table 1A.
  • the extracellular domain of the 41BB-ADR or CD38-ADR may be operably linked to one or more signaling domains that mediate downstream signaling upon effector cell activation upon the binding to the 4-1BB or CD38, respectively, of alloreactive host immune cells.
  • the ADR comprises CD3 ⁇ , represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 59 or a functional fragment thereof, or comprises a CD3 ⁇ derivative (for example, CD3 ⁇ 1XX, represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 60 or a functional fragment thereof).
  • Attorney Docket No.: FATE-173/01WO the CD3 ⁇ comprises an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 59.
  • the CD3 ⁇ comprises an amino acid sequence of at least about 95% sequence identity to SEQ ID NO: 59. In some embodiments, the CD3 ⁇ comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the CD3 ⁇ derivative comprises an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 60. In some embodiments, the CD3 ⁇ derivative comprises an amino acid sequence of at least about 95% sequence identity to SEQ ID NO: 60. In some embodiments, the CD3 ⁇ derivative comprises the amino acid sequence of SEQ ID NO: 60. CD3 ⁇ mediates downstream ITAM-derived signaling during effector T or NK cell activation.
  • ITAM-containing signaling domains may include those derived from DAP12, Fc receptors, and other CD3 subunits.
  • the intracellular domain of ADR comprising a signaling domain further comprises one, two, three, or more costimulatory domains that enhance cytokine production from
  • the costimulatory domains may be derived from the intracellular signaling domains of costimulatory proteins including, but not limited to, CD28, CD27, 4-1BB, OX40, ICOS, CD30, HVEM, CD40, and so forth.
  • the ADR comprising CD3 ⁇ further comprises a costimulatory domain derived from 4-1BB endodomain.
  • the endodomain is represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 63 or a functional fragment thereof.
  • the endodomain comprises an amino acid sequence of at least about 90% sequence identity to SEQ ID NO: 63.
  • the endodomain comprises an amino acid sequence of at least about 95% sequence identity to SEQ ID NO: 63. In some embodiments, the endo-domain comprises the amino acid sequence of SEQ ID NO: 63. In one embodiment, when the ADR comprises 4-1BBL in its extracellular domain, the costimulatory domain of the ADR is not derived from 4-1BB.
  • the intracellular domain of an ADR may be non-covalently linked to the extracellular domain of the ADR via a transmembrane domain.
  • the ADR comprises a transmembrane domain that may be of any kind so long as it allows the CD3 ⁇ component of the ADR to be located intracellularly and the extracellular domain that targets 4- 1BB or CD38 to be located extracellularly.
  • ADRs are soluble proteins that can bind to the respective ligand on activated T cells and promote cytotoxicity by crosslinking TCR (e.g., ADR-CD3 T-cell engager protein).
  • the extracellular domain is from a surface protein having a transmembrane domain, (CD40, for example)
  • the ADR may comprise the transmembrane domain from that corresponding endogenous molecule.
  • the transmembrane domain may be from the same endogenous molecule that has the costimulatory domain.
  • TMs include those from CD3, CD8a, CD27, CD28, 4-1BB, OX40, and CD4.
  • the ADR comprises a spacer between the extracellular protein and the transmembrane domain.
  • the spacer may comprise a sequence that is inert or contributes substantially little or nothing with respect to any function the ADR may have; whereas in other cases the spacer comprises a sequence that enhances a function of the ADR and/or allows it to be detectable and/or able to be targeted for inhibition.
  • the spacer comprises an encoded protein sequence that facilitates detection of cells that express the ADR.
  • the spacer may encode an Fc region or fragments thereof that would allow for surface detection of the cells expressing the ADR, such as by using anti-Fc antibodies.
  • the spacer provides separation between the ligand binding extracellular domain and the membrane to avoid potential steric hindrances.
  • the spacer can vary in sequence and/or in length, whether a function other than being a physical separation is intended or not.
  • Exemplary spacers that may be included in the ADR are commonly known in the art, including, but not limited to, IgG4 spacers, CD28 spacers, CD8 spacers, or combinations of more than one spacer.
  • the length of the spacers may also vary, from about 15 amino acids (a.a.) to about 300 a.a. or more.
  • Non-limiting exemplary spacer peptides include those represented by an amino acid sequence of at least about 80%, about Attorney Docket No.: FATE-173/01WO 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 64 or 65.
  • the spacer peptide comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 64 or 65.
  • the spacer peptide comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 64 or 65.
  • the spacer peptide comprises the amino acid sequence of SEQ ID NO: 64.
  • the spacer peptide comprises the amino acid sequence of SEQ ID NO: 65.
  • SEQ ID NO: 64 ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFL (88 a.a.)
  • SEQ ID NO: 65 ESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGKKDPK (123 a.a.
  • the 41BB-ADR is represented by an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any of SEQ ID NOs: 66-69. In some embodiments, the 41BB-ADR comprises an amino acid sequence of at least about 90% identity to any one of SEQ ID NOs: 66-69. In some embodiments, the the 41BB-ADR comprises an amino acid sequence of at least about 95% identity to any one of SEQ ID NOs: 66-69. In some embodiments, the the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 66.
  • the the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the the 41BB-ADR comprises the amino acid sequence of SEQ ID NO: 69.
  • the CD38-ADR comprises an amino acid sequence of at least about 90% sequence identity to SEQ Attorney Docket No.: FATE-173/01WO ID NO: 70. In some embodiments, the CD38-ADR comprises an amino acid sequence of at least about 95% sequence identity to SEQ ID NO: 70. In some embodiments, the CD38-ADR comprises the amino acid sequence of SEQ ID NO: 70.
  • immune cells iPSCs, and iPSC-derived effector cells comprising a solid tumor targeting backbone comprising a polynucleotide encoding a 4-1BB specific ADR or a CD38 specific ADR, among other selected components, wherein the effector cells, including the genetically engineered T and NK cells, possess alloreactive resistance to the host immune system associated with the allogeneic use of the effector cells for treatment of tumors and infectious diseases in a patient. 5.
  • Exogenously introduced cytokine signaling complex [000172] By avoiding systemic high-dose administration of clinically relevant cytokines, the risk of dose-limiting toxicities due to such a practice is reduced while cytokine-mediated cell autonomy is being established.
  • a cytokine signaling complex comprising a partial or full length peptide of one or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and/or their respective receptors may be introduced to the cell as part of the solid tumor targeting backbone to enable cytokine signaling with or without the expression of the cytokine itself, thereby maintaining or improving cell growth, proliferation, expansion, and/or effector function with reduced risk of cytokine toxicities.
  • the introduced cytokine and/or its respective native or modified receptor for cytokine signaling are Attorney Docket No.: FATE-173/01WO expressed on the cell surface.
  • the cytokine signaling is constitutively activated.
  • the activation of the cytokine signaling is inducible.
  • the activation of the cytokine signaling is transient and/or temporal.
  • the transient/temporal expression of a cell surface cytokine/cytokine receptor is through an expression construct carried by a retrovirus, Sendai virus, an adenovirus, an episome, mini-circle, or RNAs including mRNA.
  • a protein complex for signaling of one, two, or more cytokines including, but not limited to, IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18 and IL21, into the cell are provided herein.
  • the transmembrane (TM) domain can be native to the IL15 receptor or may be modified or replaced with the transmembrane domain of any other membrane bound proteins.
  • the cytokine signaling complex comprises an IL15 receptor fusion (IL15RF) comprising a full or partial length of IL15 and a full or partial length of IL15 receptor (IL15R).
  • IL15RF IL15 receptor fusion
  • IL15R ⁇ are co-expressed by using a self-cleaving peptide, mimicking trans-presentation of IL15, without eliminating cis-presentation of IL15.
  • IL15R ⁇ is fused to IL15 at the C-terminus through a linker, mimicking trans-presentation without eliminating cis-presentation of IL15 as well as ensuring that IL15 is membrane-bound.
  • IL15R ⁇ with truncated intracellular domain is fused to IL15 at the C-terminus through a linker, mimicking trans-presentation of IL15, maintaining IL15 membrane-bound, and additionally eliminating cis-presentation and/or any other potential signal transduction pathways mediated by a normal IL15R through its intracellular domain.
  • IL15R ⁇ is fused to IL15 without an intracellular domain (IL15 ⁇ ), as described in International Pub. Nos. WO 2019/191495 and WO 2019/126748, the entire disclosure of each of which is incorporated herein by reference.
  • the cytokine signaling complex comprises an IL7 receptor fusion (IL7RF) comprising a full or partial length of IL7 and a full or partial length of IL7 receptor.
  • the transmembrane (TM) domain can be native to the IL7 receptor or may be modified or replaced with a transmembrane domain of any other membrane bound proteins.
  • a native (or wildtype) or modified IL7R may be fused to IL7 at the C-terminus through a linker, enabling constitutive signaling and maintaining membrane-bound IL7.
  • such a construct comprises an amino acid sequence of at least 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 71, with transmembrane domain, signal peptide and linker being flexible and varying in length and/or sequences.
  • the IL7 construct comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 71, with Attorney Docket No.: FATE-173/01WO transmembrane domain, signal peptide and linker being flexible and varying in length and/or sequences.
  • the IL7 construct comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 71, with transmembrane domain, signal peptide and linker being flexible and varying in length and/or sequences. In some embodiments, the IL7 construct comprises the amino acid sequence of SEQ ID NO: 71.
  • cytokine signaling is provided by exogenous and soluble IL2 or IL18.
  • IL-2 is a cytokine that promotes proliferation and differentiation of T cells, while IL-18 enhances T cell effector function.
  • temporal and dose control of the exogenous cytokine can have important implications for the lymphoid lineage and T cell lineage commitment during the iPSC differentiation process.
  • the soluble cytokine expression is under the control of an endogenous TRAC promoter.
  • the soluble cytokine expression is under the control of an endogenous Tim-3 promoter.
  • the cell comprises a polynucleotide encoding a soluble IL2 (sIL2), such as a polypeptide having the sequence of SEQ ID NO: 72.
  • the cell comprises a polynucleotide encoding a soluble IL18 (sIL18), such as a polypeptide having the sequence of SEQ ID NO: 73.
  • the cytokines and/or receptors thereof may be introduced to iPSCs using one or more of the construct designs described herein, and to their derivative cells upon iPSC differentiation.
  • a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having a solid tumor targeting backbone as disclosed herein, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, which are well-defined and uniform in composition, and can be mass produced at a significant scale in a cost-effective manner.
  • Chimeric Antigen Receptor (CAR) expression Applicable to the genetically engineered immune cells, iPSCs and derivative effector cells thereof may be any CAR design known in the art.
  • CAR is a fusion protein generally including an ectodomain that comprises a target binding region (for example, an antigen recognition domain), a transmembrane domain, and an endodomain.
  • the ectodomain can further include a signal peptide or leader sequence and/or a spacer.
  • the endodomain can further comprise a signaling peptide that activates the effector cell expressing the CAR.
  • the signaling peptide of the endodomain comprises a full length or at least a portion of a polypeptide of 2B4, CD2, CD3 ⁇ , CD3 ⁇ 1XX, CD8, CD28, CD28H, CD137 (4-1BB), CS1, Attorney Docket No.: FATE-173/01WO DAP10, DAP12, DNAM1, FcERI ⁇ , IL2R ⁇ , IL7R, IL21R, IL2R ⁇ (IL15R ⁇ ), IL21, IL7, IL12, IL15, IL21, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, or NKG2D.
  • the signaling peptide of a CAR comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to at least one ITAM (immunoreceptor tyrosine-based activation motif) of CD3 ⁇ .
  • ITAM immunoglobulin-based activation motif
  • Exemplary N-terminal signal peptides include MALPVTALLLPLALLLHA (SEQ ID NO: 74; CD8asp) or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 75; IgKsp), or any signal peptide sequence or functional variants thereof known in the art.
  • the antigen recognition domain can specifically bind an antigen.
  • the CAR is suitable to activate T, NK or NKT cells expressing said CAR.
  • the CAR is NK cell specific for comprising NK-specific signaling components.
  • the CAR is NKT cell specific for comprising NKT-specific signaling components.
  • said T cells are derived from a CAR expressing iPSCs comprising a solid tumor targeting backbone as described herein, and the derivative T cells may comprise T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, ⁇ T cells, ⁇ T cells, or a combination thereof.
  • said NK cells are derived from a CAR expressing iPSCs comprising a solid tumor targeting backbone as described herein.
  • said NKT cells are derived from a CAR expressing iPSCs comprising a solid tumor targeting backbone as described herein.
  • the antigen recognition region comprises a murine antibody, a human antibody, a humanized antibody, a camel Ig, a single variable new antigen receptor (VNAR), a shark heavy-chain antibody (Ig-NAR), a chimeric antibody, a recombinant antibody, a single-domain antibody (dAb), an anti-idiotype antibody, a bi-specific-, multi- specific- or multimeric- antibody, or antibody fragment thereof.
  • Anti-idiotype antibodies are specific for binding to an idiotope of another antibody, wherein the idiotope is an antigenic determinant of an antibody.
  • a bi-specific antibody may be a BiTE (bi-specific T cell engager) or a BiKE (bi-specific killer cell engager), and a multi-specific antibody may be a TriKE (tri- specific Killer cell engager).
  • Non-limiting examples of antibody fragments include Fab, Fab’, F(ab’)2, F(ab’)3, Fv, Fabc, pFc, Fd, single chain fragment variable (scFv), tandem scFv (scFv)2, single chain Fab (scFab), disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb), camelid heavy-chain IgG and Nanobody® fragments, recombinant heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the antibody.
  • an antigen binding domain of a CAR comprises CDR1, CDR2, and CDR3 of a heavy chain (H-CDRs) of an antibody or fragments thereof.
  • the antigen binding domain of a CAR Attorney Docket No.: FATE-173/01WO comprising the H-CDRs of an antibody further comprises the CDRs of a light chain (L-CDRs) of the antibody.
  • the antigen recognition domain of a CAR specifically binds an antigen associated with a disease or pathogen.
  • the disease-associated antigen is a tumor antigen, wherein the tumor may be a liquid or a solid tumor.
  • the CAR targets antigens of hematological malignancies, which include, but are not limited to, acute and chronic leukemias (acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), lymphomas, non- Hodgkin lymphoma (NHL), Hodgkin’s disease, multiple myeloma, and myelodysplastic syndromes.
  • AML acute myelogenous leukemia
  • ALL acute lymphoblastic leukemia
  • CML chronic myelogenous leukemia
  • NHL non- Hodgkin lymphoma
  • Hodgkin’s disease multiple myeloma
  • myelodysplastic syndromes a CARs targeting solid cancer antigens
  • the antigens are associated with sarcomas and carcinomas.
  • the solid cancers suitable for CAR targeting include, but are not limited to, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, breast lung cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric/stomach cancer, head and neck cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, metastatic cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, salivary gland cancer, skin cancer, testicular tumor, thyroid tumor, urothelial cancer, and uterine/endometrial cancer.
  • the CAR targets an antigen associated to adenocarcinoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, bronchogenic carcinoma, cholangiocarcinoma, chondrosarcoma, choriocarcinoma, colon carcinoma, Ewing’s tumor, fibrosarcoma, gallbladder carcinoma, hepatocellular carcinoma, hepatoma, leiomyosarcoma, liposarcoma, lymphoid malignancy, medullary carcinoma, medullary thyroid carcinoma, melanoma, mesothelioma, myxosarcoma, non-small cell lung cancer, osteosarcoma, papillary adenocarcinoma, papillary carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, peritoneal carcinoma, renal cell carcinoma, rhabdomyosarcoma, sarcoma, seminoma, squamous cell
  • the CAR targets antigens of CNS tumors including, but not limited to, acoustic neuroma, astrocytoma, CNS lymphoma, ependymoma, hemangioblastoma, germinoma, glioma (including brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), medulloblastoma, menangioma, neuroblastoma, oligodendroglioma, pinealoma, retinoblastoma, Schwannoma craniopharyogioma, and brain metastases.
  • CNS tumors including, but not limited to, acoustic neuroma, astrocytoma, CNS lymphoma, ependymoma, hemangioblastoma, germinoma, glioma (including brainstem glioma and mixed gli
  • Non-limiting examples of antigens that may be targeted by a CAR include oncofetal antigen (h5T4), 8H9, 9D7, ACPP, ⁇ actinin-4 (ACTN4), ADAM12, ADRB3, ADGRE2/EMR2, AFP, AKAP-4, ALK, ALPP, ALPPL2, Androgen receptor, ASGR1 Attorney Docket No.: FATE-173/01WO (asialoglycoprotein receptor 1), ASGR2 (asialoglycoprotein receptor 2), AXL, B7H3, B7H6, BAGE, ⁇ -catenin, BCR, BCR-ABL, Bigh3, BING-4, BORIS, BRCA1/2, BST2, carbonic anhydrase IX (CAIX/CA9), CA125, C-C motif chemokine receptor 1 (CCR1), CCR4, carcinoembryonic antigen (CEA/CECAM5), Calcium-activated chloride channel 2 (CLCA4), Carbohydrates (Le), CD3,
  • Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites and protozoa capable of causing diseases.
  • Non-limiting examples of solid cancers with corresponding tumor antigens are provided in Table 1B.
  • Table 1B – Exemplary Solid Tumors and Solid Tumor Associated Antigens Attorney Docket No.: FATE-173/01WO Attorney Docket No.: FATE-173/01WO
  • the antigen recognition domain of a CAR comprises CDRs of the heavy chain (H-CDRs), CDRs of both the heavy and the light chains (H- and L- CDRs), the variable region of the heavy chain (VH), or a single chain of the variable regions of both the heavy and light chains (VH and VL) of the binding domains of an antibody that is specific to a tumor antigen, including those exemplified in this application.
  • the CAR is designed based on the binding domains of an antibody comprising trastuzumab, cetuximab, panitumumab, ofatumumab, belimumab, ipilimumab, pertuzumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, MDX-1105, dacetuzumab, urelumab, MPDL3280A, lambrolizumab, blinatumomab, nimotuzumab, zalutumumab, onartuzumab, patritumab, clivatuzumab, sofituzumab, edrecolomab, adecatumumab, anetumab, huDS6, lifastuzumab, sacituzumab, PR1A3, humanized PR1A3, humanized Ab2-3, claudixim
  • the antigen recognition domain of the CAR specifically binds to an antigen present on bladder cancer.
  • the CAR targeting a bladder cancer associated antigen specifically binds to HER2, MICA/B, CD207, EFNA4, LY6K, LYPD3, Nectin4, PTK7, SLITRK6, TIM-3, TNC, UPK1B, or UPK2.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- Attorney Docket No.: FATE-173/01WO CDRs, the VH, or a single chain of VH and VL of an antibody comprising enfortumab, trastuzumab, pertuzumab or SLITRK6.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on bone cancer.
  • the CAR targeting a bone cancer associated antigen specifically binds to MICA/B, ADAM12, CCR1, CD99, CD248, EPHA2, GPNMB, LRRC15, or TP-3.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising huM25, DS-8895a variant 1, DS-8895a variant 2, or glembatumab.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on brain cancer.
  • the CAR targeting a brain cancer associated antigen specifically binds to MICA/B, CD133, DLL3, EGFRvIII, or TNC.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising AMG595, ABT806, rovalpituzumab or depatuxizumab.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a breast cancer cell.
  • the CAR targeting a breast cancer associated antigen specifically binds to HER2, MICA/B, ADAM12, ADGRE2/EMR2, CCR4, CD49f, CD133, CDH3 (p-cadherin), CLDN6, c-MET, CXCR2, EFNA4, EGFR, EPCAM/EGP2, EPHA2, GPNMB, ICAM1, LAMP-1, LIV-1, LILRB2, LRRC15, LYPD3, MUC1, tMUC1, PRLR, PTK7, Sialo-epitope CA6, TNC, or TROP2.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising trastuzumab, pertuzumab, sacituzumab, ladiratuzumab, huLiv1-14, Liv1-1.7A4, huLiv1-22, huDS6, glembatumumab, PF-0664720, MEDI-547, DS-8895a variant 1, or DS-08895a variant 2.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a breast lung cancer cell.
  • the CAR targeting a breast lung cancer associated antigen specifically binds to HER2, MICA/B, ADGRE2/EMR2, EPCAM/EGP2, or ROR1.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on cervical/uterine/endometrial cancer.
  • the CAR targeting a cervical/uterine/endometrial cancer associated antigen specifically binds to MICA/B, EFNA4, LY6K, MUC1, MUC16, LYPD3, PTK7, SLC12A3, or SSTR1.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising PF-0664720, anetumumab, 4H11, 4H5, huDS6, or sofituzumab.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a cholangiocarcinoma cell.
  • the CAR targeting a cholangiocarcinoma associated antigen specifically binds to MICA/B or tMUC1.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on colorectal cancer.
  • the CAR targeting a colorectal cancer associated antigen specifically binds to HER2, MICA/B, ADAM12, CA19.9, CD3, CD49f, CD133, CEA/CECAM5, CLCA1, c-MET, EFNA4, EPHB2, GPA33, GPR35, GUCY2C, ICAM1, LGR5/GPR49, LRRC15, MS4A12, MUC12, MUC17, TIM-3, or TMEM238.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising huM25, PR1A3, humanized PR1A3, pantumumab, cetuximab, nimotuzumab, or zalutumumab.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on an esophageal cancer cell.
  • the CAR targeting an esophageal cancer associated antigen specifically binds to HER2, MICA/B, CA19.9, CD10, CEA/CECAM5, EFNA4, EPHB2, MUC21, TMEM238, TMPRSS11B, or TMPRSS11E.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a gall bladder carcinoma cell.
  • the CAR targeting a gall bladder carcinoma associated antigen specifically binds to EPCAM/EGP2.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on gastric/stomach cancer.
  • the CAR targeting a gastric/stomach cancer associated antigen specifically binds to HER2, MICA/B, CEA/CECAM5, CLDN18.2, c-MET, CR1L, EFNA4, EPHB2, LGR5/GPR49, MUC17, PSCA, TIM-3, or TMEM238.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising sofituzumab, anetumab, pertuzumab, trastuzumab, or humanized PR1A3.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a glioma cancer cell.
  • the CAR targeting a glioma cancer associated antigen specifically binds to MICA/B, ADGRE2/EMR2, CD49f, CD133, EGFR, EGFRvIII, EPHA2, HM1.24, or IL13-R ⁇ 2.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on head and neck cancer.
  • the CAR targeting a head and neck cancer associated antigen specifically binds to HER2, MICA/B, ADAM12, CD3, c-MET, EFNA4, LRRC15, LY6K, LYPD3, PTK7, or TNC.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single Attorney Docket No.: FATE-173/01WO chain of VH and VL of an antibody comprising cetuximab, panitumumab, nimtuzumab, PF- 0664720, pantumumab, cetuximab, nimotuzumab, or zalutumumab.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on kidney cancer.
  • the CAR targeting a kidney cancer associated antigen specifically binds to MICA/B, CD70, CDH6, c-MET, ENPP3, or HAVCR1.
  • the antigen recognition domain of said CAR comprises the H- CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising AGS-16M8F, AGS-16C3, the antibody of CDX-014, or onartuzumab.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on liver cancer.
  • the CAR targeting a liver cancer associated antigen specifically binds to MICA/B, ASGR1, ASGR2, C9 (CAIX), CA19.9, CEA/CECAM5, CCR1, CD3, CD133, EPCAM/EGP2, GPC3, ICAM1, LGR5/GPR49, SLC13A5, SLC22A1, SLC22A7, TIM-3, TRF2, or UGT1A1.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising codrituzumab, oportuzumab, or humanized PR1A3.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising panitumumab, cetuximab, pembrolizumab, nivolumab, atezolizumab, and nimotuzumab, lifastuzumab, anetumab, PF-0664720, farletuzumab, rovalpituzumab, lifastuzumab, sofituzumab, huDS6, ABT806, AMG595, or huM25.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a mesothelioma cell. In some embodiments, the CAR targeting a mesothelioma associated antigen specifically binds to MICA/B, FAP, or MSLN. [000204] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on a metastatic cancer cell. In some embodiments, the CAR targeting a metastatic cancer cell associated antigen specifically binds to MICA/B, MSLN, or VEGFR-II. [000205] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on a neuroblastoma cell.
  • the CAR targeting a neuroblastoma associated antigen specifically binds to MICA/B or GD2.
  • Attorney Docket No.: FATE-173/01WO In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on a non-small cell lung cancer (NSCLC) cell. In some embodiments, the CAR targeting a non-small cell lung cancer associated antigen specifically binds to MICA/B, c-MET, or EGFR. [000207] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on ovarian cancer.
  • the CAR targeting an ovarian cancer associated antigen specifically binds to HER2, MICA/B, CCR1, CD3, CD133, CLDN6, c-MET, EFNA4, EPCAM/EGP2, FAP, FOLR1, FOLR3, FR- ⁇ , FZD10, GPR27, GPR119, LRRC15, MSLN, MUC1, MUC16, PTK7, SLC34A2, sTN, TMEM238, or VTCN1.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising sofituzumab, 4H11, 4H5, huDS6, farletuzumab, anetumab, trastuzumab, pertuzumab, PF-0664720, sibrotuzumab, huM25, or lifastuzumab.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on pancreatic cancer.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising PF-0664720, clivatuzumab, 4H11, 4H5, anetumumab, huDS6, sofituzumab, huM25, or RG7841.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a peritoneal carconima cell.
  • the CAR targeting a peritoneal carconima associated antigen specifically binds to FOLR3.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on prostate cancer.
  • the CAR targeting a prostate cancer associated antigen specifically binds to MICA/B, ACPP, CD10, CD49f, CD133, EFNA4, OR51E2, PSCA, PSMA/FOLH1, PTK7, SLC30A4, SLC45A3, STEAP, TIM-3, or TMEFF2/TENB2.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising mirvetuximab, or J591 variant 1 or 2.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a renal cancer cell.
  • the CAR targeting a renal cancer associated antigen specifically binds to MICA/B, CD3, CD70, ICAM1, KISS1R, LILRB2, QRFPR, SLC6A3, or TIM-3.
  • Attorney Docket No.: FATE-173/01WO In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on a sarcoma. In some embodiments, the CAR targeting a sarcoma associated antigen specifically binds to MICA/B or LRRC15.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on a salivary gland cancer cell. In some embodiments, the CAR targeting a salivary gland cancer associated antigen specifically binds to HER2 or MICA/B. [000214] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on skin cancer. In some embodiments, the CAR targeting a skin cancer associated antigen specifically binds to CCR4, CD3, CD10, or ICAM1. [000215] In some embodiments, the antigen recognition domain of the CAR specifically binds to an antigen present on a synovial sarcoma.
  • the antigen recognition domain of the CAR specifically binds to an antigen present on uterine/endometrial cancer cell.
  • the CAR targeting a uterine/endometrial cancer associated antigen specifically binds to HER2, MICA/B, ALPP, ALPPL2, CCR1, CLDN6, EFNA4, EPHB2, FOLR1, LILRB2, LY6K, LYPD3, MUC1, MUC16, or PTK7.
  • the antigen recognition domain of said CAR comprises the H-CDRs, H- and L- CDRs, the VH, or a single chain of VH and VL of an antibody comprising PF-0664720, farletuzumab, sofituzumab, 4H11, or 4H5.
  • the antigen recognition domain of the CAR specifically binds to a tumor antigen known to be associated with three or more cancer types (sometimes referred to as “pan-tumor antigen”).
  • a non-limiting set of such pan-tumor antigens comprises at least ADAM12, ADGRE2/EMR2, CA19.9, CCR1, CCR4, CD3, CD10, CD49f, CD133, CEA/CECAM5, CLDN6, c-MET, EFNA4, EGFR, EGFRvIII, EPHA2, EPHB2, FOLR1, HER2, ICAM1, LILRB2, LRRC15, LY6K, LYPD3, MICA/B, MSLN, MUC1, tMUC1, MUC16, MUC17, PSCA, PTK7, TIM-3, TMEM238, and TNC as exemplified in Table 2.
  • the antigen recognition domain of the CAR specifically binds to tumor associated HER2, wherein an effector cell comprising said CAR and a solid tumor targeting backbone as disclosed is useful for treating one or more cancers comprising at least bladder cancer, breast cancer, breast lung cancer, colorectal cancer, esophageal cancer, gastric/stomach cancer, head and neck cancer, lung cancer, ovarian cancer, or salivary gland cancer.
  • the antigen recognition domain of the CAR specifically binds to tumor associated MSLN, wherein an effector cell comprising said CAR and a solid Attorney Docket No.: FATE-173/01WO tumor targeting backbone as disclosed is useful for treating one or more cancers comprising at least lung cancer, metastatic cancer, mesothelioma, ovarian cancer, or pancreatic cancer.
  • the antigen recognition domain of the CAR specifically binds to tumor associated MUC1, wherein an effector cell comprising said CAR and a solid tumor targeting backbone as disclosed is useful for treating one or more cancers comprising at least breast cancer, cervical cancer, lung cancer, ovarian cancer, pancreatic cancer, or uterine/endometrial cancer.
  • the antigen recognition domain of the CAR specifically binds to tumor associated PSCA, wherein an effector cell comprising said CAR and a solid tumor targeting backbone as disclosed is useful for treating one or more cancers comprising at least gastric/stomach cancer, pancreatic cancer, or prostate cancer.
  • the CARs appliable to the cells described herein include at least an ectodomain, a transmembrane domain, and an endodomain.
  • the endodomain of the CAR comprises at least one signaling domain that is activated upon antigen binding.
  • one or more co-stimulation domains is further included for optimized functionality.
  • Exemplary signal transducing proteins suitable for a CAR design include, but are not limited to, 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3 ⁇ /1XX (i.e., CD3 ⁇ or CD3 ⁇ 1XX), DAP10, DAP12, DNAM1, FcERI ⁇ , IL21R, IL-2R ⁇ (IL-15R ⁇ ), IL-2R ⁇ , IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1 and CD8.
  • the description of the exemplary signal transducing proteins, including transmembrane and cytoplasmic sequences of the proteins are provided below, and further in Table 3A.
  • the endodomain of the CAR comprises at least a first signaling domain having an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain, or a portion thereof, of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3 ⁇ , CD3 ⁇ 1XX, DAP10, DAP12, DNAM1, FcERI ⁇ IL21R, IL-2R ⁇ (IL- 15R ⁇ ), IL-2R ⁇ , IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, represented by SEQ ID NOs: 98-120, respectively.
  • the first signaling domain comprises an amino acid sequence of at least 90% identity to any of SEQ ID NOs: 98-120.
  • the first signaling domain comprises an amino acid sequence of at least 95% identity to any of SEQ ID NOs: 98-120.
  • the first signaling domain comprises the amino acid sequence of any of SEQ ID NOs: 98-120.
  • the signaling domain of the CAR comprises only a portion of the cytoplasmic domain of 2B4, 4- 1BB, CD16, CD2, CD28, CD28H, CD3 ⁇ , CD3 ⁇ 1XX, DAP10, DAP12, DNAM1, FcERI ⁇ IL21R, IL-2R ⁇ (IL-15R ⁇ ), IL-2R ⁇ , IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, represented by SEQ ID NOs: 98-120.
  • the portion of the cytoplasmic domain selected for the CAR signaling domain comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to an ITAM (immunoreceptor tyrosine-based activation motif), a YxxM motif, a TxYxxV/I motif, FcR ⁇ , hemi-ITAM, and/or an ITT-like motif.
  • ITAM immunomunoreceptor tyrosine-based activation motif
  • the endodomain of the CAR comprising a first signaling domain further comprises a second signaling domain comprising an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain, or a portion thereof, of 2B4, 4- 1BB, CD16, CD2, CD28, CD28H, CD3 ⁇ , CD3 ⁇ 1XX, DAP10, DAP12, DNAM1, FcERI ⁇ IL21R, IL-2R ⁇ (IL-15R ⁇ ), IL-2R ⁇ , IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1 or CD8, represented by SEQ ID NOs: 98-120, respectively, wherein the second signaling domain is different from the first signaling domain.
  • the second signaling domain comprises an amino acid sequence of at least 90% identity to any of SEQ ID NOs: 98-120. In some embodiments, the second signaling domain comprises an amino acid sequence of at least 95% identity to any of SEQ ID NOs: 98-120. In some embodiments, the second signaling domain comprises the amino acid sequence of any of SEQ ID NOs: 98-120.
  • the endodomain of the CAR comprising a first and a second signaling domain further comprises a third signaling domain comprising an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain, or a portion thereof, of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3 ⁇ , CD3 ⁇ 1XX, DAP10, DAP12, DNAM1, FcERI ⁇ , IL21R, IL-2R ⁇ (IL-15R ⁇ ), IL-2R ⁇ , IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, represented by SEQ ID NOs: 98-120, respectively, wherein the third signaling domain is different from the first and the second signaling domains.
  • the third signaling domain comprises an amino acid sequence of at least 90% identity to any of SEQ ID NOs: 98-120. In some embodiments, the third signaling domain comprises an amino acid sequence of at least 95% identity to any of SEQ ID NOs: 98-120. In some embodiments, the third signaling domain comprises the amino acid sequence of any of Attorney Docket No.: FATE-173/01WO SEQ ID NOs: 98-120.
  • signal transducing proteins suitable for designing a signaling domain of a CAR endodomain further comprise CD27, OX40, ICOS, PD-1, LAG-3, BTLA, or CTLA-4.
  • said endodomain comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the cytoplasmic domain or a portion thereof, of a protein including, but not limited to, DNAM1, CD28H, KIR2DS2, DAP12 or DAP10.
  • said endodomain comprises fused cytoplasmic domains, or portions thereof, in a form including, but not limited to, 2B4-CD3 ⁇ /1XX (i.e., 2B4-CD3 ⁇ or 2B4- CD3 ⁇ 1XX; same below), 2B4-DNAM1, 2B4-FcERI ⁇ , 2B4-DAP10, CD16-DNAM1, CD16- DAP10, CD16-DAP12, CD2-CD3 ⁇ /1XX, CD2-DNAM1, CD2-FcERI ⁇ , CD2-DAP10, CD28- DNAM1, CD28-FcERI ⁇ , CD28-DAP10, CD28-DAP12, CD28-CD3 ⁇ /1XX, CD28H-CD3 ⁇ /1XX, DAP10-CD3 ⁇ /1XX, DAP10-DAP12, DAP12-CD3 ⁇ /1XX, DAP12-D
  • said endodomain comprises fused cytoplasmic domains, or portions thereof, in a form including, but not limited to, 2B4-DAP10-CD3 ⁇ /1XX, 2B4-IL21R- DAP10, 2B4-IL2RB-DAP10, 2B4-IL2RB-CD3 ⁇ /1XX, 2B4-41BB-DAP10, CD16-2B4-DAP10, or KIR2DS2-2B4-CD3 ⁇ /1XX.
  • the transmembrane domain of the CAR comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a full length or a portion of the transmembrane region of CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , 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, FcERI ⁇ , IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or T cell receptor polypeptide.
  • the transmembrane domain of a CAR comprises an amino acid sequence that has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a full length or a portion of the transmembrane region of (a) 2B4, CD16, CD2, CD28, CD28H, CD3 ⁇ , DAP10, DAP12, DNAM1, FcERI ⁇ , KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CS1, or CD8, represented by SEQ ID NOs: 76, 78-86, 91-97, respectively; or of (b) 2B4, CD28, CD28H, DAP10, DNAM1, KIR2DS2, and NKG2D.
  • the transmembrane domain comprises an amino acid sequence of at least about 90% identity to any of SEQ ID NOs: 76, 78- Attorney Docket No.: FATE-173/01WO 86, 91-97. In some embodiments, the transmembrane domain comprises an amino acid sequence of at least about 95% identity to any of SEQ ID NOs: 76, 78-86, 91-97. In some embodiments, the transmembrane domain comprises the amino acid sequence of any of SEQ ID NOs: 76, 78- 86, 91-97. In some embodiments of the CAR, the transmembrane domain and its immediately linked signaling domain are from the same protein.
  • the transmembrane domain and the signaling domain that is immediately linked are from different proteins.
  • CAR constructs comprising a transmembrane domain (TM) and an endodomain (labelled as: TM-(endodomain)) are shown in Table 3B.
  • the illustrated CAR constructs each comprise a transmembrane domain, and an endodomain comprising one or more signaling domains derived from the cytoplasmic region of one or more signal transducing proteins.
  • a transmembrane domain is a three-dimensional protein structure which is thermodynamically stable in a membrane such as the phospholipid bilayer of a biological membrane (e.g., a membrane of a cell or cell vesicle).
  • a membrane such as the phospholipid bilayer of a biological membrane (e.g., a membrane of a cell or cell vesicle).
  • the transmembrane domain of the CAR applicable to the cells provided herein comprises a single alpha helix, a stable complex of several transmembrane alpha helices, a transmembrane beta barrel, a beta-helix of gramicidin A, or any combination thereof.
  • the transmembrane domain of the CAR comprises all or a portion of a “transmembrane protein” or “membrane protein” that is within the membrane.
  • transmembrane protein or “membrane protein” is a protein located at and/or within a membrane.
  • transmembrane proteins that are suitable for providing a transmembrane domain comprised in a CAR according to some embodiments of the invention include, but are not limited to, a receptor, a ligand, an immunoglobulin, a glycophorin, or any combination thereof.
  • the transmembrane domain comprised in the CAR comprises all or a portion of a transmembrane domain of 2B4, 4-1BB, BTLA, CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, CS1, CTLA-4, DNAM1, DAP10, DAP12, FcERI ⁇ , ICOS, ICAM-1, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, LAG3, PD1, NKp30, NKp44, NKp46, NKG2C, NKG2D, OX40, T cell receptor polypeptide (such as TCR ⁇ and/or TCR ⁇ ), a nicotinic acetylcholine receptor, a GABA receptor, or any combination thereof.
  • T cell receptor polypeptide such as TCR ⁇ and/or TCR ⁇
  • one or more signaling domains comprised in the CAR endodomain are derived from the same or a different protein from which the TM is derived. As shown in Table 3B, the portion representing the transmembrane domain of the CAR is underlined, the domains comprised in the endodomain appear in parenthesis, “()”, with each of the TM and signaling domains designated by the name of the signal transducing protein from which the domain sequence is derived.
  • the amino acid sequence of each TM or Attorney Docket No.: FATE-173/01WO signaling domains may be of about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a full length or a portion of the corresponding transmembrane or cytoplasmic regions of the designated signal transducing protein.
  • Exemplary CAR constructs comprising a transmembrane domain and an endodomain as provided herein include, but are not limited to: NKG2D-(2B4-IL2RB-CD3 ⁇ ), CD8-(41BB-CD3 ⁇ 1XX), CD28-(CD28-2B4-CD3 ⁇ ), CD28-(CD28-CD3 ⁇ 1XX), CD28H-(CD28H-CD3 ⁇ ), DNAM1-(DNAM1-CD3 ⁇ ), DAP10- (DAP10-CD3 ⁇ ), KIR2DS2-(KIR2DS2-CD3 ⁇ ), KIR2DS2-(KIR2DS2-DAP10), KIR2DS2- (KIR2DS2-2B4), CD16-(CD16-2B4-DAP10), CD16-(CD16-DNAM1), NKp46-(NKp46-2B4), NKp46-(NKp46-2B4-CD3 ⁇ ), NKp46-(NKp46-CD2-DAP10), CD2-(CD2-CD
  • each of the above exemplary CAR constructs comprising a transmembrane domain and an endodomain comprises an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to a sequence represented by each of SEQ ID NOs: 121-139 in Table 3B.
  • the CAR comprises an amino acid sequence of at least about 90% identity to any of SEQ ID NOs: 121-139.
  • the CAR comprises an amino acid sequence of at least about 95% identity to any of SEQ ID NOs: 121- 139.
  • the CAR comprises the amino acid sequence of any of SEQ ID NOs: 121-139.
  • the illustrative sequence for each construct provided in Table 3B has text formatted to match the formatting of the corresponding region in the illustration at left of the sequence (i.e., underlined, normal, or bolded text).
  • the TM is the first sequence region; however, constructs may include an extracellular domain preceeding the TM (see, e.g., Construct 7 in Table 3B), and may be from the same or a different protein as the TM.
  • two or more signaling domains comprised in the CAR endodomain may be separated by one or more additional sequences, such as a spacer or a linker.
  • the ectodomain can further include a signal peptide or leader sequence and/or a spacer/hinge.
  • Exemplary spacers that may be included in a CAR or an ADR are commonly known in the art, including, but not limited to, IgG4 spacers, CD28 spacers, CD8 spacers, or combinations of more than one spacer.
  • the length of the spacers may also vary, from about 15 amino acids (a.a.) to about 300 a.a. or more.
  • a spacer of less than around 80 a.a., for example 10-80 a.a. is considered short; a spacer of about 80-180 a.a. is considered medium; and a spacer of more than 180 a.a. is considered long.
  • Non-limiting exemplary spacer peptides include those represented by an amino acid sequence of at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any of SEQ ID NOs: 140-144.
  • the spacer peptide comprises an amino acid sequence of at least about 90% identity to any of SEQ ID NOs: 140-144.
  • the spacer peptide comprises an amino acid sequence of at least about 95% identity to any of SEQ ID NOs: 140-144.
  • the spacer peptide comprises the amino acid sequence of any of SEQ ID NOs: 140-144.
  • SEQ ID NO: 140 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (39 a.a.) SEQ ID NO: 141 ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFL (88 a.a.) SEQ ID NO: 142 ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFQSTYRVVSVLT (89 a.a.) SEQ ID NO: 143 ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE
  • the CAR comprising a co-stimulatory domain derived from CD28, and a native or modified ITAM1 of CD3 ⁇ also comprises a hinge domain (or “spacer”) and trans- membrane domain derived from CD28, wherein an scFv may be connected to the transmembrane domain through the hinge, and the CAR comprises an amino acid sequence of at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 145, wherein the spacer may vary in length and sequence.
  • the CAR comprises an amino acid sequence of at least 80% to SEQ ID NO: 145, wherein the spacer may vary in length and sequence.
  • the CAR comprises an amino acid sequence of at least 90% to SEQ ID NO: 145, wherein the spacer may vary in length and sequence. In some embodiments, the CAR comprises an amino acid sequence of at least 95% to SEQ ID NO: 145, wherein the spacer may vary in length and sequence. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 145.
  • the CAR applicable to the cells provided herein comprises a transmembrane domain derived from NKG2D,
  • the CAR comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 146. In some embodiments, the CAR comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 146. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 146.
  • said CAR comprising a transmembrane domain derived from NKG2D, a co- stimulatory domain derived from 2B4, and a signaling domain comprising the native or modified CD3 ⁇ may further comprise a hinge.
  • the genetically engineered immune cells, iPSCs and derivative effector cells comprise a solid tumor targeting backbone as disclosed herein and a CAR comprising an antigen recognition region specific to a tumor cell surface HER2 antigen.
  • the antigen binding domain of the HER2-CAR in this application is based on the CDRs of CasMab250, a HER2 cancer-specific monoclonal antibody (CasMab), and this CasMab250 based HER2-CAR is also referred to “CasMab250-CAR” from time to time in this application.
  • the antigen binding domain of the HER2-CAR comprises a single chain variable fragment (scFV) having a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in-between, when compared to the exemplary sequences represented by SEQ ID NO: 153 or SEQ ID NO: 154, wherein each of SEQ ID NOs: 153 and 154 comprise a linker that can vary in length and/or sequence.
  • the scFV comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 153 or 154.
  • the scFV comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 153 or 154. In some embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 153. In some embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 154.
  • the CAR comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 155, wherein the linker in the ectodomain and the spacer between the ectodomain and transmembrane domain may vary in length and sequence. In some embodiments, the CAR comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 155, wherein the linker in the ectodomain and the spacer between the ectodomain and transmembrane domain may vary in length and sequence. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the CAR provided herein recognizes a HER2 antigen specific to cells of solid tumors.
  • the CAR provided herein recognizes a HER2 antigen of a tumor comprising breast cancer, ovary cancer, endometrium cancer, lung cancer, esophageal cancer, salivary gland cancer, bladder cancer, gastric cancer, colorectal cancer, or head and neck cancer.
  • the CAR provided herein recognizes a HER2 antigen of a tumor and does not respond, or has a low level of response, to HER2 expressed on non-cancer or normal cells.
  • the antigen recognition region is a scFV that specifically binds to the conserved ⁇ 3 domain of MICA and MICB.
  • the scFV comprises a variable region of the heavy chain and a variable region of the light chain, respectively represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to SEQ ID NO: 156 and 157.
  • the scFV is represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to any of SEQ ID NOs: 158 and 159.
  • the scFV comprises an amino acid sequence of at least 90% identity to SEQ ID NO: 158 or 159.
  • the scFV comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 158 or 159.
  • the scFV comprises the amino acid sequence of SEQ ID NO: 158 or 159.
  • MICA/B scFV light chain (LC)) SEQ ID NO: 158 MDFQVQIFSFLLISASVIMSRQIQLVQSGPELKKPGETVKVSCKASGYMFTNYAMNWVKQAPEKGLKWMG WINTHTGDPTYADDFKGRIAFSLETSASTAYLQINNLKNEDTATYFCVRTYGNYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVKLLIYDTSILHL GVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKFPRTFGGGTTLEIK (MICA/B scFV; HC-Linker-LC; Signal peptide/Leader – other signal peptides are also possible; Linker – other linkers are also possible) SEQ ID NO: 159 MDFQVQIFSFLLISASVIMSRDIQMTQTTSSLSASLGDRVTIS
  • the antigen recognition region is a scFV that specifically binds to the extracellular domain of CD269.
  • the scFV comprises a VH and a VL, represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to SEQ ID NO: 160 and SEQ ID NO: 161, respectively; or SEQ ID NO: 162 and SEQ ID NO: 163, or SEQ ID NO: 164 and SEQ ID NO: 165, respectively.
  • the scFV is represented by an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to any of SEQ ID NOs: 166-171.
  • the scFV comprises an amino acid sequence of at least 90% identity to any of SEQ ID NOs: 166- 171.
  • the scFV comprises an amino acid sequence of at least 95% identity to any of SEQ ID NOs: 166-171.
  • the scFV comprises an amino acid sequence of any of SEQ ID NOs: 166-171.
  • SEQ ID NO: 160 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVRQAPGKGLVWVGEINPSSSTINYAPSLKDKFTI SRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS (BCMA scFV heavy chain-1 (VH)) SEQ ID NO: 161 EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQKPGQAPRALIYSASLRFSGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELK (BCMA scFV light chain-1 (VL)) SEQ ID NO: 162 QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQKFTGRVTM TRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWYFDVWGQGTMVTV
  • the CAR comprises a recombinant heavy-chain-only antibody (VHH) that specifically binds to B7H3.
  • VHH recombinant heavy-chain-only antibody
  • the CAR comprises a binding domain comprising an amino acid sequence that is of at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identity to any of SEQ ID NOs: 172-177.
  • the binding domain comprises an amino acid sequence of at least about 90% identity to any of SEQ ID NOs: 172-177.
  • the binding domain comprises an amino acid sequence of at least about 95% identity to any of SEQ ID NOs: 172-177.
  • the binding domain comprises the sequence of any of SEQ ID Nos: 172-177.
  • the CAR comprises a binding domain comprising a variant of SEQ ID NO: 172, and wherein the variant has one or more mutations at positions comprising 1, 40, 46, 79, 87, 88, 89, 97, 98, and 117 of SEQ ID NO: 172.
  • the CAR comprises an amino acid sequence represented by a variant of SEQ ID NO: 172, wherein the variant has one or more substitutions comprising Q1E, T40A, E46V, G79L, K87R, P88A, D89E, V97A, S98R, and Q117L according to SEQ ID NO: 172.
  • SEQ ID NO: 172 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQTPGKGLEWVSTINRDGSATWYADSVKGRFTI SRDNAKNTGYLQMNSLKPDDTAVYYCVSDPDNYSSDEMVPYWGQGTQVTVSS (122 a.a.
  • VHH camelid B7H3 Attorney Docket No.: FATE-173/01WO SEQ ID NO: 173 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQAPGKGLVWVSTINRDGSATWYADSVKGRFTI SRDNAKNTLYLQMNSLRAEDTAVYYCARDPDNYSSDEMVPYWGQGTLVTVSS (122 a.a.
  • VHH1 SEQ ID NO: 174 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQAPGKGLVWVSTINRDGSATWYADSVKGRFTI SRDNAKNTLYLQMNSLRAEDTAVYYCVSDPDNYSSDEMVPYWGQGTLVTVSS (122 a.a. VHH2) SEQ ID NO: 175 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQTPGKGLVWVSTINRDGSATWYADSVKGRFTI SRDNAKNTLYLQMNSLRAEDTAVYYCVSDPDNYSSDEMVPYWGQGTLVTVSS (122 a.a.
  • VHH3 SEQ ID NO: 176 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQAPGKGLEWVSTINRDGSATWYADSVKGRFTI SRDNAKNTLYLQMNSLRAEDTAVYYCVSDPDNYSSDEMVPYWGQGTLVTVSS (122 a.a. VHH4) SEQ ID NO: 177 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQTPGKGLEWVSTINRDGSATWYADSVKGRFTI SRDNAKNTGYLQMNSLRPEDTAVYYCVSDPDNYSSDEMVPYWGQGTLVTVSS (122AA.
  • the antigen binding domain of the CAR comprises a VH and a VL domain having a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in-between, when compared to the exemplary sequences represented by SEQ ID NOs: 178 and 179, or SEQ ID NOs: 180 and 181, or SEQ ID NOs: 182 and 183.
  • SEQ ID NOs: 178 and 179 or SEQ ID NOs: 180 and 181, or SEQ ID NOs: 182 and 183.
  • the antigen binding domain of the CAR comprises a single chain variable fragment (scFV) having a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in-between, when compared to the exemplary sequence represented by SEQ ID NOs: 184, 185, or 186, wherein SEQ ID NOs: 184, 185, or 186 comprises a linker that can vary in length and/or sequence.
  • the scFV comprises an amino acid sequence of at least 90% identity to any of SEQ ID NOs: 184-186.
  • the scFV comprises an amino acid sequence of at least 95% identity to any of SEQ ID NOs: 184- 186. In some embodiments, the scFV comprises the amino acid sequence of any of SEQ ID NOs: 184-186.
  • Pat No.8,409,577 or a tandem CAR (see for example, Hegde et al., J Clin Invest.2016;126(8):3036-3052); an inducible CAR (see for example, U.S. Pub. Nos.2016/0046700, 2016/0058857, and 2017/0166877); a switchable CAR (see for example, U.S. Pub. No.2014/0219975); and any other designs known in the art.
  • the polynucleotide encoding a CAR as disclosed is operatively linked to an endogenous promoter.
  • the polynucleotide encoding a CAR as disclosed is operatively linked to an exogenous promoter.
  • the promoters may be inducible, or constitutive, and may be temporal-, tissue- or cell type- specific. Suitable constitutive promoters for methods disclosed herein include, but are not limited to, cytomegalovirus (CMV), elongation factor 1 ⁇ (EF1 ⁇ ), phosphoglycerate kinase (PGK), hybrid CMV enhancer/chicken ⁇ -actin (CAG) and ubiquitin C (UBC) promoters.
  • the exogenous promoter is CAG.
  • the cells comprising a solid tumor targeting backbone comprise a polynucleotide encoding a CAR and/or one or more additional modified modalities provided herein.
  • a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one phenotype as provided herein, including but not limited to, a solid tumor targeting backbone as described herein and a CAR, wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derivative NK and T cells, which are well- defined and uniform in composition, and can be mass produced at significant scale in a cost- effective manner.
  • TCE T Cell Enhancer
  • TCE edits have the ability to activate one or more of cytokine signaling (e.g., soluble cytokines, or cytokine-receptor fusion constructs), costimulatory signals (e.g., over-expression of costimulatory receptors or other important signal transduction proteins), or gene expression profiles (e.g., over-expression of transcription factors).
  • cytokine signaling e.g., soluble cytokines, or cytokine-receptor fusion constructs
  • costimulatory signals e.g., over-expression of costimulatory receptors or other important signal transduction proteins
  • gene expression profiles e.g., over-expression of transcription factors
  • the TCE edits are compatible with T cell differentiation process.
  • TCF1 and CD27 are suitable TCEs for potentiating iPSC derived T cells.
  • the TCF1 has a sequence of SEQ ID NO: 187.
  • the CD27 has a sequence of SEQ ID NO: 188.
  • the polynucleotide encoding a TCE as disclosed is operatively linked to an exogenous promoter.
  • the promoters may be inducible, or constitutive, and may be temporal-, tissue- or cell type- specific. Suitable constitutive promoters for methods disclosed herein include, but are not limited to, cytomegalovirus (CMV), elongation factor 1 ⁇ (EF1 ⁇ ), phosphoglycerate kinase (PGK), hybrid CMV enhancer/chicken ⁇ -actin (CAG) and ubiquitin C (UBC) promoters.
  • the exogenous promoter is CAG. 8.
  • CD38 knockout [000252] The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma and a CD20 negative B-cell malignancy, which makes it an attractive target for antibody Attorney Docket No.: FATE-173/01WO therapeutics to deplete cancer cells.
  • Antibody mediated cancer cell depletion is usually attributable to a combination of direct cell apoptosis induction and activation of immune effector mechanisms such as ADCC (antibody-dependent cell-mediated cytotoxicity).
  • the immune effector mechanisms in concert with the therapeutic antibody may also include antibody-dependent cell-mediated phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC).
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • CD38 is also expressed on plasma cells, as well as on NK cells and activated T and B cells. During hematopoiesis, CD38 is expressed on CD34 + stem cells and lineage-committed progenitors of lymphoid, erythroid, and myeloid, and during the final stages of maturation which continues through the plasma cell stage.
  • CD38 As a type II transmembrane glycoprotein, CD38 carries out cell functions as both a receptor and a multifunctional enzyme involved in the production of nucleotide-metabolites. As an enzyme, CD38 catalyzes the synthesis and hydrolysis of the reaction from NAD + to ADP-ribose, thereby producing secondary messengers CADPR and NAADP which stimulate release of calcium from the endoplasmic reticulum and lysosomes, critical for the calcium dependent process of cell adhesion. As a receptor, CD38 recognizes CD31 and regulates cytokine release and cytotoxicity in activated NK cells.
  • CD38 is also reported to associate with cell surface proteins in lipid rafts, to regulate cytoplasmic Ca 2+ flux, and to mediate signal transduction in lymphoid and myeloid cells.
  • systemic use of CD38 antigen binding receptor transduced T cells has been shown to lyse the CD38 + fractions of CD34 + hematopoietic progenitor cells, monocytes, NK cells, T cells and B cells, leading to incomplete treatment responses and reduced or eliminated efficacy because of the impaired recipient immune effector cell function.
  • CD38-specific antibody NK cell reduction in both bone marrow and peripheral blood was observed, although other immune cell types, such as T cells and B cells, were unaffected despite their CD38 expression (Casneuf et al., Blood Advances.2017; 1(23):2105-2114).
  • the present application includes a strategy to leverage the full potential of CD38 targeted cancer treatment by knocking out CD38 in the effector cell, thereby overcoming CD38-specific antibody and/or CD38 antigen binding domain- induced effector cell depletion or reduction through fratricide.
  • a CD38-specific Attorney Docket No.: FATE-173/01WO antibody a secreted CD38-specific engager or a CD38-CAR (chimeric antigen receptor) against activation of recipient T, Treg, NK, and/or B cells can be used as a replacement for lymphodepletion using chemotherapy such as Cy/Flu (cyclophosphamide/fludarabine) prior to adoptive cell transferring.
  • chemotherapy such as Cy/Flu (cyclophosphamide/fludarabine) prior to adoptive cell transferring.
  • CD38 + T and pbNK cells when targeting CD38 + T and pbNK cells using CD38- effector cells in the presence of anti-CD38 antibodies or CD38 inhibitors, the depletion of CD38 + alloreactive cells increases the NAD + (nicotinamide adenine dinucleotide, a substrate of CD38) availability and decreases NAD + consumption related cell death, which, among other advantages, boosts effector cell responses in an immunosuppressive tumor microenvironment and supports cell rejuvenation in aging, degenerative or inflammatory diseases.
  • NAD + nicotinamide adenine dinucleotide, a substrate of CD38
  • CD38 knockout is compatible with other components and processes contemplated for establishing a solid tumor targeting backbone as disclosed in this application, thereby providing an immune cell, an iPSC and differentiated effector cell therefrom comprising a CD38 knockout with additional backbone edits.
  • the solid tumor targeting backbone comprised in the iPSC line or a derivative thereof comprises an TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X- C-motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, among other edits as provided herein.
  • the provided CD38 neg iPSC line optionally comprises one or more additional engineered modalities described herein, and as shown in Table 4.
  • CD38 neg derivative effector cells comprising a solid tumor targeting backbone are protected against fratricide and allorejection when CD38 targeted therapeutic moieties are employed with the effector cells, among other advantages including improved metabolic fitness, increased resistance to oxidative stress and inducing a protein expression program in the effector cell that enhances cell activation and effector function.
  • anti-CD38 monoclonal antibody therapy significantly depletes a patient’s activated immune system without adversely affecting the patient’s hematopoietic stem cell compartment.
  • a CD38 neg derivative cell has the ability to resist CD38 antibody mediated depletion, and may be effectively administered in combination with an anti-CD38 antibody or CD38-CAR without the use of toxic conditioning agents, thereby reducing and/or replacing chemotherapy-based lymphodepletion.
  • the CD38 knockout in an iPSC line is a bi- allelic knockout.
  • knocking out CD38 at the same time as inserting one or more transgenes, including a C-X-C-motif chemokine receptor or a variant thereof, a TGF ⁇ - SRR, and/or a CD16 variant as provided herein, at a selected position in CD38 can be achieved, for example, by a CD38-targeted knock-in/knockout (CD38-KI/KO) construct.
  • a CD38-targeted knock-in/knockout construct CD38-KI/KO
  • the construct comprises a pair of CD38 targeting homology arms for position-selective insertion within the CD38 locus.
  • the preselected targeting site is within an exon of CD38.
  • the CD38-KI/KO constructs provided herein allow the transgene(s) to express either under the CD38 endogenous promoter or under an exogenous promoter comprised in the construct.
  • a linker sequence for example, a 2A linker or IRES, is placed between any two transgenes.
  • the 2A linker encodes a self-cleaving peptide derived from FMDV, ERAV, PTV-I, and TaV (referred to as “F2A”, “E2A”, “P2A”, and “T2A”, respectively), allowing for separate proteins to be produced from a single translation.
  • insulators are included in the construct to reduce the risk of transgene and/or exogenous promoter silencing.
  • the exogenous promoter comprised in a CD38-KI/KO construct may be CAG, or other constitutive, inducible, temporal-, tissue-, or cell type- specific promoters including, but not limited to CMV, EF1 ⁇ , PGK, and UBC.
  • said iPSC is capable of directed differentiation to produce functional derivative hematopoietic cells including, but not limited to, mesodermal cells with definitive hemogenic endothelium (HE) potential, definitive HE, CD34 + hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPP), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages.
  • the CD38 negative effector cells are NK lineage cells derived from iPSCs.
  • the CD38 negative effector cells are T lineage cells derived from iPSCs.
  • the iPSC and derivative cells thereof comprise a solid tumor targeting backbone comprising CD38 neg and at least two of: a polynucleotide encoding a C-X-C motif chemokine receptor or a variant thereof, a polynucleotide encoding a TGF ⁇ -SRR, and a polynucleotide encoding a CD16 variant, and optionally include one or more additional genomic edits as described herein. 9. Stealth related edits [000260] Multiple HLA class I and class II proteins must be matched for histocompatibility in allogeneic recipients to avoid allogeneic rejection problems.
  • HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or deletion or disruption of HLA class-I associated genes including, but not limited to, beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene and Tapasin.
  • B2M beta-2 microglobulin
  • TAP1 gene TAP1 gene
  • TAP2 gene TAP2 gene
  • Tapasin the B2M gene encodes a common subunit essential for cell surface expression of all HLA class I heterodimers.
  • B2M Attorney Docket No.: FATE-173/01WO negative cells are HLA-I deficient.
  • HLA class II deficiency can be achieved by functional deletion or disruption of HLA class II associated genes including, but not limited to, RFXANK, CIITA, RFX5 and RFXAP.
  • CIITA is a transcriptional coactivator, functioning through activation of the transcription factor RFX5 required for class II protein expression.
  • CIITA negative cells are HLA-II deficient.
  • this application provides an iPSC and derivative cells therefrom comprising HLA-I and/or HLA-II deficiency, for example by lacking B2M and/or CIITA expression, wherein the obtained derivative effector cells enable allogeneic cell therapies by eliminating the need for MHC (major histocompatibility complex) matching, and avoiding recognition and killing by host (allogeneic) T cells.
  • MHC major histocompatibility complex
  • HLA-E, HLA-G or other non-classical HLA-I proteins may be optionally knocked in to avoid NK cell recognition and killing of the HLA-I deficient effector cells derived from an engineered iPSC.
  • the provided HLA-I deficient iPSC and its derivative cells further comprise HLA-G knock-in.
  • the provided HLA-I deficient iPSC and its derivative cells further comprise one or both of CD58 knockout and CD54 knockout.
  • CD58 and CD54 are adhesion proteins initiating signal-dependent cell interactions, and facilitating cell, including immune cell, migration. It was previously shown that CD58 and/or CD54 disruption effectively reduces the susceptibility of HLA-I deficient iPSC-derived effector cells to allogeneic NK cell killing. While it was shown that CD58 knockout has a higher efficiency in reducing allogeneic NK cell activation than CD54 knockout, double knockout of both CD58 and CD54 was shown to provide the most enhanced reduction of NK cell activation. In some observations, the CD58 and CD54 double knockout is even more effective than HLA-G overexpression for HLA-I deficient cells in overcoming “missing-self” effect.
  • the iPSC and its derivative cells comprising a solid tumor targeting backbone comprising two or more of: a C-X-C-motif chemokine receptor or a variant thereof, a TGF ⁇ -SRR, and a CD16 variant.
  • said iPSC and its derivative cells are CD58 negative.
  • said iPSC and its derivative cells are CD54 negative.
  • said iPSC and its derivative cells are CD54 negative and CD58 negative.
  • the engineering for HLA-I and/or HLA-II deficiency may be bypassed, or kept intact, by expressing an inactivation CAR targeting an upregulated surface Attorney Docket No.: FATE-173/01WO protein in activated recipient immune cells to avoid allorejection.
  • the upregulated surface protein in the activated recipient immune cells includes, but is not limited to, CD38, CD25, CD69, CD44, 4-1BB, OX40, or CD40L.
  • the cell expresses such an inactivation CAR, it is preferable that the cell does not express, or has knockout of, the same surface protein targeted by CAR.
  • the inactivation CAR comprises at least one of a CD38-CAR, a CD25-CAR, a CD69-CAR, a CD44-CAR, a 4-1BB-CAR, an OX40- CAR, and a CD40L-CAR.
  • a master cell bank comprising single cell sorted and expanded clonal engineered iPSCs having at least one phenotype as provided herein, including but not limited to, a solid tumor targeting backbone as described herein and HLA modification (“HLA” in Table 4: HLA-I and/or HLA-II deficiency with or without HLA-E or HLA-G knock in, or with knockout of one or both of CD58 and CD54), wherein the cell bank provides a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, including but not limited to derivative NK and T cells, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective manner.
  • HLA HLA modification
  • the present application provides an immune cell, an iPSC, an iPS cell line cell, or a population thereof, and a derivative functional cell obtained from differentiating the iPSC, wherein each cell comprises a solid tumor targeting backbone comprising an TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X-C-motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, among other modifications as described in the application, wherein the cell is an eukaryotic cell, an animal cell, a human cell, an induced pluripotent cell (iPSC), an iPSC-derived effector cell, an immune cell, or a feeder cell.
  • iPSC induced pluripotent cell
  • said cells are suitable for homing or migration of the effectors to tumor sites for CAR targeted tumor killing.
  • the tumor cells at the tumor sites secrete or overexpress a chemokine that binds to a C-X-C-motif chemokine receptor or a variant thereof.
  • the C-X-C motif chemokine receptor comprises CXCR2 or CXCR3.
  • the secreted or overexpressed chemokine by the tumor cells at the tumor sites comprises IL8 (CXCL8).
  • the functional derivative cells are hematopoietic cells including, but not limited to, mesodermal cells with definitive hemogenic endothelium (HE) potential, definitive HE, CD34 + hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitors (MPP), T cell progenitors, NK cell Attorney Docket No.: FATE-173/01WO progenitors, myeloid cells, neutrophil progenitors, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutrophils, dendritic cells, and macrophages.
  • HE definitive hemogenic endothelium
  • MPP hematopoietic multipotent progenitors
  • NK cell Attorney Docket No.: FATE-173/01WO progenitors, myeloid cells, neutrophil progenitors, T lineage cells, NKT lineage cells, NK lineage cells, B lineage cells, neutr
  • the functional derivative hematopoietic cells comprise effector cells having one or more functional features that are not present in a counterpart primary T, NK, NKT, and/or B cell.
  • Said effector cells have improved ability to home or migrate to, and remain in, turmor sites which include solid tumors, and provide a tumor antigen dual targeting mechanism to tackle tumor antigen heterogeneity and tumor antigen escape.
  • the dual targeting through CAR binding and CD16-mediated ADCC further increases tumor targeting precision, enhancing tumor killing and minimizing the impact of tumor antigen escape.
  • the iPSC, iPS cell line cell, or clonal population thereof, and/or derivative effector cells therefrom comprising a solid tumor targeting backbone as described herein, wherein the solid tumor targeting backbone further comprises CD38 knockout, and said cells are suitable for a subject undergoing an adoptive cell therapy.
  • the subject may additionally receive a tumor sensitizing procedure (e.g., administration of a sensitizing agent, such as a chemotherapeutic agent, radiation, or radiotherapeutic) to upregulate tumor cell chemokine expression including, but not limited to, CXCL8 overexpression, to further enhance C-X-C motif chemokine receptor overexpressing effector cell homing, trafficking and retention, and cytotoxicity at the tumor sites.
  • a tumor sensitizing procedure e.g., administration of a sensitizing agent, such as a chemotherapeutic agent, radiation, or radiotherapeutic
  • a sensitizing agent such as a chemotherapeutic agent, radiation, or radiotherapeutic
  • said effector cells comprise T lineage cells.
  • said effector cells comprise NK lineage cells.
  • the iPSCs and their derivative cells that comprise a solid tumor targeting backbone comprising an TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X-C-motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, among other modifications as described herein.
  • said cells have the ADR and/or a cytokine inserted in a TCR constant region (TRAC or TRBC), leading to TCR knockout, and optionally placing ADR and/or cytokine expression under the control of the endogenous TCR promoter to promote T cell differentiation, T cell identity and lineage commitment, and to avoid T cell exhaustion.
  • TCR neg cells do not require HLA matching, have reduced alloreactivity, and are able to prevent GvHD (Graft versus Host Disease) when used in allogeneic adoptive cell therapies.
  • Additional insertion sites of polynucleotides of interest include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, NKG2A, NKG2D, CD25, CD38, Attorney Docket No.: FATE-173/01WO CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, ASB2, and TIGIT.
  • the polynucleotides of interest comprise those encoding a CAR, a TGF ⁇ -SRR, a C-X-C-motif chemokine receptor or a variant thereof, a CD16 variant, a TCE.
  • the effector cell, the iPSC and its derivative NK or T cell described herein comprises one or more CARs, where the CAR is inserted in the CD58, TIM3, TIGIT, NKG2A or NKG2D locus, leading to CD58, TIM3, TIGIT, NKG2A or NKG2D knockout.
  • the effector cell, the iPSC and its derivative NK or T cell described herein comprises co-expressed TGF ⁇ -SRR, C-X-C-motif chemokine receptor or a variant thereof, and a CD16 variant at a CD38 locus, wherein the CD38 is knocked out upon the insertion of the transgenes.
  • an iPSC comprising a solid tumor targeting backbone comprising an TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, wherein the polynucleotide encoding an interleukin cytokine or a cytokine signaling complex (IL) comprising a full or partial length of cytokine and/or a full or partial length of a cytokine receptor to enable cytokine signaling contributing to cell survival, persistence and/or expansion, wherein the iPSC line is capable of directed differentiation to produce functional derivative hematopoietic cells having improved survival, persistency, expansion, and effector cell function, as well as homing, trafficking, tumor site retention and cytotoxicity.
  • IL cytokine signaling complex
  • the exogenously introduced IL cytokine signaling(s) comprise the signaling of any one, two, or more of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21.
  • the introduced IL cytokine signaling complex is for IL15 signaling in the cell, and the cell is optionally an NK lineage cell.
  • the introduced IL cytokine signaling complex is for IL2, IL7 or IL18 signaling in the cell, and the cell is optionally a T lineage cell.
  • the introduced IL cytokine signaling complex is expressed on the cell surface.
  • the introduced IL cytokine is soluble.
  • the IL cytokine signaling is constitutively activated.
  • the IL cytokine signaling is under the control of temporal and dose regulation of an endogenous or exogeneous promoter.
  • activation of the IL cytokine signaling is inducible.
  • activation of the IL cytokine signaling is transient and/or temporal.
  • the transient/temporal expression of a cell surface or soluble cytokine/cytokine receptor is through a retrovirus, Sendai virus, an adenovirus, an episome, mini-circle, or RNAs including mRNA.
  • effector cells comprising a solid tumor targeting backbone comprising polynucleotides encoding an TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X-C- motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, and optionally one or more additional genetic modifications as provided in Table 4 and throughout the application are capable of maintaining or improving cell growth, proliferation, expansion, and/or effector function autonomously without contacting additionally supplied soluble cytokines in vitro or in vivo, as well as enhanced homing, trafficking, and retention at
  • the present application provides iPSCs and their functional derivative hematopoietic cells, which comprise any one of the following genotypes in Table 4.
  • “TRAC_” or “CD38_” in the genotype indicates the insertion locus.
  • insertion locus is not specified, the insertion takes place in any locus as provided in this application.
  • “ ⁇ ” is used to separate transgenes at different loci.
  • the genetically modified modalities further comprise one or more of: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins promoting engraftment, trafficking, homing, viability, self-renewal, persistence, immune response regulation and modulation, and/or survival of the iPSCs or derivative cells thereof.
  • the genetically modified iPSC and the derivative cells thereof comprise a genotype listed in Table 4.
  • the iPSC and its derivative effector cells comprising any one of the genotypes in Table 4 may additionally comprise disruption of at least one of TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, and any gene in the chromosome 6p21 region; or introduction of at least one of HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, antigen-specific TCR, an Fc receptor, an engager, and a surface triggering receptor for coupling with bi-, multi- specific or universal engagers.
  • Engagers are fusion proteins consisting of two or more single-chain variable fragments (scFvs) of different antibodies, with at least one scFv that binds to an effector cell surface molecule or surface triggering receptor, and at least another to a target cell via a target cell specific surface molecule.
  • engagers include, but are not limited to, bi-specific T cell engagers (BiTEs), bi-specific killer cell engagers (BiKEs), tri-specific killer cell engagers (TriKEs), multi-specific killer cell engagers, or universal engagers compatible with multiple immune cell types.
  • engagers can be bi-specific or multi-specific.
  • Such bi-specific or multi-specific engagers are capable of directing an effector cell (e.g., a T cell, a NK cell, an NKT cell, a B cell, a macrophage, and/or a neutrophil) to a tumor cell and activating the immune effector cell, and have shown great potential to maximize the benefits of CAR-T cell therapy.
  • the engager is used in combination with a population of the effector cells comprising a solid tumor targeting backbone as described herein by concurrent or consecutive administration, wherein the effector cells comprise a surface molecule, or surface triggering receptor, that is recognized by the engager.
  • the engager is a bi-specific antibody expressed by a derivative effector cell through genetically engineering an iPSC comprising a solid tumor targeting backbone as described herein, and directed differentiation of the engineered iPSC.
  • exemplary effector cell surface molecules, or surface triggering receptors that can be used for bi- or multi- specific engager recognition, or coupling, include, but are not limited to, CD3, CD28, CD5, CD16, NKG2D, CD64, CD32, CD89, NKG2C, and a chimeric Fc receptor as disclosed herein.
  • the exogenous CD16 expressed on the surface of the derivative effector cells for Attorney Docket No.: FATE-173/01WO engager recognition is a hnCD16, comprising a CD16 (containing F176V and optionally S197P) or CD64 extracellular domain, and native or non-native transmembrane, stimulatory and/or signaling domains as described herein.
  • the exogenous CD16 expressed on the surface of effector cells for engager recognition is a CD16-based chimeric Fc receptor (CFcR).
  • Antibodies for immunotherapy in addition to the genomically engineered effector cells comprising a solid tumor targeting backbone as provided herein, additional therapeutic agents comprising an antibody, or an antibody fragment that targets an antigen associated with a condition, a disease, or an indication may be used with these effector cells in a combinational therapy.
  • the antibody is used in combination with a population of the effector cells comprising a solid tumor targeting backbone as described herein by concurrent or consecutive administration to a subject.
  • such antibody or a fragment thereof may be expressed by the effector cells by genetically engineering an iPSC using an exogenous polynucleotide sequence encoding said antibody or fragment thereof, and directing differentiation of the engineered iPSC.
  • the antibodies suitable for combinational treatment as an additional therapeutic agent to the administered iPSC-derived effector cells further include bi-specific or multi-specific antibodies that target more than one antigen or epitope on a target cell or recruit effector cells (e.g., T cells, NK cells, or macrophage cells) toward target cells while targeting the target cells.
  • effector cells e.g., T cells, NK cells, or macrophage cells
  • Such bi-specific or multi-specific antibodies function as engagers capable of directing an effector cell (e.g., a T cell, a NK cell, an NKT cell, a B cell, a macrophage, and/or a neutrophil) to a tumor Attorney Docket No.: FATE-173/01WO cell and activating the immune effector cell, and have shown great potential to maximize the benefits of antibody therapy.
  • the iPSC-derived effector cells comprise hematopoietic lineage cells comprising a genotype listed in Table 4. In some embodiments, the iPSC-derived effector cells comprise NK cells comprising a genotype listed in Table 4. In some embodiments, the iPSC-derived effector cells comprise T cells comprising a genotype listed in Table 4.
  • the combination comprises iPSC-derived NK or T cells comprising a solid tumor targeting backbone comprising an TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X-C-motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, and optionally one or more additional genetic modifications as provided herein; and a therapeutic antibody as described above.
  • the combination comprises iPSC-derived NK or T cells comprising a solid tumor targeting backbone as described herein, and optionally TCR knockout, a CAR, a cytokine signaling complex, exogenous CD16 or a variant thereof, and CD38 knockout; and a therapeutic antibody as described above.
  • the combination comprises iPSC-derived NK or T cells comprising a solid tumor targeting backbone as described herein, and optionally TCR knockout, a CAR, IL cytokine signaling complex, exogenous CD16 or a variant thereof, CD38 knockout, and HLA-I and/or HLA-II deficiency; and a therapeutic antibody as described above.
  • the CAR targets a solid tumor antigen as set forth herein.
  • the exogenous CD16 is hnCD16.
  • hnCD16 provides enhanced ADCC of the monoclonal antibody, whereas the CAR not only targets a specific tumor antigen but also prevents tumor antigen escape using a dual targeting strategy in combination with a monoclonal antibody targeting a different tumor antigen.
  • Checkpoint inhibitors are cell molecules, often cell surface molecules, capable of suppressing or downregulating immune responses when not inhibited. It is now clear that tumors co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens.
  • Checkpoint inhibitors are antagonists capable of reducing checkpoint gene expression or gene products, or deceasing activity of checkpoint molecules, thereby blocking inhibitory checkpoints, and restoring immune system function.
  • the development of checkpoint inhibitors targeting PD1/PDL1 or CTLA4 has Attorney Docket No.: FATE-173/01WO transformed the oncology landscape, with these agents providing long term remissions in multiple indications.
  • 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 iPSC-derived cells as provided herein in a combination therapy with CI.
  • the iPSC-derived cells are NK cells.
  • the iPSC-derived cells are T cells.
  • the derivative NK cells provided herein have been shown to resist PDL1-PD1 mediated inhibition, and to have the ability to enhance T cell migration, to recruit T cells to the tumor microenvironment, and to augment T cell activation at the tumor site. Therefore, the tumor infiltration of T cells facilitated by the functionally potent genomically engineered derivative NK cells indicate that said NK cells are capable of synergizing with T cell targeted immunotherapies, including the checkpoint inhibitors, to relieve local immunosuppression and to reduce tumor burden.
  • the checkpoint inhibitor is used in combination with a population of the effector cells comprising a solid tumor targeting backbone as described herein by concurrent or consecutive administration thereof to a subject.
  • the checkpoint inhibitor is expressed by the effector cells by genetically engineering an iPSC using an exogenous polynucleotide sequence encoding said checkpoint inhibitor, or a fragment or variant thereof, and directing differentiation of the engineered iPSC.
  • Some embodiments of the combination therapy with the effector cells comprising a solid tumor targeting backbone as described herein comprise at least one checkpoint inhibitor to target at least one checkpoint molecule; wherein the effector cells have a genotype listed in Table 4.
  • the iPSC-derived effector cell for checkpoint inhibitor combination therapy comprises a solid tumor targeting backbone comprising an TCR promoter- driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X-C-motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, among other modifications as provided herein.
  • the above derivative effector cell comprising a solid tumor targeting backbone as provided herein comprises knock-out of CD54, CD58, TIM3, TIGIT.
  • said derivative effector cell may additionally comprise deletion, disruption, or reduced expression of at least one of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region; or introduction of at least one of HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, CAR, Attorney Docket No.: FATE-173/01WO Fc receptor, and surface triggering receptor for coupling with bi-, multi- specific or universal engagers.
  • Suitable checkpoint inhibitors for combination therapy with the derivative NK or T cells as provided herein include, but are not limited to, antagonists of PD1 (Pdcdl, CD279), PDL- 1 (CD274), TIM3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG3 (CD223), CTLA4 (CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A2AR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA/B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A/HLA-
  • the antagonist inhibiting any of the above checkpoint molecules is an antibody.
  • the checkpoint inhibitory antibodies may be murine antibodies, human antibodies, humanized antibodies, a camel Ig, a single variable new antigen receptor (VNAR), a shark heavy-chain-only antibody (Ig NAR), chimeric antibodies, recombinant antibodies, or antibody fragments thereof.
  • Non-limiting examples of antibody fragments include Fab, Fab′, F(ab′)2, F(ab′)3, Fv, single chain antigen binding fragments (scFv), (scFv)2, disulfide stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb, Nanobody), recombinant heavy-chain-only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be more cost-effective to produce, more easily used, or more sensitive than the whole antibody.
  • the one, or two, or three, or more checkpoint inhibitors comprise 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 antibody), IPH43 (anti-MICA antibody), IPH33 (anti-TLR3 antibody), lirimumab (anti-KIR antibody), monalizumab (anti-NKG2A antibody), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and any derivatives, functional equivalents, or biosimilars thereof.
  • atezolizumab anti-PDL1 mAb
  • avelumab anti-PDL1 mAb
  • durvalumab anti-PDL1 m
  • the antagonist inhibiting any of the above checkpoint molecules is microRNA-based, as many miRNAs are found as regulators that control the expression of immune checkpoints (Dragomir et al., Cancer Biol Med.2018, 15(2):103-115).
  • the 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.
  • Some embodiments of the combination therapy with the provided iPSC-derived effector cells comprise at least one checkpoint inhibitor to target at least one checkpoint Attorney Docket No.: FATE-173/01WO molecule; wherein the iPSC-derived cells have a genotype listed in Table 4.
  • Some other embodiments of the combination therapy with the provided derivative effector cells comprise two, three or more checkpoint inhibitors such that two, three, or more checkpoint molecules are targeted.
  • said checkpoint inhibitor is an antibody, or a humanized or Fc modified variant or fragment, or a functional equivalent or biosimilar thereof, and said checkpoint inhibitor is produced by the iPSC-derived cells by expressing an exogenous polynucleotide sequence encoding said antibody, or a fragment or variant thereof.
  • the exogenous polynucleotide sequence encoding the antibody, or a fragment or a variant thereof that inhibits a checkpoint is co-expressed with a CAR, either in separate constructs or in a bi- or tri- cistronic construct comprising both the CAR and the sequence encoding the antibody, or the fragment thereof.
  • the sequence encoding the antibody or the fragment thereof can be linked to either the 5’ or the 3’ end of a CAR expression construct through a self-cleaving 2A coding sequence, illustrated as, for example, CAR-2A-CI or CI-2A-CAR.
  • the coding sequences of the checkpoint inhibitor and the CAR may be in a single open reading frame (ORF).
  • the checkpoint inhibitor When the checkpoint inhibitor is delivered, expressed and secreted as a payload by the derivative effector cells capable of infiltrating the tumor microenvironment (TME), it counteracts the inhibitory checkpoint molecule upon engaging the TME, allowing activation of the effector cells by activating modalities such as CAR or activating receptors.
  • TME tumor microenvironment
  • the checkpoint inhibitor co-expressed with CAR inhibits at least one of the checkpoint molecules: PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2AR, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA/B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A/HLA-E, and inhibitory KIR.
  • the checkpoint inhibitor co-expressed with CAR inhibits at least one of the checkpoint molecules: PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1
  • the checkpoint inhibitor co-expressed with CAR in a derivative cell having a genotype listed in Table 4 comprises atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, or their humanized, or Fc modified variants, fragments and their functional equivalents or biosimilars.
  • the checkpoint inhibitor co-expressed with CAR is atezolizumab, or its humanized, or Fc modified variants, fragments or their functional equivalents or biosimilars.
  • the checkpoint inhibitor co-expressed with CAR is nivolumab, or its humanized, or Fc modified variants, fragments or their functional equivalents or biosimilars.
  • the checkpoint inhibitor co-expressed Attorney Docket No.: FATE-173/01WO with CAR is pembrolizumab, or its humanized, or Fc modified variants, fragments or their functional equivalents or biosimilars.
  • the combination therapy comprising the iPSC- derived cells comprising a solid tumor targeting backbone as provided herein and at least one antibody inhibiting a checkpoint molecule
  • said antibody is not produced by, or in, the iPSC- derived cells and is additionally administered before, with, or after the administering of the derivative cells having a genotype listed in Table 4.
  • the administering of one, two, three or more checkpoint inhibitors in a combination therapy with the provided derivative effector cells are simultaneous or sequential.
  • the checkpoint inhibitor included in the treatment is one or more of atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc modified variants, fragments and their functional equivalents or biosimilars.
  • the checkpoint inhibitor included in the treatment is atezolizumab, or its humanized or Fc modified variant, fragment and its functional equivalent or biosimilar. In some embodiments of the combination treatment comprising derived NK cells or T cells having a genotype listed in Table 4, the checkpoint inhibitor included in the treatment is nivolumab, or its humanized or Fc modified variant, fragment or its functional equivalent or biosimilar. In some embodiments of the combination treatment comprising derived NK cells or T cells having a genotype listed in Table 4, the checkpoint inhibitor included in the treatment is pembrolizumab, or its humanized or Fc modified variant, fragment or its functional equivalent or biosimilar. 14.
  • a CFR is a fusion protein with a receptor functionality.
  • a CFR enables an effector cell to initiate an appropriate signal transduction cascade through CFR binding with a selected agonist for enhanced therapeutic properties of the effector cell expressing the CFR.
  • a CFR comprises an ectodomain, a transmembrane domain, and an endodomain, wherein the ectodomain, the transmembrane domain and the endodomain do not comprise any endoplasmic reticulum (ER) retention signals or endocytosis signals.
  • the ectodomain of the CFR is for initiating signal transduction upon binding to an engager; the transmembrane domain is for membrane anchoring of the CFR; and the endodomain comprises at least one signaling domain that regulates (e.g., activates or deactivates) a signaling pathway of choice for enhancing cell therapeutic properties including, but not limited to, tumor killing, persistence, mobility, differentiation, TME counteracting, and/or controlled apoptosis.
  • the elimination of ER retention signals from the CFR permits CFR cell surface presentation by itself when expressed, and the elimination of endocytosis signals from the CFR reduces CFR internalization and surface downregulation.
  • the domains of the CFRs as provided by some embodiments herein are modular, meaning for a given endodomain of a CFR, the ectodomain of the CFR is switchable depending on the binding specificity of a selected agonist, such as an antibody, a BiTE, a TriKE, or any other type of engager, to be used with said CFR; and for a given ectodomain and a specificity matching agonist, the endodomain is switchable depending on the desired signaling pathway to be activated.
  • a selected agonist such as an antibody, a BiTE, a TriKE, or any other type of engager
  • the three integration sites are TRAC, CD38, and TIGIT, with all three endogenous genes knocked out upon transgene insertion.
  • the three integration sites are TRAC, CD38, and PH12, with endogenous genes at TRAC and CD38 knocked out upon transgene insertion.
  • the three integration sites are TRAC, CD38, and H11, with endogenous genes at TRAC and CD38 knocked out upon transgene insertion.
  • Suitable constructive promoters for methods of the invention include, but not limited to, cytomegalovirus (CMV), elongation factor 1 ⁇ (EF1 ⁇ ), Attorney Docket No.: FATE-173/01WO phosphoglycerate kinase (PGK), hybrid CMV enhancer/chicken ⁇ -actin (CAG) and ubiquitin C (UBC) promoters.
  • the exogenous promoter is CAG.
  • the exogenous polynucleotides integrated by the method described herein may be driven by endogenous promoters in the host genome, at the integration site.
  • the one or more exogenous polynucleotides comprised in the construct for the methods of targeted integration are driven by one promoter.
  • the construct comprises one or more linker sequences between two adjacent polynucleotides driven by the same promoter to provide greater physical separation between the moieties and maximize the accessibility to enzymatic machinery.
  • the linker peptide of the linker sequences may consist of amino acids selected to make the physical separation between the moieties (exogenous polynucleotides, and/or the protein or peptide encoded therefrom) more flexible or more rigid depending on the relevant function.
  • the linker sequence may be cleavable by a protease or cleavable chemically to yield separate moieties.
  • the linker may be predominantly comprised of amino acids with small side chains, such as glycine, alanine, and serine, to provide for flexibility. In some embodiments about 80 to 90 percent or greater of the linker sequence comprises glycine, alanine, or serine Attorney Docket No.: FATE-173/01WO residues, particularly glycine and serine residues.
  • a G4S linker peptide separates the end-processing and endonuclease domains of the fusion protein.
  • a 2A linker sequence allows for two separate proteins to be produced from a single translation. Suitable linker sequences can be readily identified empirically.
  • linker sequences also can be determined by conventional computer modeling techniques.
  • the linker sequence encodes a self-cleaving peptide.
  • the self-cleaving peptide is 2A.
  • the linker sequence provides an Internal Ribosome Entry Sequence (IRES).
  • IRS Internal Ribosome Entry Sequence
  • any two consecutive linker sequences are different.
  • the above method for targeted integration in a preselected locus is used to insert any polynucleotide of interest, for example, polynucleotides encoding safety switch proteins, targeting modality, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and proteins promoting engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of stem cells and/or progenitor cells.
  • the construct comprising one or more exogenous polynucleotides further comprises one or more marker genes.
  • the exogenous polynucleotide in a construct of the invention is a suicide gene encoding a safety switch protein.
  • the method of introducing into cells a construct comprising exogenous polynucleotides for targeted integration can be achieved using a method of gene transfer to cells known per se.
  • the construct comprises backbones of viral vectors such as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, or Sendai virus vectors.
  • the plasmid vectors are used for delivering and/or expressing the exogenous polynucleotides to target cells (e.g., pAl- 11, pXTl, pRc/CMV, pRc/RSV, pcDNAI/Neo) and the like.
  • the episomal vector is used to deliver the exogenous polynucleotide to target cells.
  • recombinant adeno- associated viruses rAAV
  • rAAV can be used for genetic engineering to introduce insertions, deletions or substitutions through homologous recombination. Unlike lentiviruses, rAAVs do not integrate into the host genome.
  • an AAV6 or AAV2 vector is used to introduce insertions, deletions or substitutions in a target site in the genome of iPSCs.
  • Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), RNA-guided CRISPR (Clustered Regular Interspaced Short Palindromic Attorney Docket No.: FATE-173/01WO Repeats) systems.
  • ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain.
  • a “zinc finger DNA binding domain” or “ZFBD” it is meant a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers.
  • a zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
  • a “designed” zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
  • 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 trap or hybrid selection. ZFNs are described in greater detail in U.S. Pat.
  • TALEN a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain.
  • a targeted nuclease that finds use in the subject methods is a targeted Spo11 nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA binding domain, e.g., a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. that has specificity for a DNA sequence of interest.
  • targeted nucleases suitable for embodiments of the present invention include, but not limited to Bxb1, phiC31, R4, PhiBT1, and W ⁇ /SPBc/TP901-1, whether used individually or in combination.
  • targeted nucleases include naturally occurring and recombinant nucleases; CRISPR related nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like.
  • CRISPR/Cas9 requires two major components: (1) a Cas9 endonuclease and (2) the crRNA-tracrRNA complex.
  • DICE-mediated insertion uses a pair of recombinases, for example, phiC31 and Bxb1, to provide unidirectional integration of an exogenous DNA that is tightly restricted to each enzymes’ own small attB and attP recognition sites.
  • One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genome integration.
  • 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 to cells to enable site specific homologous recombination by the cell host enzymatic machinery.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a ZFN-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a TALEN-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cas9 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cas9-mediated insertion.
  • the method of targeted Attorney Docket No.: FATE-173/01WO integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases to a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing an expression cassette for DICE recombinases, to enable DICE-mediated targeted integration.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a ZFN-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR ⁇ or ⁇ 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.
  • the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP,
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a TALEN- mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR ⁇ or ⁇ 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.
  • the desired integration site comprises AAVS1, CCR5, ROSA26, collagen,
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cas9 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cas9-mediated insertion, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR ⁇ or ⁇ 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.
  • the desired integration site comprises AAVS1, CCR5, ROSA26, collagen
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases to a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing an expression cassette for DICE recombinases, to enable DICE-mediated targeted integration, wherein the desired integration site comprises AAVS1, CCR5, ROSA26, collagen, HTRP, H11, PH12, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR ⁇ or ⁇ constant region, NKG2A, NKG2D, CD25, CD38, CD44, Attorney Docket No.: FATE-173/01WO CD54, CD56, CD58, CD69, CD71, OX40, 4-1BB, CIS, CBL-B, SOCS2, PD
  • the genomically modified iPSCs and their derivative cells obtained using the methods and compositions described herein comprise at least one genotype listed in Table 4.
  • III. Method of Obtaining and Maintaining Genome-engineered iPSCs [000319]
  • the present invention also provides a method of obtaining and maintaining genome-engineered iPSCs comprising one or more targeted edits (e.g., multiplex engineering) at one or more desired sites, wherein the one or more targeted edits remain intact and functional in expanded genome-engineered iPSCs or the iPSC-derived non- pluripotent cells at the respective selected editing sites.
  • targeted edits e.g., multiplex engineering
  • the targeted editing introduces into the genome of the iPSC, and derivative cells thereof, insertions, deletions, and/or substitutions (i.e., targeted integration and/or in/dels at selected sites).
  • the many benefits of obtaining genomically-engineered derivative cells through editing and differentiating iPSC as provided herein include, but are not limited to: unlimited source for engineered effector cells; no need for repeated manipulation of the effector cells, especially when multiple engineered modalities are involved; the obtained effector cells are rejuvenated for having elongated telomere and experiencing less exhaustion; the effector cell population is homogeneous in terms of editing site, copy number, and void of allelic variation, random mutations and expression variegation, largely due to the enabled clonal selection in engineered iPSCs as provided herein.
  • the genome-engineered iPSCs comprising one or more targeted edits at one or more selected sites are maintained, passaged and expanded as single cells for an extended period in cell maintenance culture medium (FMM), wherein the iPSCs retain the targeted editing and functional modification at the selected site(s).
  • FMM cell maintenance culture medium
  • the iPSCs cultured in FMM have been shown to continue to maintain their undifferentiated, and ground or na ⁇ ve, profile; provide genomic stability without the need for culture cleaning or selection; and readily to give rise to all three somatic lineages, in vitro differentiation via embryoid bodies or monolayer (without formation of embryoid bodies); and by in vivo differentiation via teratoma formation. See, for example, International Pub. No.
  • the genome-engineered iPSCs comprising one or more targeted integrations and/or in/dels are maintained, passaged and expanded in a medium (FMM) comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and free of, or essentially Attorney Docket No.: FATE-173/01WO free of, TGF ⁇ receptor/ALK5 inhibitors, wherein the iPSCs retain the intact and functional targeted edits at the selected sites.
  • FMM medium
  • FATE-173/01WO free of, TGF ⁇ receptor/ALK5 inhibitors wherein the iPSCs retain the intact and functional targeted edits at the selected sites.
  • Another aspect of the invention provides a method of generating genome- engineered iPSCs through targeted editing of iPSCs; or through first generating genome- engineered non-pluripotent cells by targeted editing, and then reprogramming the selected/isolated genome-engineered non-pluripotent cells to obtain iPSCs comprising the same targeted editing as the non-pluripotent cells.
  • a further aspect of the invention provides genome- engineering non-pluripotent cells which are concurrently undergoing reprogramming by introducing targeted integration and/or targeted in/dels to the cells, wherein the contacted non- pluripotent cells are under sufficient conditions for reprogramming, and wherein the conditions for reprogramming comprise contacting non-pluripotent cells with one or more reprogramming factors and small molecules.
  • the targeted integrations and/or targeted in/dels may be introduced to the non-pluripotent cells prior to, or essentially concomitantly with, initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally one or more small molecules.
  • the targeted integrations and/or in/dels may also be introduced to the non- pluripotent cells after the multi-day process of reprogramming is initiated by contacting the non- pluripotent cells with one or more reprogramming factors and small molecules, and wherein the vectors carrying the constructs are introduced before the reprogramming cells present stable expression of one or more endogenous pluripotent genes including but not limited to, SSEA4, Tra181 and CD30.
  • the reprogramming is initiated by contacting the non- pluripotent cells with at least one reprogramming factor, and optionally a combination of a TGF ⁇ receptor/ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor.
  • the genome-engineered iPSCs produced through any methods above are further maintained and expanded using a mixture comprising a combination of a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor.
  • the master cell bank is subsequently cryopreserved, providing a platform for additional iPSC engineering and a renewable source for manufacturing off-the-shelf, engineered, homogeneous cell therapy products, which are well-defined and uniform in composition, and can be mass produced at significant scale in a cost-effective manner.
  • the reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1, and any combinations thereof as disclosed in International Pub. Nos.
  • autoimmune disorders include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), some forms of juvenile idiopathic arthritis, glomerulonephritis, Graves’ disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, some forms of myocarditis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis, Sjögren’s syndrome, systemic lupus, erythematosus, some forms of thyroiditis, some forms of uveitis, vitiligo, granulomatosis with poly
  • the antibody, or antibody fragment specifically binds to a tumor antigen.
  • the tumor or viral specific antigen activates the administered iPSC-derived hematopoietic lineage cells to enhance their killing ability.
  • the antibodies suitable for combinational treatment as an additional therapeutic agent to the administered iPSC-derived hematopoietic lineage cells include, but are not limited to, anti-CD20 antibodies (e.g., rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab), anti-HER2 antibodies (e.g., trastuzumab, pertuzumab), anti-CD52 antibodies (e.g., alemtuzumab), anti-EGFR antibodies (e.g., cetuximab), anti-GD2 antibodies (e.g., dinutuximab), anti-PDL1 antibodies (e.g., anti-CD20 antibodies (e.g
  • Some embodiments of the combination therapy comprising the provided derivative effector cells further comprise at least one inhibitor targeting a checkpoint molecule. Some other embodiments of the combination therapy with the provided derivative effector cells comprise two, three or more inhibitors such that two, three, or more checkpoint molecules are targeted.
  • the effector cells for combination therapy as described herein are derivative NK cells as provided.
  • the effector cells for combination therapy as described herein are derivative T cells.
  • the derivative NK or T cells for combination therapies are functionally enhanced as provided herein.
  • the two, three or more checkpoint inhibitors may be administered in a combination therapy with, before, or after the administering of the derivative effector cells.
  • the chemotherapeutic agent comprises an anthracycline, an alkylating agent, an alkyl sulfonate, an aziridine, an ethylenimine, a methylmelamine, a nitrogen mustard, a nitrosourea, an antibiotic, an antimetabolite, a folic acid analog, a purine analog, a pyrimidine analog, an enzyme, a podophyllotoxin, a platinum-containing agent, an interferon, and an interleukin.
  • Additional agents include aminglutethimide, cisplatin, carboplatin, mitomycin, altretamine, cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan (CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, celecoxib, cetuximab, cladribine, clofurabine, cytarabine, dacarbazine, denileukin diftitox, diethlstilbestrol, docetaxel, Attorney Docket No.: FATE-173/01WO dromostanolone, epirubicin, erlotinib, estramustine, etoposide, ethinyl estradiol
  • compositions suitable for administration to a subject/patient can further include one or more pharmaceutically acceptable carriers (additives) and/or diluents (e.g., pharmaceutically acceptable medium, for example, cell culture medium), or other pharmaceutically acceptable components.
  • pharmaceutically acceptable carriers and/or diluents are determined in part by the particular composition being administered, as well as by the particular method used to administer the therapeutic composition.
  • the therapeutic composition comprises the iPSC-derived T cells made by the methods and composition disclosed herein.
  • the therapeutic composition comprises the pluripotent cell derived NK cells made by the methods and composition disclosed herein.
  • the therapeutic composition comprises the iPSC-derived CD34 + HE cells made by the methods and composition disclosed herein.
  • the therapeutic composition comprises the pluripotent cell derived HSCs made by Attorney Docket No.: FATE-173/01WO the methods and composition disclosed herein.
  • the therapeutic composition comprises the pluripotent cell derived MDSC made by the methods and composition disclosed herein.
  • a therapeutic composition comprising a population of iPSC-derived hematopoietic lineage cells as disclosed herein can be administered separately by intravenous, intraperitoneal, enteral, or tracheal administration methods or in combination with other suitable compounds to affect the desired treatment goals.
  • These pharmaceutically acceptable carriers and/or diluents can be present in amounts sufficient to maintain a pH of the therapeutic composition of between about 3 and about 10.
  • a buffering agent can be as much as about 5% on a weight to weight basis of the total composition.
  • Electrolytes such as, but not limited to, sodium chloride and potassium chloride can also be included in the therapeutic composition.
  • the pH of the therapeutic composition is in the range from about 4 to about 10.
  • the pH of the therapeutic composition is in the range from about 5 to about 9, from about 6 to about 9, or from about 6.5 to about 8.
  • the therapeutic composition includes a buffer having a pH in one of said pH ranges.
  • the therapeutic composition has a pH of about 7.
  • the therapeutic composition has a pH in a range from about 6.8 to about 7.4.
  • the therapeutic composition has a pH of about 7.4.
  • the invention also provides, in some embodiments, the use of a pharmaceutically acceptable cell culture medium in particular compositions and/or cultures disclosed herein. Such compositions are suitable for administration to human subjects. Generally speaking, any medium that supports the maintenance, growth, and/or health of the iPSC-derived effector cells in accordance with embodiments of the invention are suitable for use as a pharmaceutical cell culture medium.
  • the pharmaceutically acceptable cell culture medium is a serum free, and/or feeder-free medium.
  • the serum-free medium is animal-free, and can optionally be protein-free.
  • the medium can contain biopharmaceutically acceptable recombinant proteins.
  • Animal-free medium refers to medium wherein the components are derived from non-animal sources.
  • the iPSC-derived hematopoietic lineage cells can have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, proT cells, proNK cells, CD34 + HE cells, HSCs, B cells, myeloid-derived suppressor cells (MDSCs), Attorney Docket No.: FATE-173/01WO regulatory macrophages, regulatory dendritic cells, or mesenchymal stromal cells.
  • One aspect of the present application provides a method of treating a subject in need by administering one or more therapeutic doses of effector cells comprising an TCR promoter-driven ADR and/or a cytokine, and a constitutively expressed TCE, in addition to edits comprising a CAR, a TGF ⁇ -SRR, a C-X-C-motif chemokine receptor or a variant thereof, a CD16 variant, and a CD38 knockout, among other modifications as provided herein, or a genotype listed in Table 4.
  • the number of derived hematopoietic lineage cells in the therapeutic composition is at least 0.1 x 10 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 x 10 9 cells, per dose.
  • the number of derived hematopoietic lineage cells in the therapeutic composition is about 0.1 x 10 5 cells to about 1 x 10 6 cells, per dose; about 0.5 x 10 6 cells to about 1x 10 7 cells, per dose; about 0.5 x 10 7 cells to about 1 x 10 8 cells, per dose; about 0.5 x 10 8 cells to about 1 x 10 9 cells, per dose; about 1 x 10 9 cells to about 5 x 10 9 cells, per dose; about 0.5 x 10 9 cells to about 8 x 10 9 cells, per dose; about 3 x 10 9 cells to about 3 x 10 10 cells, per dose, or any range in- between.
  • the number of derived hematopoietic lineage cells in the therapeutic composition is the number of immune cells in a partial or single cord of blood, or is at least 0.1 x 10 5 cells/kg of bodyweight, at least 0.5 x 10 5 cells/kg of bodyweight, at least 1 x 10 5 cells/kg of bodyweight, at least 5 x 10 5 cells/kg of bodyweight, at least 10 x 10 5 cells/kg of bodyweight, at least 0.75 x 10 6 cells/kg of bodyweight, at least 1.25 x 10 6 cells/kg of bodyweight, at least 1.5 x 10 6 cells/kg of bodyweight, at least 1.75 x 10 6 cells/kg of bodyweight, at least 2 x 10 6 cells/kg of bodyweight, at least 2.5 x 10 6 cells/kg of bodyweight, at least 3 x 10 6 cells/kg of bodyweight, at least 4
  • a dose of derived hematopoietic lineage cells is delivered to a subject.
  • the effective amount of cells provided to a subject is at least 2 x 10 6 cells/kg, at least 3 x 10 6 cells/kg, at least 4 x 10 6 cells/kg, at least 5 x 10 6 cells/kg, at least 6 x 10 6 cells/kg, at least 7 x 10 6 cells/kg, at least 8 x 10 6 cells/kg, at least 9 x 10 6 cells/kg, or at least 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of cells provided to a subject is about 2 x 10 6 cells/kg, about 3 x 10 6 cells/kg, about 4 x 10 6 cells/kg, about 5 x 10 6 cells/kg, about 6 x 10 6 cells/kg, about 7 x 10 6 cells/kg, about 8 x 10 6 cells/kg, about 9 x 10 6 cells/kg, or about 10 x 10 6 cells/kg, or more cells/kg, including all intervening doses of cells.
  • the effective amount of cells provided to a subject is from about 2 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 3 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 4 x 10 6 cells/kg to about 10 x 10 6 cells/kg, about 5 x 10 6 cells/kg to about 10 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 7 x 10 6 cells/kg, 3 x 10 6 cells/kg to about 8 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/kg to about 6 x 10 6 cells/kg, 4 x 10 6 cells/kg to about
  • the therapeutic use of derived hematopoietic lineage cells is a single-dose treatment.
  • the therapeutic use of derived hematopoietic lineage cells is a multi-dose treatment.
  • the multi-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, or every 50 days, or any number of days in-between.
  • the multi-dose treatment comprises three, four, or five, once-weekly doses.
  • compositions comprising a population of derived hematopoietic lineage cells of embodiments of the invention can be sterile, and can be suitable and ready for administration (i.e., can be administered without any further processing) to human patients/subjects.
  • a cell- based composition that is ready for administration means that the composition does not require Attorney Docket No.: FATE-173/01WO any further processing or manipulation prior to transplant or administration to a subject.
  • the invention provides an isolated population of derived hematopoietic lineage cells that are expanded and/or modulated prior to administration with one or more agents including small chemical molecules.
  • the compositions and methods for modulating immune cells including iPSC-derived effector cells are described in greater detail, for example, in International Pub. No. WO2017/127755, the relevant disclosure of which is incorporated herein by reference.
  • the cells can be activated and expanded using methods as described, for example, in U.S. Patents 6,352,694.
  • the primary stimulatory signal and the co-stimulatory signal for the derived hematopoietic lineage cells can be provided by different protocols.
  • the agents providing each signal can be in solution or coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (i.e., in "cis” formation) or to separate surfaces (i.e., in "trans” formation).
  • one agent can be coupled to a surface and the other agent in solution.
  • the agent providing the co-stimulatory signal can be bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.
  • the agents can be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents such as disclosed in U.S. Pub. Nos.2004/0101519 and 2006/0034810, the disclosures of which are incorporated by reference, for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T lymphocytes in embodiments of the present invention.
  • aAPCs artificial antigen presenting cells
  • hiPSC Maintenance in Small Molecule Culture hiPSCs were routinely passaged as single cells once confluency of the culture reached 75%–90%. For single-cell dissociation, hiPSCs were washed with PBS (Mediatech) and treated with Accutase (Millipore). The single- cell suspension was then mixed with conventional medium, centrifuged, resuspended in FMM, and plated on Matrigel-coated surface. Passages were typically 1:6–1:8, and fed every 2–3 days with FMM. Cell cultures were maintained in a humidified incubator set at 37°C and 5-10% CO2.
  • iPSCs with genomic targeted editing using ZFN or CRISPR-Cas9 were bulk sorted and clonal sorted of GFP + SSEA4 + TRA181 + iPSCs. Single cell dissociated targeted iPSC pools were resuspended in staining buffer made fresh for optimal performance. Conjugated primary antibodies, including SSEA4-PE, TRA181-Alexa Fluor-647 (BD Biosciences), were added to the cell solution.
  • a TCE has the capability to promote T cell identity, function and performance of iPSC derived T cells (iT cell).
  • lentiviral- transduced iT cells having an exemplary CAR specific to a tumor target GPRC5D for illustration purposes
  • IncuCyte based restimulation assay in vitro at a 1:1 ratio of effector to H929-NLR, a target cell line endogenously expressing the tumor target of interest.
  • the study was performed for 3 rounds with partial media change every 48 hr followed by cell transferring to a new plate with target cells. No cytokine support was given throughout the study.
  • the readout was the target cell count by IncuCyte.
  • the H929-NLR cell count was normalized to time point 0 and the relative H929 count was plotted on the y axis across different time points for individual rounds.
  • TGI tumor growth inhibition
  • CD27 contributed to increased TGI for two rounds and the effect of TCF1 (the Transcription Factor 1 protein, encoded by the human gene TCF7) lasted further beyond the first two rounds to round 3.
  • TCF1 and CD27 therefore were advanced for further analyses as potential TCEs for CAR-iT functionalities.
  • Candidate edits were then placed in different engineering configurations and in combination with an exemplary KLK2 CAR.
  • the engineering configurations included (a) TRAC knockout and insertion (KO/I) with CAR alone (1 of FIG.3), (b) TRAC KO/I with a bicistron (2-4 of FIG.3), (c) TRAC KO/I with a tricistron (6-9 of FIG.3), or (d) TRAC KO/I with a bicistron in combination with a constitutively expressed TCE candidate at a different insertion site (10-11 of FIG.3; and CD38 KO/I in this example).
  • the primary CAR T cells comprising the same CAR but without TRAC knockout were used as control. [000391] All editing constructs were introduced to iPSCs and followed by T cell differentiation.
  • the iT identity marked by CAR expression and intracellular CD3 expression in each differentiated cell group confirmed the proper T cell differentiation from engineered iPSCs having the edits as indicated (FIG.3).
  • the various groups of iT cells were then used as effector cells for subsequent functional tests, including in vivo studies.
  • EXAMPLE 3 – T Cell Enhancer (TCE) Function Analysis [000392] Two million of antigen-expressing tumor cells PC3 were implanted subcutaneously to NSG mice on D0. Tumor volume measurement was performed to assess tumor cell engraftment in each group of mice that was randomly assigned to a treatment arm.
  • the bi- cistronic configurations that co-express sIL2 or IL15RF with a CAR had better control of tumor growth than that co-expresses IL7RF. It is also observed that the TRAC regulated sIL2 expression enhances tumor cell growth control as compared to CAR-T cell without the sIL2 driven by the TRAC endogenous promoter. Notably, when in combination with constitutively expressed TCF7 or CD27, the bi-cistronic construct co-expressing IL7RF has improved tumor volume reduction when compared to without the constitutively expressed TCE (FIGS.4A-4B).
  • TCEs in a form of tri-cistronic configuration did not improve tumor control of TRAC CAR co-expressing IL7RF (FIGS.4A-4B).
  • the tumor mass, spleen, bone marrow of NSG mice of indicated groups from the above study were harvested for ex vivo function evaluation on D8 (3 days post effector dosing) or D15 (10 days post effector dosing).
  • D8 3 days post effector dosing
  • D15 10 days post effector dosing
  • FIGS.6A-6B the total effector cell count in tumor mass is plotted on the x axis to show the effector cell expansion, with the total tumor cell count plotted on y axis for each individual mouse of the indicated group to show the effectiveness of the tumor cell killing.
  • sIL2 iT cells not only have as good iT expansion as effector cells having, for example, engineering edits 3, 4 or 5, as indicated in FIGS.6A-6B, the sIL2 iT cells further demonstrated better tumor clearance in comparison. It is also notable that iT cells having engineering edits 6 (TCF7 iT) led to the greatest tumor cell count reduction, thus a highly effective tumor cell killing, despite that these cells showed less cell expansion, suggesting better potency of these iT cells.
  • PH12 is a newly discovered and confirmed genomic safe harbor (GSH) locus in a non-coding region on human chromosome 17, having a coordinate of chr17:44210001-44271500 in human reference genome assembly hg38.
  • GSH genomic safe harbor
  • the sequence of the human reference genome hg38 is available from Genome Reference Consortium, with a reference number GRCh38/hg38, and also available in the Genome Browser provided by The UC Santa Cruz Genomics Institute.
  • a previously known GSH locus H11 was used as a control for confirmatory study of PH12 for engineering various cell types, including primary cells, iPSC obtained from reprograming fibroblast (FiPSC) or T cells (TiPSC).
  • Polynucleotides encoding CD19-CAR, BCMA-CAR, or EGFR were engineered into PH12 for the purpose of verifying the site’s suitability as a genomic safe harbor locus.
  • the insertion of various polynucleotides at PH12 in various cells using CRISPR showed a cutting efficiency more than 80%, with the knock-in efficiency (both bi-allelic and mono-allelic) ranging from about 40% to about 80%.
  • the EGFR or CAR expression after insertion at PH12 is stable during prolonged culturing and at a level comparable to that of insertion at H11 locus.
  • Both the mono-allelic and bi-allelic PH12-tageted iPSC clones maintained pluripotency and had iPSC marker expression patterns well preserved.
  • the engineered iPSCs were further differentiated to NK cells and expanded for phenotype and function evaluation.
  • the differentiated cells maintained expected phenotype, function, and genome stability.
  • the suitability of PH12 as a genomic safe harbor locus was thereby confirmed for inserting exogenous polynucleotides encoding one or more proteins or peptides driven by constitutive or inducible promoter as desired.
  • the exogenous polynucleotide can be any one of TGFbR redirector, CXCR2, CD16, CAR, TCE (CD27 or TCF1), or any other gene of interest as disclosed herein.
  • ADR Alloimmune Defense Receptor
  • 41BB-ADR was previously configured for constitutive and strong expression, for example driven by the synthetic promoter CAG engineered into the CD38 locus. It was later discovered that, in some cases, such a configuration is not optimally compatible with Attorney Docket No.: FATE-173/01WO iPSC-derived T cell differentiation processes. It was observed that CAG-driven expression of 41BB-ADR can negatively impact iT cell differentiation for having limited lymphoid commitment shown by low expression of CD7 and a suppressed T cell lineage commitment shown by few CD4+ single positive, CD8+ single positive and CD4+CD8+ double positive cells.
  • T cell derived iPSCs engineered to express either constitutive ADR engineered into CD38 loci (+ADR) or control engineered cells that lacked ADR (No ADR) were differentiated into CAR-T cells. Lymphoid and T cell commitment was evaluated by flow cytometry, assessing CD45, CD7, CD8 ⁇ , and CD4 expression.
  • CAR-iT cells with constitutively expressed ADR demonstrated aberrant lymphoid commitment with 63.7% of cells lacking CD7 expression and 88.4% lacking either CD4 and/or CD8 ⁇ expression.
  • 41BB-ADR was inserted at TRAC locus to be driven by TRAC endogenous promoter.
  • the TRAC targeted 41BB-ADR (TRAC_41BB-ADR) iPSCs are differentiated alongside control TRAC_CAR iPSC and CAG_41BB-ADR iPSC towards CD34-expressing hemogenic endothelium (iHE) followed by differentiation towards hematopoietic cells, lymphoid cells and subsequently T cells using the compositions and methods described herein.
  • the temporal control of 41BB-ADR expression is demonstrated by similar kinetics of ADR expression to the kinetics of CAR expression observed in the control TRAC_CAR-derived iT cells (FIG.8).
  • TRAC inserted 41BB-ADR iPSC with iT cell differentiation is demonstrated by the similar expression level of 41BB-ADR to the CAR expression in the control TRAC_CAR iPSC-derived iT cells, which iT cells of both groups have the hematopoietic marker CD45, the lymphoid marker CD7 and the T cell lineage marker intracellular CD3.
  • flow cytometry was performed confirming (top row) appropriate lymphoid commitment (99% CD45+CD7+) for both TRAC control (left) and TRAC ADR (right) engineered CAR iT cells.
  • the co-cultures are monitored for (1) a reduction in ADR-negative iT cell counts compared to the ADR-positive iT Attorney Docket No.: FATE-173/01WO cell counts, and (2) the expansion of alloreactive T cells in co-cultures with ADR-negative iT cells compared to a decrease in alloreactive T cell counts in co-cultures with ADR-positive iT cells.
  • iPSC derived CAR-T cells engineered to express either IL7RF or soluble IL2 (sIL2) were evaluated in two separate prostate and ovarian cancer xenograft models.
  • CAR-iT cells secreting IL-2 show enhanced CAR and ADCC activity in vivo, and that ADR in combination with sIL2 leads to improved performance in a tri-culture assay with tumor, primed allo T cells, and iT cells.
  • IL-2 production by iPSC derived CAR-T cells (CAR iT) engineered to express soluble IL2 from the TRAC locus were evaluated under steady state and under CAR activation conditions and compared to CAR iT cells that lacked sIL2 or primary, healthy donor derived CAR-T cells.
  • CAR iT iPSC derived CAR-T cells
  • FIG.13A limited but enhanced IL2 production (1.5-2.0 pg/ml) was observed by TRAC sIL2 engineered CAR iT cells compared to primary CAR-T cells and control CAR iT cells after 48 and 72 hours of culture.
  • CAR iT cells engineered to produce a lower level of IL2 under TRAC regulation demonstrate sustained anti-tumor activity compared to similar IL-2 engineering into TCL1 or TCL2.
  • engineering of IL2 into alternative “T cell loci” that produce higher level of IL2 after CAR activation leads to poorer anti-tumor activity of the effector cells in vivo. Without being bound by theory, it seems that when IL2 expression level is too low it leads to poor effector cell persistence, however if the IL2 expression level is too high, it leads to effector cell exhaustion.
  • TRAC regulated IL2 expression is at a level uniquely beneficial to both persistence and efficacy of CAR iT cells.
  • TRAC regulated IL2 supports hnCD16 and ADR mediated efficacy.
  • iPSC derived T (iT) cells were engineered to constitutively express TGF ⁇ - SRR, hnCD16, and CXCR2 with either TRAC regulated ADR and IL7RF (“ADR 7”) or TRAC regulated ADR and soluble IL-2 (“ADR 2”) (FIG.15A).
  • ADR 7 TRAC regulated ADR and IL7RF
  • ADR 2 TRAC regulated ADR and soluble IL-2
  • NSG mice previously challenged subcutaneously with SKOV3 target cells were administered Herceptin alone, Herceptin and ADR 2 iT cells, or Herceptin and ADR 7 iT cells, and tumor burden was evaluated up to 50 days post tumor challenge.
  • ADR 2 and ADR 7 iT cells demonstrated specific in vivo antibody dependent cytotoxicity when combined with Herceptin, ADR 2 (0 mm 3 Avg tumor burden; 5 of 5 tumor clear) outperformed ADR 7 (64.8 mm 3 Avg tumor burden; 0 of 5 tumor clear) iT cells.
  • ADR 7 and ADR 2 iT cells co-cultured with SKOV3 tumor cells and pAT in the presence of Herceptin; and the tumor burden was evaluated where indicated via luminescence assay.
  • FIG.15C unlike ADR 7 iT cells which lost anti-tumor control after three days of tri-culture, reaching ⁇ 7x10 4 RLU (relative luminescence unit) on Day 8, ADR 2 iT cells demonstrated sustained anti-tumor activity in the presence of primed allogeneic T cells out to Day 8 of tri-culture (3x10 2 RLU).
  • T cell derived iPSCs engineered to express a constitutive CAR engineered into CD58 along with ADR and soluble IL2 in the TRAC locus and constitutive TGF ⁇ -SRR, hnCD16, and CXCR2 from CD38, were differentiated into constitutive CAR iT cells (ADR/IL2 + Const. CAR).
  • the CAR expression by the constitutive CAR iT cell population was evaluated prior to tumor challenge and at the end of each round of tumor challenge. Geometric mean fluorescent intensities are shown as a heat map indicative of CAR expression level by each effector cell population (FIG.16A). As expected, the constitutive CAR expression level is much higher than the TRAC controlled CAR expression. Previously, a TRAC controlled CAR was preferred with the belief that lower CAR expression level would decrease the level of cell exhaustion, and that the temporal controlled CAR expression also steers progenitor cells to commit to T cell lineage during the iPSC differentiation.
  • CAR iT cells engineered to express (i) TRAC regulated CAR and soluble IL2 (TRAC CAR/IL2), or (ii) constitutive CAR along with TRAC regulated ADR and soluble IL2 (Const. CAR + ADR/IL2) in a prostate xenograft model was evaluated.
  • the NSG mice previously challenged subcutaneously with PC3 tumor cells were administered (A) 1x10 7 or (B) 5x10 6 TRAC CAR/IL2 or (C) 5x10 6 Constitutive CAR + ADR/IL2, with the tumor burden measured out to 45 days post tumor challenge.
  • the insertion is at CD38.
  • flow cytometric staining for TCF1 in control CAR-iT cells compared to over-expression of TCF1 (trace “2”) is shown in FIG.18A.
  • the progenitor iT cells (around D24 iPSC differentiation) were profiled by flow cytometry for expression of the GATA3 and Bcl11b, two representative transcription factors that are regulated by TCF1 and are responsible for preventing differentiation toward B, myeloid, and innate cell lineages.
  • FIG.18B over- expression of TCF1 results in a significantly increased proportion of GATA3+ Bcl11b+ cells (43% vs 13%).
  • the end-of-process iT cells (around D42 differentiation) were profiled by flow cytometry for expression of the T cell markers CD7 and CD5. While both the control and TCF1-overexpressing iT cells were >99% CD7 positive, overexpression of TCF1 increased the proportion of iT cells that were double positive for CD7 and CD5 (59% vs 13%), demonstrating the reinforced T cell lineage commitment with TCF1 overexpression in the CAR iT cells.
  • FIG.19A is a schematic showing an ADR_2 backbone (TRAC_ADR-IL2 and CD38KO_TGF ⁇ R2-hnCD16-CXCR2) engineered to also express a CAG-promoter driven bicistronic cassette (CAR-2a-TCF1) that was engineered into the CD58 locus (CD58KO_CAR- TCF1).
  • FIG.19B represents BLI data from mice that were given 1e5 Nalm6 tumor on day 0 and treated with the indicated effector cells on day 1. It is shown that CAR-iT cells expressing TCF1 had complete tumor growth inhibition on day 21, whereas CAR-iT cells that did not overexpress TCF1 were starting to show tumor rebound past day 15.
  • FIG.20A shows the flow cytometric measurement of the indicated iT cells co- cultured with primed allo T cells. It was shown that over-expression of TCF1 leads to enhanced persistence of the ADR_2 iT cells when co-cultured with primed allo T cells (pAT) that target the iT cells. In a tri-culture setting where iT cells need to eliminate tumor while also protecting themselves from primed allo T cell attack, the TCF1 positive ADR_2 iT cells showed increased persistence over the TCF1 negative ADR_2 iT cells, and the flow cytometric measurement of the indicated iT cells are shown in FIG.20B.
  • pAT primed allo T cells
  • the increased persistence of the TCF1 positive ADR_2 iT cells also showed improved control of tumor growth in a tri-culture setting, as shown by the luciferase-based readout of tumor cells in the tri-culture assay over time (FIG.20C).
  • the general design of the master cell bank (MCB) for deriving effector cells through iPSC differentiation includes generation of two intermediary engineered iPS cell lines (pre-MCBs) having multiplexed genome editing at various genome engineering sites for gene knock-ins and knockouts.
  • a series of bi-cistronic and tri-cistronic constructs were designed and prepared for engineering cells at multiple loci for endogenous gene knockout and at the same time to insert (KO/KI) multiple functional modalities in desired combinations and desired placement in order to explore a functional backbone arrangement that better equips effector cells for solid tumor immuno-therapies.
  • a first pre-MCB comprises knock-in of one or more of TGF ⁇ R redirector (TGF ⁇ -SRR), CXCR2 and hnCD16 at a preselected site (site 1).
  • Suitable genome engineering sites for the knock-in include, but are not limited to, a TCR locus (e.g., TRAC or TRBC), CD38, CD54, CD56, CD58, TIM3, TIGIT, H11 and PH12.
  • CD38 was selected as site 1 for pre-MCB1 generation. While inserting at CD38 two or more of CD16, CXCR2 and a TGF ⁇ -SRR using a bi- or tri- cistronic construct to address challenges of the solid tumor environment, the endogenous CD38 gene is knocked out as a result such that an anti- CD38 monoclonal antibody or a CD38-CAR may be applied to selectively deplete CD38- expressing activated host immune cells.
  • the TGF ⁇ R redirector, CXCR2 and/or hnCD16 are driven by an exogenous promoter for constitutive expression.
  • the TGF ⁇ R2 redirector is a fusion protein of TGF ⁇ R2 with one of IL12Rb, IL18R, IL21R, or fragments thereof, referred to as TGF ⁇ R2-IL12Rb, TGF ⁇ R2-IL18R, TGF ⁇ R2-IL21R specifically or as TGF ⁇ -SRR in general.
  • the TGF ⁇ -SRR comprises IL18R.
  • the pre-MCB1 iPSCs were differentiated into CAR iT or CAR iNK cells to verify the pluripotency and was used for further genomic editing to generate pre-MCB2.
  • a second pre-MCB comprises additional edits introduced into pre- MCB1 cells at TRAC locus.
  • PreMCB2 comprises an insertion of IL7RF or IL2 at TRAC, optionally, co-expressed with 41BB-ADR.
  • the TRAC gene is knocked out by the insertion, and the expression of the inserted gene(s) is subjected to control of TRAC endogenous promoter.
  • the pre-MCB2 iPSCs having both CD38 and TRAC sites editing were used for further genomic editing to generate MCB.
  • an antigen-specific CAR was not included at the TRAC locus as compared to the TRAC_CAR configuration, which was shown to improve CAR- T cell efficacy and delay CAR-T cell exhaustion upon activation by tuning down the CAR expression level through TRAC endogenous promoter (Eyquem et al. Nature.2017 Mar 2; 543(7643): 113–117).
  • the pre-MCB2 clone was engineered at a preselected site (site 2) to knock in a first CAR (CAR1) and TCE, optionally with an additional CAR (CAR2) to generate MCB for effector cell differentiation.
  • sites CD58, TIM3 and PH12 are each being used as site 2 in parallel experimentations.
  • the endogenous genes are knocked out by targeted insertion.
  • CAR1 and CAR2 have different targeting specificity and are driven by CAG promoter as an example.
  • BCMA-CAR and CD38- CAR were used where two CARs are in the design.
  • TCF1 is the selected TCE for illustration purposes. It was observed that the combination of IL7RF and TCF1 enhanced antitumor activity of CAR-iT cells in a subcutaneous xenograft tumor model.
  • FIG.21 illustrates the engineering designs and process to generate MCB for effector cell differentiation through pre-MCB1 and pre-MCB2.
  • FIGS.22A-22C show the flow cytometry confirmation of CAR1 expression after CD58 targeted editing of pre-MCB2.
  • tri-site edited MCB iPSCs were evaluated for their lymphoid commitment by assessing their CD45 and CD7 expression via flow cytometry on individual clones from each cell group having indicated backbone configurations.
  • suitable temporally regulated endogenous promoters include, but are not limited to Tim-3, TIGIT, and ASB2.
  • sIL2, IL7RF and/or IL18 improves in vivo persistence and expansion of the engineered iT cell independent of cytokine support.
  • the ability to expand independently of cytokine support is also useful for in vitro cell selection during stepwise cell engineering because the cytokine autonomy allows modified cells to expand while unmodified cells remain inactive.
  • Such outgrowth of modified cells during culture alleviates the need for sorting, which increases the screening throughput and scale for a large number of cell positive for intended edits.

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Abstract

L'invention concerne des procédés et des compositions pour obtenir des cellules immunitaires et des cellules effectrices dérivées fonctionnellement améliorées obtenues à partir d'une différenciation dirigée de CSPi génétiquement modifiées. L'invention concerne également des cellules dérivées ayant une édition génomique stable et fonctionnelle qui permet d'obtenir des effets thérapeutiques améliorés ou accrus. L'invention concerne en outre des compositions thérapeutiques et leur utilisation faisant appel aux cellules effectrices dérivées fonctionnellement améliorées seules, ou avec des anticorps ou des inhibiteurs de points de contrôle dans des polythérapies.
PCT/US2024/040194 2023-08-02 2024-07-30 Squelette de ciblage de tumeur solide favorisant la différenciation et la fonction de cellules effectrices Pending WO2025029810A1 (fr)

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