WO2022120334A1 - Genetically engineered cells and uses thereof - Google Patents

Genetically engineered cells and uses thereof Download PDF

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Publication number
WO2022120334A1
WO2022120334A1 PCT/US2021/072646 US2021072646W WO2022120334A1 WO 2022120334 A1 WO2022120334 A1 WO 2022120334A1 US 2021072646 W US2021072646 W US 2021072646W WO 2022120334 A1 WO2022120334 A1 WO 2022120334A1
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WO
WIPO (PCT)
Prior art keywords
seq
cell
ipsc
amino acid
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2021/072646
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English (en)
French (fr)
Inventor
Michael Naso
Mark WALLET
Barry Morse
Luis Borges
Buddha GURUNG
Hillary QUINN
Liam Campion
Jill Carton
Heidi Jessup
Kenneth Brasel
Lucas THOMPSON
John Wheeler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Century Therapeutics Inc
Original Assignee
Century Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Century Therapeutics Inc filed Critical Century Therapeutics Inc
Priority to CA3201621A priority Critical patent/CA3201621A1/en
Priority to JP2023534338A priority patent/JP2023553419A/ja
Priority to KR1020237022356A priority patent/KR20230118887A/ko
Priority to EP21844163.2A priority patent/EP4255922A1/en
Priority to CN202180092723.7A priority patent/CN116981685A/zh
Priority to AU2021392032A priority patent/AU2021392032A1/en
Priority to IL303269A priority patent/IL303269A/en
Priority to MX2023006395A priority patent/MX2023006395A/es
Publication of WO2022120334A1 publication Critical patent/WO2022120334A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
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    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
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    • A61K40/31—Chimeric antigen receptors [CAR]
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    • C07K16/2863—Immunoglobulins [IG], 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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Definitions

  • This application provides genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof. Also provided are uses of the iPSCs or derivative cells thereof to express a chimeric antigen receptor for allogenic cell therapy. Also provided are related vectors, polynucleotides, and pharmaceutical compositions.
  • iPSCs genetically engineered induced pluripotent stem cells
  • This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “CNTY-001- WO-01_SequenceListing_ST25” and a creation date of November 1, 2021 and having a size of 113 kb.
  • the sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
  • Chimeric antigen receptors significantly enhance anti-tumor activity of immune effector cells.
  • CARs are engineered receptors typically comprising an extracellular targeting domain that is linked to a linker peptide, a transmembrane (TM) domain, and one or more intracellular signaling domains.
  • the extracellular domain consists of an antigen binding fragment of an antibody (such as a single chain Fv, scFv) that is specific for a given tumor-associated antigen (TAA) or cell surface target.
  • TAA tumor-associated antigen
  • the extracellular domain confers the tumor specificity of the CAR, while the intracellular signaling domain activates the T cell that has been genetically engineered to express the CAR upon TAA/target engagement.
  • the engineered immune effector cells are re-infused into cancer patients, where they specifically engage and kill cells expressing the TAA target of the CAR (Maus et al., Blood. 2014 Apr 24;123(17):2625-35; Curran and Brentjens, J Clin Oncol. 2015 May 20;33(15): 1703-6).
  • CAR-T technology and its wider application is also limited due to a number of other key shortcomings, including, e.g., a) an inefficient anti -tumor response in solid tumors, b) limited penetration and susceptibility of adoptively transferred CAR T cells to an immunosuppressive tumor microenvironment (TME), c) poor persistence of CAR-T cells in vivo, d) serious adverse events in the patients including cytokine release syndrome (CRS) and graft-versus-host disease (GVHD) mediated by the CAR-T, and e) the time required for manufacturing.
  • TEE immunosuppressive tumor microenvironment
  • CRS cytokine release syndrome
  • GVHD graft-versus-host disease
  • a genetically engineered induced pluripotent stem cell or a derivative cell thereof.
  • the cell comprises: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope, preferably a truncated epithelial growth factor (tEGFR) variant, and an interleukin 15 (IL-15), wherein the inactivated cell surface receptor and the IL- 15 are operably linked by an autoprotease peptide, such as an autoprotease peptide of a porcine teseho virus- 1 2A (P2A) peptide; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RF
  • an iPSC cell or a derivative cell thereof comprising, i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) targeting a CD 19 antigen; (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant and an interleukin 15 (IL- 15), wherein the tEGFR variant and the IL-15 are operably linked by an autoprotease peptide, such as the autoprotease peptide of a porcine tesehovirus-1 2A (P2A) peptide; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably a deletion or reduced expression of B2M and CIITA genes.
  • CAR chimeric antigen receptor
  • IL- 15 interle
  • the iPSC cell or the derivative cell thereof further comprises a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) or human leukocyte antigen G (HLA-G).
  • HLA-E human leukocyte antigen E
  • HLA-G human leukocyte antigen G
  • one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are of one or more genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl 1, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided that at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes and the integration
  • the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the iPSC is derived from a reprogrammed T-cell.
  • the CAR comprises: (i) a signal peptide, such as a signal peptide comprising or being a GMCSFR signal peptide; (ii) an extracellular domain comprising a binding domain specifically binds a CD 19 antigen; (iii) a hinge region, such as a hinge region comprising a CD28 hinge region; (iv) a transmembrane domain, such as transmembrane domain comprising a CD28 transmembrane domain; (v) an intracellular signaling domain, such as an intracellular signaling domain comprising a CD3 ⁇ intracellular domain; and (vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain.
  • a signal peptide such as a signal peptide comprising or being a GMCSFR signal peptide
  • an extracellular domain comprising a binding domain specifically binds a CD 19 antigen
  • a hinge region such as a hinge region comprising a CD28
  • the extracellular domain comprises a scFv derived from an antibody that specifically binds the CD 19 antigen.
  • the CAR comprises: (i) a signal peptide; (n) an extracellular domain comprising a binding domain that specifically binds an antigen;
  • a hinge region (iii) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain.
  • the signal peptide comprises or is a GMCSFR signal peptide.
  • the extracellular domain comprises a VHH domain.
  • the hinge region comprises a CD28 hinge region.
  • the transmembrane domain comprises a CD28 transmembrane domain.
  • the intracellular signaling domain comprises a CD3 ⁇ intracellular domain.
  • the co-stimulatory domain comprises a CD28 signaling domain.
  • the CAR comprises:
  • the signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1;
  • the extracellular domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7;
  • the hinge region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22;
  • transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24;
  • the intracellular signaling domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6;
  • the co-stimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20.
  • the CAR comprises:
  • the signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1;
  • the hinge region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22;
  • transmembrane domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24;
  • the intracellular signaling domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6;
  • the co-stimulatory domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20.
  • the CAR comprises: (i) the signal peptide comprising the amino acid sequence of SEQ ID NO: 1; (ii) an extracellular domain comprising a scFV or a VHH domain; (iii) the hinge region comprising the amino acid sequence of SEQ ID NO: 22; (iv) the transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24; (v) the intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6; and (vi) the co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 20.
  • the CAR comprises: (i) the signal peptide comprising the amino acids sequence of SEQ ID NO: 1; (ii) the extracellular domain comprising the amino acid sequence of SEQ ID NO: 7; (iii) the hinge region comprising the amino acid sequence of SEQ ID NO: 22; (iv) the transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24; (v) the intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6; and (vi) the co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 20.
  • the inactivated cell surface protein is selected from the group of monoclonal antibody specific epitopes selected from epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, den
  • the inactivated cell surface protein is a truncated epithelial growth factor (tEGFR) variant.
  • tEGFR truncated epithelial growth factor
  • the tEGFR variant has or consists of, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71.
  • the tEGFR variant has or consists of the amino acid sequence of SEQ ID NO: 71.
  • the IL-15 has the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72.
  • the IL-15 comprises the amino acid sequence of SEQ ID NO: 72.
  • the autoprotease peptide has the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 73.
  • the autoprotease peptide has the amino acid sequence of SEQ ID NO: 73.
  • the iPSC or derivative has a deletion or reduced expression of one or more of the B2M and/or CIITA genes.
  • the tEGFR variant consists of the amino acid sequence of SEQ ID NO: 71
  • the autoprotease peptide has the amino acid sequence of SEQ ID NO: 73
  • the IL-15 comprises the amino acid sequence of SEQ ID NO:
  • the HLA-E has the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66.
  • the HLA-E has the amino acid sequence of SEQ ID NO: 66.
  • the HLA-G has the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69.
  • the HLA-G has the amino acid sequence of SEQ ID NO: 69.
  • a genetically engineered iPSC or a derivative cell thereof comprises:
  • a first exogenous polynucleotide encoding a CAR having: (i) a signal peptide comprising the amino acids sequence of SEQ ID NO: 1; (ii) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 7; (iii) the hinge region comprising the amino acid sequence of SEQ ID NO: 22; (iv) the transmembrane domain comprising the amino acid sequence of SEQ ID NO: 24; (v) the intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 6; and (vi) the co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 20; and
  • a second exogenous polynucleotide encoding a tEGFR variant consisting of the amino acid sequence of SEQ ID NO: 71 and an IL-15 comprising the amino acid sequence of SEQ ID NO: 72, wherein the tEGFR variant and the IL-15 are operably linked by an autoprotease peptide comprising the amino acid sequence of SEQ ID NO: 73, wherein the first and second exogenous polynucleotides are integrated at loci of two genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes, preferably the B2M and CIITA genes, and the integrations result in a deletion or reduced expression of the two genes.
  • the second exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 75; and (ii) the third exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 67.
  • the first exogenous polynucleotide is integrated at a locus of AAVS1 gene; (i) the second exogenous polypeptide is integrated at a locus of CIITA gene; and (ii) the third exogenous polypeptide is integrated at a locus of B2M gene; wherein integration of the exogenous polynucleotides deletes or reduces expression of CIITA and B2M, preferably, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.
  • the derivative cell is a natural killer (NK) cell or a T cell.
  • the genetically engineered iPSC or the derivative cell thereof further comprises a third exogenous polynucleotide encoding an HLA-E having the amino acid sequence of SEQ ID NO: 66 or an HLA-G having the amino acid sequence of SEQ ID NO: 69.
  • the third exogenous polynucleotide is integrated at a locus of a gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl 1, GAPDH, RUNX1, TAPI, TAP2, Tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT genes, preferably of the AAVS1 gene.
  • a gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl 1, GAPDH, RUNX1, TAPI, TAP2, Tapasin, NLRC5, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38,
  • the first exogenous polynucleotide comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 62.
  • the second exogenous polynucleotide comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 75.
  • the third exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 67.
  • the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.
  • the first exogenous polynucleotide is integrated at a locus of AAVS1 gene; the second exogenous polynucleotide is integrated at a locus of CIITA gene; and the third exogenous polynucleotide is integrated at a locus of B2M gene; wherein integration of the exogenous polynucleotides deletes or reduces expression of CIITA and B2M genes, preferably, the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62, the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75, and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.
  • the derivative cell is a natural killer (NK) cell or a T cell.
  • NK natural killer
  • iPSC induced pluripotent stem cell
  • NK natural killer
  • T cell derived from the iPSC i.e., iNK or iT
  • a first exogenous polynucleotide encoding a CAR comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 61;
  • a second exogenous polynucleotide encoding a tEGFR variant comprising or consisting of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71, an autoprotease peptide comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 73, and an IL-15 comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72, wherein the tEGFR is operably linked to the IL-15 via the autoprotease peptide; and
  • a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66, or an HLA-G comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69; wherein the first, second and third exogenous polynucleotides are integrated at loci of AAVS1, CIITA and B2M genes, to thereby delete or reduce expression of the CIITA and B2M genes.
  • HLA-E human leukocyte antigen E
  • an iPSC, an NK cell or a T cell of the application comprises:
  • a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; wherein the first, second and third exogenous polynucleotides are integrated at loci of AAVS1, CIITA and B2M genes, respectively, to thereby delete or reduce expression of the CIITA and B2M genes.
  • HLA-E human leukocyte antigen E
  • the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.
  • an iPSC a natural killer (NK) cell or a T cell comprising:
  • a second exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and
  • tEGFR truncated epithelial growth factor
  • a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; wherein the first, second and third exogenous polynucleotides are integrated at loci of AAVS1, CIITA and B2M genes, to thereby delete or reduce expression of CIITA and B2M.
  • HLA-E human leukocyte antigen E
  • the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and (ii) the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67, and the first, second and third exogenous polynucleotides are integrated at loci of AAVS1, CIITA and B2M genes, respectively.
  • composition comprising the cells of the application.
  • a composition of the application can further comprise, or be used in combination with, one or more other therapeutic agents.
  • other therapeutic agents include, but are not limited to, a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).
  • a method of treating cancer in a subject in need thereof the method comprising administering a cell of the application or a composition of the application to the subject in need thereof.
  • the cancer is non-Hodgkin’s lymphoma (NHL).
  • the method comprises introducing to an iPSC cell the first, second, and optionally third, exogenous polynucleotides to thereby obtain the genetically engineered iPSC.
  • Any genetic engineering method can be used to obtain the genetically engineered iPSC of the application.
  • the genetic engineering comprises targeted editing, and more preferably the targeted editing comprises deletion, insertion, or in/del, and wherein the targeted editing is carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods.
  • a method of differentiating an induced pluripotent stem cell (iPSC) into an NK cell by subjecting the cells to a differentiation protocol including the addition of recombinant human IL- 12 for the final 24 hours of culture.
  • the recombinant IL-12 comprises or is IL12p70.
  • HPC hematopoietic progenitor cell
  • iPSC induced pluripotent stem cell
  • a CD34+ hematopoietic progenitor cell derived from an induced pluripotent stem cell (iPSC) comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody- specific epitope and an interleukin 15 (IL- 15), wherein the inactivated cell surface receptor and the IL- 15 are operably linked by an autoprotease peptide; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.
  • inventions of the application include a genetically engineered iPSC or a derivative cell thereof for use in treating a cancer in a subject in need thereof.
  • the engineered iPSCs derivative cells of the invention have improved persistency, increased resistance to immune cells, or increased immune-resistance; or the genome-engineered iPSCs may have increased resistance to T and/or NK cells.
  • the IL-15 transgene of the invention once transfected into the iPSC’s and differentiated into NK cells in accordance with the invention, demonstrate increased persistency, decreased exhaustion and increased serial killing when compared to NK cells derived from iPSC’s cells without the IL-15 transgene of the invention.
  • the genome-engineered iPSCs of the invention have the potential to differentiate into non-pluripotent cells comprising hematopoietic lineage cells having the same functional targeted genomic editing.
  • the genome- engineered iPSCs of the invention have the potential to differentiate into mesodermal cells, CD34 cells, hemogenic endothelium cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, or B cells.
  • a polynucleotide encoding an artificial cell death polypeptide.
  • the polynucleotide encodes an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope and an interleukin 15 (IL-15), wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide.
  • IL-15 interleukin 15
  • the inactivated cell surface receptor is selected from the group of monoclonal antibody specific epitopes selected from epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, den
  • the inactivated cell surface receptor is a truncated epithelial growth factor (tEGFR) variant.
  • tEGFR truncated epithelial growth factor
  • the autoprotease peptide comprises or is a porcine tesehovirus-1 2 A (P2A) peptide.
  • the tEGFR variant consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71, preferably SEQ ID NO: 71.
  • the IL- 15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72, preferably SEQ ID NO: 72.
  • the autoprotease peptide comprises an ammo acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 73, preferably SEQ ID NO: 73.
  • the polynucleotide consists of operably linked polynucleotides encoding a truncated epithelial growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and an interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO: 72.
  • tEGFR truncated epithelial growth factor
  • IL-15 interleukin 15
  • a polynucleotide encoding an inactivated cell surface receptor that comprises an epitope specifically recognized by an antibody selected from the group consisting of cetuximab, matuzumab, necitumumab, panitumumab, polatuzumab vedotin, rituximab and trastuzumab, and an IL- 15, wherein the epitope and the cytokine are operably linked by a P2A sequence.
  • the inactivated cell surface receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74, 79, 81, and 83.
  • iPSC induced pluripotent stem cell
  • the vector further comprises:
  • the left homology sequence comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO:
  • the right homology sequence comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polynucleotide sequence of SEQ ID NO:
  • the vector comprises a polynucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 86.
  • FIGs 1A-1C show schematics of vectors (plasmids) according to embodiments of the application.
  • FIG. 1A shows CIITA targeting transgene plasmid with a CMV early enhancer/chicken ⁇ actin (CAG) promoter, SV40 terminator / poly adenylation signal, and tEGFR-IL15 coding sequence.
  • FIG. IB shows AAVS1 targeting transgene plasmid with a CAG promoter, SV40 terminator / poly adenylation, and anti-CD19 scFv chimeric antigen receptor (CAR) coding sequence.
  • FIG. 1C shows B2M targeting transgene plasmid with a CAG promoter, SV40 terminator / polyadenylation, and Peptide-B2M-HLA-E coding sequence.
  • FIG. 2 shows a graph demonstrating CAR- iNK cell-mediated target cell cytotoxicity over time in Reh cells and CD 19 knockout (CD19KO) Reh cells.
  • FIGs. 3A-C show functionality of iNK cell expressing CAR-IL15 compared to iNK cells expressing CAR alone.
  • FIG. 3A shows a graph demonstrating IL- 15 concentration (pg/ml/le6 cells/ 24 hours) released from CAR iNK cells and CAR/IL15 iNK cells.
  • FIG. 3B shows graphs demonstrating percentage iNK cells after 20 days in the blood and lungs of mice injected with CAR iNK cells or C AR- IL 15 iNK cells.
  • FIG. 3C shows graphs demonstrating percentage iNK cells in the lungs of mice injected with CAR iNK cells or CAR-IL15 iNK cells and with and without recombinant IL-15.
  • FIGs. 4A-C show the proliferation and serial killing of CAG-CAR-IL-15 iNK cells.
  • FIG. 4A shows a graph demonstrating the serial killing of CD 19+ Reh cells by CAG-CAR/IL15-iNK cells over time.
  • FIG. 4B shows a graph demonstrating the increased proliferation of CAG-CAR/IL-15 iNK cells compared to CAG-CAR iNK cells.
  • FIG. 4C shows a graph demonstrating the increased target serial killing of CD 19+ Raji cells over time by CAG-CAR/IL-15 iNK cells compared to CAG-CAR iNK cells.
  • FIGs. 5A-5B show cytotoxicity of CAG-CAR-IL15 expressing 1NK cells with and without human recombinant IL 12.
  • FIG. 5A shows a graph demonstrating Raji cell death over time when cultured with CAG-CAR-IL15 iNK cells with and without IL 12.
  • FIG. 5B shows a graph demonstrating tumor growth measured as mean whole body luminescent average radiance of mice infused with IL12-primed and unprimed CAG-CAR-IL15 iNK cells.
  • FIGs. 6A-6B show Cetuximab-induced cell elimination in CAG-CAR expressing iNK cells and CAG-CAR-IL15-tEGFR expressing iNK cells.
  • FIG. 6A shows a graph demonstrating percentage Annexin-V staining in CAG-CAR expressing cells.
  • FIG. 6B shows a graph demonstrating percentage Annexin-V staining in CAG-CAR-IL15-tEGFR expressing cells.
  • FIGs. 7A-7C show an Incucyte based assay measuring the loss of Nuclight Red K562 cells over time with effector to target ratios of (A) 20: 1, (B) 10: 1, and (C) 1 : 1. Normalized target cell count as a percentage of target cell only count for four iNK1248-iPSC611 and the average of 3 PB-NKs. Each data point is the average of 3 replicates and error bars represent standard error of the mean.
  • FIG. 8 shows a flow based NK purity check of PB-NKs isolated from three PBMC donors and iNK1248-iPSC611.
  • FIGs. 9A-D show an Incucyte based assay measuring the loss of Nuclight Red target cells over time with four effector to target ratios. Normalized target cell count in Reh and Reh-CD19KO co-cultured with iNK1248-iPSC611 at effector to target ratios of (A) 10: 1, (B) 5: 1, (C) 1 : 1, and (D) 1:5 as a percentage of target cell only counts. Each data point is the average of 3 replicates and error bars represent standard error of the mean.
  • FIGs. 10A-D show an Incucyte based assay measuring the loss of Nuclight Red target cells over time with four effector to target ratios.
  • Normalized target cell count in NALM6 and NALM6-CD19KO co-cultured with iNK1248-iPSC611 at effector to target ratios of (A) 10 : 1 , (B) 5 : 1 , (C) 1: 1, and (D) 1 : 5 as a percentage of target cell only counts.
  • Each data point is the average of 3 replicates and error bars represent standard error of the mean.
  • FIG. 11 shows cumulative fold expansion of iNK1248-iPSC611 and WT iNK1487-iPSC005 over a 21-day persistence assay without exogenous IL2 support.
  • Cells were cultured in basal NKCM for 14 days at 37 °C with 5% CO2. Every 3-4 days, all conditions were harvested, counted on the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and then replated. After 21 days, cumulative fold change was calculated.
  • FIGs. 12A-F show cumulative fold expansion of iNK1248-iPSC611 and WT iNK1487-iPSC005 over a 21-day persistence assay.
  • Cells were cultured in NKCM containing one of six IL2 concentrations: (A) 10 nM, (B) 3 nM, (C) 1 nM, (D) 0.3 nM, (E) 0.1 nM, (F) 0 nM for 21 days at 37 °C with 5% CO2. Every 3-4 days, all conditions were harvested, counted on the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and then replated. After 21 days, cumulative fold change was calculated.
  • FIG. 13 shows a gating strategy for ADCC assays.
  • Cells were gated on lymphocytes, followed by exclusion of doublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finally on LIVE/DEADTM Near-IR+ to determine % of dead therapeutic iNK targets.
  • CTV CellTrace Violet
  • NIR Near-IR.
  • FIG. 14 shows EGFR staining on therapeutic iNK cells.
  • EGFR PE levels on therapeutic iNK stained with EGFR (black histogram) compared with unstained therapeutic iNK (gray histogram) or an unedited WT iNK (dashed line).
  • FIG. 15 shows cetuximab-mediated ADCC of therapeutic iNK cells.
  • Percent specific cell lysis of therapeutic iNK cells mediated by Cetuximab black triangles
  • human IgGl isotype control open triangles
  • IL-2 activated PBMC were co-cultured with therapeutic iNK at a 25: 1 E:T ratio for 16 hours and percent specific cell death of iNK determined.
  • Each data point is a mean of triplicate wells, error bars ⁇ standard deviation.
  • FIG. 16 shows select sensitivity of WT iNK cells to anti -HLA- ABC Ab- mediated complement cytotoxicity.
  • FIG. 17 shows a gating strategy for allo-evasion CTL cytotoxicity and activation assays.
  • Cells were gated on quantitation beads and lymphocytes. Within lymphocytes exclusion of doublets, followed by gating on LIVE/DEADTM Near-IR negative, followed by CTV to identify iNK cells and TCR ⁇ to identify T cells. Within T cells, CD4-negative, CD8-positive cells, followed by CD25 to identify activated CD8+ T cells. Key assay parameters Quantitation beads, live iNK cells and activated CD8+ T cells are indicated.
  • FIGs. 18A-B show CTL-mediated lysis of iNK cells. Assessment of specific iNK lysis by FACS.
  • FIG. 18A shows gating on iNK and T cells.
  • FIG. 18B shows specific lysis of iNK cells co-cultured with CTL at 5: 1 CTLtiNK ratio. Each symbol represents one donor, open bar is the parental wild-type iNK cells, and the shaded bar is the edited ⁇ 2MKO iNK cells.
  • FIGs. 19A-B show activation of iNK-specific CTL in co-cultures.
  • FIG. 19A shows a histogram plot of CD25 expression of CD8+ T cells. Dashed line indicates T cells cultured alone, the solid open histogram indicates T cells co-cultured with parental wild-type iNK cells, and the shaded histogram indicates T cells co-cultured with edited ⁇ 2MKO iNK cells.
  • FIG. 19B shows frequencies of activated T cells in co- cultures with parental iNK cells (open bar), ⁇ 2MKO iNK cells (shaded bar), or T cells alone with no targets (hatched bar). Each symbol represents one donor.
  • FIG. 20 shows a gating strategy for all-evasion cytotoxicity assays.
  • Cells were gated on lymphocytes, followed by exclusion of doublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finally on LIVE/DEADTM Near-IR+ to determine % of dead iNK targets.
  • CTV CellTrace Violet
  • NIR Near-IR.
  • FIG. 21 shows HLA-E staining on therapeutic iNK cells.
  • HLA-E open histogram
  • mouse IgGl isotype control gray filled histogram.
  • FIG. 22 shows NKG2A staining on PBMCs.
  • PBMC samples were gated on viable lymphocytes (data not shown), followed by a gate on CD3-CD56+ cells (“NK cells”).
  • NK cells CD3-CD56+ cells
  • Frequencies of NKG2A-expressing NK cells were then determined based on an FMO.
  • FIG. 23 shows cell death of therapeutic iNK cells (gray bars) relative to WT (black bars) compared with iNK lacking ⁇ 2M (white bars). Freshly thawed PBMC were co-cultured with therapeutic iNK at a 25: 1 E:T ratio in the presence of 10 ng/mL IL- 15 for 72 hours and cell death of edited iNK relative to WT determined. Each data point is a mean of triplicate wells.
  • FIG. 24 shows mean percent body weight change of untreated mice ( ⁇ ), or mice treated intravenously with iPSC611 at 10x10 6 ( ⁇ ) and 15x10 6 ( ⁇ ) (cryogenic) cells. Means are plotted where ⁇ 50% of the treatment group are present. Arrows represent dosing days.
  • FIG. 25 shows mean whole body average radiance of untreated mice ( ⁇ ), and mice treated intravenously weekly for three doses with iPSC611 at 10x10 6 ( ⁇ ) and 15x10 6 ( ⁇ ) (cryogenic) cells. Groups are plotted until Day 21, the last imaging timepoint where the untreated control group remained and the timepoint at which %TGI was calculated. Arrows represent dosing days.
  • FIG. 26 shows percent survival of NALM6-bearing mice treated with iPSC611. Mice were left untreated, or treated intravenously weekly for three doses with iPSC611 at 10x10 6 and 15x10 6 cryogenic cells. Mice were humanely euthanized when in moribund condition and exhibiting signs of excessive tumor burden, as a surrogate for survival.
  • FIG. 27 shows persistence of iPSC611 in lungs and blood of NALM6-bearing mice. Mice were left untreated, or received a single intravenous dose of iPSC611 at 15x10 6 cryogenic cells. One-week post-injection, lungs and blood were harvested for FACS analysis. Number of iNK per 100,000 lymphocytes is plotted for individual mice (o), and average per group represented by bars.
  • FIG. 28 shows mean percent body weight change of mice treated intravenously with iPSC611 at 15x10 6 cells receiving IP PBS ( ⁇ ), or cetuximab at 40 mg/kg ( ⁇ ).
  • FIG. 29 shows presence of iPSC611 in lungs and blood of NSG mice.
  • Mice were left untreated (naive), or received a single intravenous dose of iPSC611 at 15x10 6 cells on Day 1.
  • mice were treated IP with 20 mL/kg PBS ( ⁇ ), or 40 mg/kg cetuximab ( ⁇ ). All mice received rhIL-2 on Days 1 and 3.
  • lungs and blood were sampled and processed for FACS analysis and detection of iPSC611.
  • Data is represented as the Number of iNK per 100,000 lymphocytes per mouse, with mean ⁇ SD plotted.
  • any numerical values such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non- exclusive or open-ended.
  • a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • “or” refers to an inclusive or and not to an exclusive or.
  • a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together.
  • subject means any animal, preferably a mammal, most preferably a human.
  • mammal encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.
  • nucleic acids or polypeptide sequences e.g., CAR polypeptides and the CAR polynucleotides that encode them
  • sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat ’I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
  • isolated means a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues.
  • Nucleic acids, peptides, proteins, and cells that have been “isolated” thus include nucleic acids, peptides, proteins, and cells purified by standard purification methods and purification methods described herein.
  • isolated nucleic acids, peptides, proteins, and cells can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • polynucleotide synonymously referred to as “nucleic acid molecule,” “nucleotides,” “nucleic acids,” or “polynucleic acids,” refers to any polyribonucleotide or poly deoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • 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.
  • the term “vector” as used herein 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 vector, viral vectors, cosmids, and artificial chromosomes.
  • Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, and the like.
  • integration it is meant that one or more nucleotides of a construct is stably inserted into the cellular genome, i.e., covalently linked to the nucleic acid sequence within the cell's chromosomal DNA.
  • target integration it is meant that the nucleotide(s) of a construct is inserted into the cell's chromosomal or mitochondrial DNA at a pre-selected site or “integration site”.
  • integration as used herein further refers to a process involving insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of an endogenous sequence or nucleotide at the integration site. In the case, where there is a deletion at the insertion site, “integration” can further comprise replacement of the endogenous sequence or a nucleotide that is deleted with the one or more inserted nucleotides.
  • the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into, or non-native to, the host cell.
  • the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non- chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell.
  • the term “endogenous” refers to a referenced molecule or activity that is present in the host cell in its native form. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid natively contained within the cell and not exogenously introduced.
  • 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 nnRNA, 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.
  • “Operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter).
  • Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • the term encompasses the transcription of a gene into RNA.
  • the term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications.
  • the expressed CAR can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.
  • peptide can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art.
  • the conventional one-letter or three-letter code for amino acid residues is used herein.
  • peptide can be used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural ammo acids, etc.), as well as other modifications known in the art.
  • the peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C- terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.
  • engineered immune cell refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • the average length of P2A peptides is 18-22 amino acids.
  • a P2A peptide was first identified in a foot-and- mouth disease virus (FMDV), a member of the picomavirus (Ryan et al., J Gen Virol, 1991, 72(Pt 11): 2727-2732).
  • An exemplary P2A peptide useful for the application comprises an amino acid sequence at least 90%, such as 90%, 91%, 92%, 93%, 04%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73.
  • the P2A peptide useful for the application comprises the amino acid sequence of SEQ ID NO: 73.
  • IPCs Induced Pluripotent Stem Cells (IPSCs) And Immune Effector Cells
  • IPSCs have unlimited self-renewing capacity.
  • Use of iPSCs enables cellular engineering to produce a controlled cell bank of modified cells that can be expanded and differentiated into desired immune effector cells, supplying large amounts of homogeneous allogeneic therapeutic products.
  • IPSCs and derivative cells thereof.
  • the selected genomic modifications provided herein enhance the therapeutic properties of the derivative cells.
  • the derivative cells are functionally improved and suitable for allogenic off-the-shelf cell therapies following a combination of selective modalities being introduced to the cells at the level of iPSC through genomic engineering. This approach can help to reduce the side effects mediated by CRS/GVHD and prevent long-term autoimmunity while providing excellent efficacy.
  • the term "differentiation” is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell.
  • Specialized cells include, for example, a blood cell or a muscle cell.
  • a differentiated or differentiation- induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell.
  • the term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • pluripotent refers to the ability of a cell to form all lineages of the body or soma or the embryo proper.
  • embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm.
  • Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell).
  • reprogramming or “dedifferentiation” refers 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.
  • induced pluripotent stem cells or, iPSCs, means that the stem cells are produced from differentiated adult, neonatal or fetal cells that have been induced or changed or reprogrammed into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm.
  • the iPSCs produced do not refer to cells as they are found in nature.
  • hematopoietic stem and progenitor cells refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation.
  • Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors.
  • 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, NK cells).
  • myeloid monocytes and macrophages
  • neutrophils neutrophils
  • basophils basophils
  • eosinophils neutrophils
  • eosinophils neutrophils
  • basophils basophils
  • eosinophils neutrophils
  • erythrocytes erythrocytes
  • megakaryocytes/platelets dendritic cells
  • dendritic cells lymphoid lineages
  • CD34+ hematopoietic progenitor cell refers to an HPC that expresses CD34 on its surface.
  • immune cell or “immune effector cell” refers to a cell that is involved in an immune response. Immune response includes, for example, the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.
  • NK natural killer
  • T lymphocyte and “T cell” are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and that has various roles in the immune system.
  • a T cell can have the roles including, e.g., the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells.
  • a T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal.
  • the T cell can be CD3+ cells.
  • the T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulator T cells, gamma delta T cells (gd T cells), and the like.
  • helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells.
  • T cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells).
  • the 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
  • the T cell can also be differentiated from a stem cell or progenitor 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 the secretion profiles following stimulation, which may include secretion of 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 complex) class Il-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 molecules 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” refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of the T cell receptor (TCR chains).
  • the NK cell can also refer to a genetically engineered NK cell, such as aNK cell modified to express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the NK cell can also be differentiated from a stem cell or progenitor cell.
  • 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 specific cell types including any hematopoietic lineage cells.
  • the source cell derived iPSCs, and differentiated cells therefrom are sometimes collectively called “derived” or “derivative” cells depending on the context.
  • derivative effector cells or derivative NK or “iNK” cells or derivative T or “iT” cells, as used throughout this application are cells differentiated from an iPSC, as compared to their primary counterpart obtained from natural/native sources such as peripheral blood, umbilical cord blood, or other donor tissues.
  • the genetic imprint(s) conferring a preferential therapeutic attribute is incorporated into the iPSCs either through reprogramming a selected source cell that is donor-, disease-, or treatment response- specific, or through introducing genetically modified modalities to iPSC using genomic editing.
  • the induced pluripotent stem cell (iPSC) parental cell lines may be generated from peripheral blood mononuclear cells (PBMCs) or T-cells using any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmid-based process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference.
  • the reprogramming factors may be in a form of polynucleotides, and thus are introduced to the non-pluripotent cells by vectors such as a retrovirus, a Sendai virus, an adenovirus, an episome, and a mini-circle.
  • the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector.
  • the one or more polynucleotides are introduced by a Sendai viral vector.
  • the iPSC’s are clonal iPSC’s or are obtained from a pool of iPSCs and the genome edits are introduced by making one or more targeted integration and/or in/del at one or more selected sites.
  • the iPSC’s are obtained from human T cells having antigen specificity and a reconstituted TCR gene (hereinafter, also refer to as "T-iPS” cells) as described in US Pat. Nos. 9206394, and 10787642 hereby incorporated by reference into the present application..
  • the application relates to an induced pluripotent stem cell (iPSC) cell or a derivative cell thereof comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant and an interleukin 15 (IL-15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide, such as a porcine tesehovirus-1 2A (P2A) peptide; and (iii) a deletion or reduced expression of B2M and CIITA genes.
  • CAR chimeric antigen receptor
  • IL-15 interleukin 15
  • an iPSC cell or a derivative cell thereof comprises a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), such as a CAR targeting a tumor antigen.
  • CAR chimeric antigen receptor
  • the CAR targets a CD 19 antigen.
  • chimeric antigen receptor refers to a recombinant polypeptide comprising at least an extracellular domain that binds specifically to an antigen or a target, a transmembrane domain and an intracellular signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell.
  • CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and/or production of molecules that can mediate cell death of the target antigen- expressing cell in a major histocompatibility (MHC)-independent manner.
  • MHC major histocompatibility
  • signal peptide refers to a leader sequence at the amino-terminus (N-terminus) of a nascent CAR protein, which co-translationally or post-translationally directs the nascent protein to the endoplasmic reticulum and subsequent surface expression.
  • extracellular antigen binding domain refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target or ligand.
  • hinge region or “hinge domain” refers to the part of a CAR that connects two adjacent domains of the CAR protein, i.e., the extracellular domain and the transmembrane domain of the CAR protein.
  • transmembrane domain refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane.
  • intracellular signaling domain refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal.
  • the term “stimulatory molecule” refers to a molecule expressed by an immune cell (e.g., NK cell or T cell) that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of receptors in a stimulatory way for at least some aspect of the immune cell signaling pathway.
  • Stimulatory molecules comprise two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation (referred to as “primary signaling domains”), and those that act in an antigen-independent manner to provide a secondary of co-stimulatory signal (referred to as “co-stimulatory signaling domains”).
  • the extracellular domain comprises an antigen binding domain and/or an antigen binding fragment.
  • the antigen binding fragment can, for example, be an antibody or antigen binding fragment thereof that specifically binds a tumor antigen.
  • the antigen binding fragments of the application possess one or more desirable functional properties, including but not limited to high-affinity binding to a tumor antigen, high specificity to a tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and/or antibody-dependent cellular-mediated cytotoxicity (ADCC) against cells expressing a tumor antigen, and the ability to inhibit tumor growth in subjects in need thereof and in animal models when administered alone or in combination with other anti-cancer therapies.
  • CDC complement-dependent cytotoxicity
  • ADPC antibody-dependent phagocytosis
  • ADCC antibody-dependent cellular-mediated cytotoxicity
  • antibody is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e. , IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub- classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
  • the antibodies of the application can be of any of the five major classes or corresponding sub-classes.
  • the antibodies of the application are IgGl, IgG2, IgG3 or IgG4.
  • Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains.
  • the antibodies of the application can contain a kappa or lambda light chain constant domain.
  • the antibodies of the application include heavy and/or light chain constant regions from rat or human antibodies.
  • antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2, and CDR3).
  • the light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.
  • an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the specific tumor antigen is substantially free of antibodies that do not bind to the tumor antigen).
  • an isolated antibody is substantially free of other cellular material and/or chemicals.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts.
  • the monoclonal antibodies of the application can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods.
  • the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.
  • the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab') 2 , an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain (V H H) of a camelid antibody, or any other antibody fragment that bind
  • single-chain antibody refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).
  • single domain antibody refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
  • humanized antibody refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced.
  • chimeric antibody refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.
  • multispecific antibody refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes do not overlap or do not substantially overlap.
  • the first and second epitopes are on different antigens, e.g, the different proteins (or different subunits of a multimeric protein).
  • a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain.
  • a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.
  • the term “bispecific antibody” refers to a multispecific antibody that binds no more than two epitopes or two antigens.
  • a bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope.
  • a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody comprises a V H H having binding specificity for a first epitope, and a VHH having binding specificity for a second epitope.
  • an antigen binding domain or antigen binding fragment that “specifically binds to a tumor antigen” refers to an antigen binding domain or antigen binding fragment that binds a tumor antigen, with a KD of 1 ⁇ 10 ⁇ 7 M or less, preferably 1 ⁇ 10 ⁇ 8 M or less, more preferably 5 ⁇ 10 ⁇ 9 M or less, 1 ⁇ 10 ⁇ 9 M or less, 5 ⁇ 10 ⁇ 10 M or less, or 1 ⁇ 10 ⁇ 10 M or less.
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods in the art in view of the present disclosure.
  • the KD of an antigen binding domain or antigen binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.
  • the smaller the value of the KD of an antigen binding domain or antigen binding fragment the higher affinity that the antigen binding domain or antigen binding fragment binds to a target antigen.
  • antibodies or antibody fragments suitable for use in the CAR of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals (“SMIPsTM”), intrabodies, minibodies, single domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (including, e.g., anti- id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above.
  • Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable
  • the antigen-binding fragment is an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single-domain antibody (sdAb) or nanobody, a dAb fragment, a Fd' fragment, a Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody.
  • the antigen-binding fragment is an scFv fragment.
  • the antigen-binding fragment is a VHH.
  • At least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or nanobody. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.
  • the extracellular tag-binding domain and the tag each comprise a VHH.
  • the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH.
  • At least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises an scFv. In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv.
  • the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a scFv.
  • Alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high-affinity and specific binding of target biomolecules, may also be used in the CARs of the present disclosure. Such scaffolds have been shown to yield molecules with improved characteristics, such as greater stability or reduced immunogenicity.
  • Non-limiting examples of alternative scaffolds that may be used in the CAR of the present disclosure include engineered, tenascin- derived, tenascin type III domain (e.g., CentyrinTM); engineered, gamma-B crystallin- derived scaffold or engineered, ubiquitin-derived scaffold (e.g., Affilins); engineered, fibronectin-derived, 10th fibronectin type III (10Fn3) domain (e.g., monobodies, AdNectinsTM, or AdNexinsTM);; engineered, ankyrin repeat motif containing polypeptide (e.g., DARPinsTM); engineered, low-density-lipoprotein-receptor-derived, A domain (LDLR-A) (e.g., AvimersTM); lipocalin (e.g., anticalins); engineered, protease inhibitor-derived, Kunitz domain (e.g., EETI-II/AGRP,
  • the alternative scaffold is Affilin or Centyrin.
  • the first polypeptide of the CARs of the present disclosure comprises a leader sequence.
  • the leader sequence may be positioned at the N-terminus the extracellular tag-binding domain.
  • the leader sequence may be optionally cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cellular membrane. Any of various leader sequences known to one of skill in the art may be used as the leader sequence.
  • Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), Fc ⁇ R, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8 ⁇ , or any of various other proteins secreted by T cells.
  • the leader sequence is compatible with the secretory pathway of a T cell.
  • the leader sequence is derived from human immunoglobulin heavy chain (HC).
  • the leader sequence is derived from GMCSFR.
  • the GMCSFR leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 1.
  • the first polypeptide of the CARs of the present disclosure comprise a transmembrane domain, fused in frame between the extracellular tag-binding domain and the cytoplasmic domain.
  • the transmembrane domain may be derived from the protein contributing to the extracellular tag-binding domain, the protein contributing the signaling or co- signaling domain, or by a totally different protein.
  • the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex.
  • the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid binding of proteins naturally associated with the transmembrane domain.
  • the transmembrane domain includes additional amino acids to allow for flexibility and/or optimal distance between the domains connected to the transmembrane domain.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Non-limiting examples of transmembrane domains of particular use in this disclosure may be derived from (i.e. comprise at least the transmembrane region(s) of) the a. ⁇ or ⁇ chain of the T-cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8 ⁇ , CD9, CD 16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154.
  • TCR T-cell receptor
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and/or valine can be found at each end of a synthetic transmembrane domain.
  • transmembrane domain of the ⁇ , ⁇ or Fc ⁇ R1 ⁇ chains which contain a cysteine residue capable of disulfide bonding so that the resulting chimeric protein will be able to form disulfide linked dimers with itself, or with unmodified versions of the ⁇ , ⁇ or Fc ⁇ R1 ⁇ chains or related proteins.
  • the transmembrane domain will be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • transmembrane domain of ⁇ , ⁇ or Fc ⁇ R1 ⁇ and - ⁇ , MB1 (Iga.), B29 or CD3- ⁇ , ⁇ , or ⁇ in order to retain physical association with other members of the receptor complex.
  • the transmembrane domain is derived from CD8 or CD28.
  • the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 23.
  • the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 24.
  • the first polypeptide of the CAR of the present disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, wherein the tag-binding domain, linker, and the transmembrane domain are in frame with each other.
  • spacer region generally means any oligo- or polypeptide that functions to link the tag-binding domain to the transmembrane domain.
  • a spacer region can be used to provide more flexibility and accessibility for the tag-binding domain.
  • a spacer region may comprise up to 300 ammo acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • a spacer region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the spacer region may be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or may be an entirely synthetic spacer region sequence.
  • Non-limiting examples of spacer regions which may be used in accordance to the disclosure include a part of human CD8 ⁇ chain, partial extracellular domain of CD28, Fey Rllla receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof.
  • additional linking amino acids are added to the spacer region to ensure that the antigen-binding domain is an optimal distance from the transmembrane domain.
  • the spacer when the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.
  • the spacer region comprises a hinge domain.
  • the hinge domain may be derived from CD8 ⁇ , CD28, or an immunoglobulin (IgG).
  • IgG immunoglobulin
  • the IgG hinge may be from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.
  • the hinge domain comprises an immunoglobulin IgG hinge or functional fragment thereof.
  • the IgG hinge is from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.
  • the hinge domain comprises the CHI, CH2, CH3 and/or hinge region of the immunoglobulin.
  • the hinge domain comprises the core hinge region of the immunoglobulin.
  • core hinge can be used interchangeably with the term “short hinge” (a.k.a “SH”).
  • Non-limiting examples of suitable hinge domains are the core immunoglobulin hinge regions include EPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgGl, ERKCCVECPPCP (SEQ ID NO: 58) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP) 3 (SEQ ID NO: 59) from IgG3, and ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes).
  • the hinge domain is a fragment of the immunoglobulin hinge.
  • the hinge domain is derived from CD8 or CD28.
  • the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 21.
  • the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 22.
  • the transmembrane domain and/or hinge domain is derived from CD8 or CD28. In some embodiments, both the transmembrane domain and hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and hinge domain are derived from CD28.
  • the first polypeptide of CARs of the present disclosure comprise a cytoplasmic domain, which comprises at least one intracellular signaling domain.
  • cytoplasmic domain also comprises one or more co- stimulatory signaling domains.
  • the cytoplasmic domain is responsible for activation of at least one of the normal effector functions of the host cell (e.g., T cell) in which the CAR has been placed in.
  • effector function refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is thus meant to include any truncated portion of the signaling domain sufficient to transduce the effector function signal.
  • Non-limiting examples of signaling domains which can be used in the CARs of the present disclosure include, e.g., signaling domains derived from DAP10, DAP12, Fc epsilon receptor I ⁇ chain (FCER1G), FcR , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ CD5, CD22, CD226, CD66d, CD79A, and CD79B.
  • FCER1G Fc epsilon receptor I ⁇ chain
  • FcR CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ CD5, CD22, CD226, CD66d, CD79A, and CD79B.
  • the cytoplasmic domain comprises a CD3 ⁇ signaling domain.
  • the CD3 ⁇ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 6, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 6.
  • the cytoplasmic domain further comprises one or more co-stimulatory signaling domains.
  • the one or more co-stimulatory signaling domains are derived from CD28, 41BB, IL2Rb, CD40, 0X40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM.
  • the co-stimulatory signaling domain is derived from 41BB.
  • the 41BB co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 8.
  • the co-stimulatory signaling domain is derived from IL2Rb .
  • the IL2Rb co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 9.
  • the co-stimulatory signaling domain is derived from CD40.
  • the CD40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 10.
  • the co-stimulatory signaling domain is derived from 0X40.
  • the 0X40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 11.
  • the co-stimulatory signaling domain is derived from CD80.
  • the CD80 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 12.
  • the co-stimulatory signaling domain is derived from CD86.
  • the CD86 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 13.
  • the co-stimulatory signaling domain is derived from CD27.
  • the CD27 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 14.
  • the co-stimulatory signaling domain is derived from ICOS.
  • the ICOS co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 15.
  • the co-stimulatory signaling domain is derived from NKG2D.
  • the NKG2D co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16.
  • the co-stimulatory signaling domain is derived from DAP 10.
  • the DAP 10 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 17.
  • the co-stimulatory signaling domain is derived from DAP 12.
  • the DAP 12 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 18.
  • the co-stimulatory signaling domain is derived from 2B4 (CD244).
  • the 2B4 (CD244) co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 19.
  • the CAR of the present disclosure comprises one costimulatory signaling domains. In some embodiments, the CAR of the present disclosure comprises two or more costimulatory signaling domains. In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more costimulatory signaling domains.
  • the signaling domain(s) and costimulatory signaling domain(s) can be placed in any order.
  • the signaling domain is upstream of the costimulatory signaling domains.
  • the signaling domain is downstream from the costimulatory signaling domains. In the cases where two or more costimulatory domains are included, the order of the costimulatory signaling domains could be switched.
  • Non-limiting exemplary CAR regions and sequences are provided in Table 1. Table 1.
  • the antigen-binding domain of the second polypeptide binds to an antigen.
  • the antigen-binding domain of the second polypeptide may bind to more than one antigen or more than one epitope in an antigen.
  • the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more antigens.
  • the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more epitopes in the same antigen.
  • antigen-binding domain may depend upon the type and number of antigens that define the surface of a target cell.
  • the antigen-binding domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state.
  • the CARs of the present disclosure can be genetically modified to target a tumor antigen of interest by way of engineering a desired antigen-binding domain that specifically binds to an antigen (e.g., on a tumor cell).
  • Non-limiting examples of cell surface markers that may act as targets for the antigen-binding domain in the CAR of the disclosure include those associated with tumor cells or autoimmune diseases.
  • the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen. In some embodiments, the antigen-binding domain binds to at least one tumor antigen. In some embodiments, the antigen-binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors. In some embodiments, the antigen-binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen-binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.
  • the tumor antigen is associated with glioblastoma, ovarian cancer, cervical cancer, head and neck cancer, liver cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, or hematologic malignancy.
  • tumor antigen associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBOland IL13R ⁇ 2.
  • tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, Mesothelin, CA125, EpCAM, EGFR, PDGFR ⁇ , Nectin-4, and B7H4.
  • Non-limiting examples of the tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, Mesothelin, HER2, and EGFR.
  • Non-limiting examples of tumor antigen associated with liver cancer include Claudin 18.2, GPC-3, EpCAM, cMET, and AFP.
  • Non-limiting examples of tumor antigens associated with hematological malignancies include CD22, CD79, BCMA, GPRC5D, SLAM F7, CD33, CLL1, CD123, and CD70.
  • Non-limiting examples of tumor antigens associated with bladder cancer include Nectin-4 and SLITRK6.
  • antigens that may be targeted by the antigen-binding domain include, but are not limited to, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, carbonic anhydrase EX, CD1, CDla, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD123, CD138, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAl 0, EphBl, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic go
  • the antigen targeted by the antigen-binding domain is CD 19.
  • the antigen-binding domain comprises an anti-CD19 scFv.
  • the anti-CD19 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 2.
  • VH heavy chain variable region
  • the anti-CD19 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 4, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 4.
  • VL light chain variable region
  • the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 7, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 7.
  • the antigen is associated with an autoimmune disease or disorder.
  • Such antigens may be derived from cell receptors and cells which produce “self ’-directed antibodies.
  • the antigen is associated with an autoimmune disease or disorder such as Rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, Systemic lupus erythematosus, sarcoidosis, Type 1 diabetes mellitus, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn's disease or ulcerative colitis.
  • RA Rheumatoid arthritis
  • autoimmune antigens that may be targeted by the CAR disclosed herein include but are not limited to platelet antigens, myelin protein antigen, Sm antigens in snRNPs, islet cell antigen, Rheumatoid factor, and anticitrullinated protein, citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclical citrullinated peptides), fibrinogen, fibrin, vimentin, fillaggrin, collagen I and II peptides, alpha- enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage such as collagen II, IX, and XI, circulating serum proteins such as RFs (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33/hnRNP A2, Sm, eukaryotic trasnlation elogation factor 1 alpha
  • the scFv fragment used in the CAR of the present disclosure may include a linker between the VH and VL domains.
  • the linker can be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included into the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His and The.
  • the linker should have a length that is adequate to link the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity, such as binding to an antigen.
  • the linker may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long.
  • the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10- 20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long.
  • Exemplary linkers that may be used are Gly rich linkers, Gly and Ser containing linkers, Gly and Ala containing linkers, Ala and Ser containing linkers, and other flexible linkers.
  • the linker is a Whitlow linker.
  • the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 3.
  • the linker is a (G 4 S) 3 linker.
  • the (G 4 S) 3 linker comprises the amino acid sequence set forth in SEQ ID NO: 25, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 25.
  • linker sequences may include portions of immunoglobulin hinge area, CL or CHI derived from any immunoglobulin heavy or light chain isotype.
  • Exemplary linkers that may be used include any of SEQ ID NOs: 26-56 in Table 1. Additional linkers are described for example in Int. Pat. Publ. No. WO2019/060695, incorporated by reference herein in its entirety.
  • an iPSC cell or a derivative cell thereof comprises a second exogenous polynucleotide encoding an artificial cell death polypeptide.
  • artificial cell death polypeptide refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of a cell therapy.
  • the artificial cell death polypeptide 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 artificial cell death polypeptide is activated by an exogenous molecule, e.g. an antibody, that when activated, triggers apoptosis and/or cell death of a therapeutic cell.
  • an artificial cell death polypeptide comprises an inactivated cell surface receptor that comprises an epitope specifically recognized by an antibody, particularly a monoclonal antibody, which is also referred to herein as a monoclonal antibody-specific epitope.
  • an antibody particularly a monoclonal antibody, which is also referred to herein as a monoclonal antibody-specific epitope.
  • the inactivated cell surface receptor When expressed by iPSCs or derivative cells thereof, the inactivated cell surface receptor is signaling inactive or significantly impaired, but can still be specifically recognized by an antibody.
  • the specific binding of the antibody to the inactivated cell surface receptor enables the elimination of the iPSCs or derivative cells thereof by ADCC and/or ADCP mechanisms, as well as, direct killing with antibody drug conjugates with toxins or radionuclides.
  • the inactivated cell surface receptor comprises an epitope that is selected from epitopes specifically recognized by an antibody, including but not limited to, ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, den
  • Epidermal growth factor receptor also known as EGFR, ErbBl and HER1
  • EGFR epidermal growth factor receptor
  • ErbBl ErbBl
  • HER1 is a cell-surface receptor for members of the epidermal growth factor family of extracellular ligands.
  • truncated EGFR “tEGFR,” “short EGFR” or “sEGFR” refers to an inactive EGFR variant that lacks the EGF-binding domains and the intracellular signaling domains of the EGFR.
  • An exemplary tEGFR variant contains residues 322-333 of domain 2, all of domains 3 and 4 and the transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope.
  • tEGFR variant on the cell surface enables cell elimination by an antibody that specifically binds to the tEGFR, such as cetuximab (Erbitux®), as needed. Due to the absence of the EGF-binding domains and intracellular signaling domains, tEGFR is inactive when expressed by iPSCs or derivative cell thereof.
  • An exemplary inactivated cell surface receptor of the application comprises a tEGFR variant.
  • expression of the inactivated cell surface receptor in an engineered immune cell expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cell when the cell is contacted with an anti-EGFR antibody.
  • CAR chimeric antigen receptor
  • a subject who has previously received an engineered immune cell of the present disclosure that comprises a heterologous polynucleotide encoding an inactivated cell surface receptor comprising a tEGFR variant can be administered an anti-EGFR antibody in an amount effective to ablate in the subject the previously administered engineered immune cell.
  • the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably the anti-EGFR antibody is cetuximab.
  • the tEGFR variant comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 71, preferably the amino acid sequence of SEQ ID NO: 71.
  • the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab v edotin.
  • the CD79b epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78, preferably the amino acid sequence of SEQ ID NO: 78.
  • the inactivated cell surface receptor comprises one or more epitopes of CD20, such as an epitope specifically recognized by rituximab.
  • the CD20 epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80, preferably the amino acid sequence of SEQ ID NO: 80.
  • the inactivated cell surface receptor comprises one or more epitopes of Her 2 receptor or ErbB, such as an epitope specifically recognized by trastuzumab.
  • the monoclonal antibody-specific epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82, preferably the amino acid sequence of SEQ ID NO: 82.
  • the inactivated cell surface receptor further comprises a cytokine, such as interleukin- 15 or interleukin-2.
  • Interleukin- 15 refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof.
  • a “functional portion” (“biologically active portion”) of a cytokine refers to a portion of the cytokine that retains one or more functions of full length or mature cytokine. Such functions for IL-15 include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells.
  • the sequence of a variety of IL-15 molecules are known in the art.
  • the IL- 15 is a wild-type IL-15.
  • the IL- 15 is a human IL-15.
  • the IL- 15 comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 72, preferably the amino acid sequence of SEQ ID NO: 72.
  • Inter! eukin-2 refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof.
  • the IL-2 is a wild-type IL-2.
  • the IL-2 is a human IL-2.
  • the IL-2 comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 76, preferably the amino acid sequence of SEQ ID NO: 76.
  • an inactivated cell surface receptor comprises a monoclonal antibody-specific epitope operably linked to a cytokine, preferably by an autoprotease peptide.
  • the autoprotease peptide include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus-1 2 A (P2A), a foot-and-mouth disease virus (FMDV) 2 A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2 A (BmCPV2A), a Flacherie Virus 2 A (BmIFV2A), and a combination thereof.
  • P2A porcine teschovirus-1 2 A
  • FMDV foot-and-mouth disease virus
  • E2A Equine Rhinitis A Virus
  • T2A a cytoplasmic polyhedrosis virus 2 A
  • the autoprotease peptide comprises or is an autoprotease peptide of a porcine tesehovirus-1 2A (P2A) peptide.
  • the autoprotease peptide comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 73, preferably the amino acid sequence of SEQ ID NO: 73.
  • an inactivated cell surface receptor comprises a truncated epithelial growth factor (tEGFR) variant operably linked to an interleukin- 15 (IL- 15) or IL-2 by an autoprotease peptide.
  • the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 74, preferably the amino acid sequence of SEQ ID NO: 74.
  • an inactivated cell surface receptor further comprises a signal sequence.
  • the signal sequence comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 77, preferably the amino acid sequence of SEQ ID NO: 77.
  • an inactivated cell surface receptor further comprises a hinge domain.
  • the hinge domain is derived from CD8.
  • the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 21.
  • an inactivated cell surface receptor further comprises a transmembrane domain.
  • the transmembrane domain is derived from CD8.
  • the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 23.
  • an inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody in its extracellular domain, a transmembrane region and a cytoplasmic domain.
  • the inactivated cell surface receptor further comprises a hinge region between the epitope(s) and the transmembrane region.
  • the inactivated cell surface receptor comprises more than one epitopes specifically recognized by an antibody, the epitopes can have the same or different amino acid sequences, and the epitopes can be linked together via a peptide linker, such as a flexible peptide linker have the sequence of (GGGGS)n, wherein n is an integer of 1-8 (SEQ ID NO: 25).
  • the inactivated cell surface receptor further comprises a cytokine, such as an IL- 15 or IL-2.
  • the cytokine is in the cytoplasmic domain of the inactivated cell surface receptor.
  • the cytokine is operably linked to the epitope(s) specifically recognized by an antibody, directly or indirectly, via an autoprotease peptide, such as those described herein.
  • the cytokine is indirectly linked to the epitope(s) by connecting to the transmembrane region via the autoprotease peptide.
  • Non-limiting exemplary inactivated cell surface receptor regions and sequences are provided in Table 2.
  • the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 79, preferably the amino acid sequence of SEQ ID NO: 79.
  • the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 81, preferably the amino acid sequence of SEQ ID NO: 81.
  • the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 83, preferably the amino acid sequence of SEQ ID NO: 83.
  • an iPSC or derivative cell thereof of the application can be further modified by introducing a third exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).
  • a third exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).
  • disruption of the B2M gene eliminates surface expression of all MHC class I molecules, leaving cells vulnerable to lysis by NK cells through the “missing self’ response.
  • Exogenous HLA-E expression can lead to resistance to NK-mediated lysis (Gomalusse et al., Nat Biotechnol. 2017 Aug; 35(8): 765-772).
  • the iPSC or derivative cell thereof comprises a third exogenous polypeptide encoding at least one of a human leukocyte antigen E (HLA- E) and human leukocyte antigen G (HLA-G).
  • HLA-E comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65.
  • the HLA-G comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 68, preferably SEQ ID NO: 68.
  • the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-E via a linker.
  • the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 66.
  • the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-G via a linker.
  • the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 69.
  • the genomic editing at one or more selected sites may comprise insertions of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, 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, and/or survival of the genome-engineered iPSCs or derivative cells thereof.
  • the exogenous polynucleotides for insertion are operatively linked to (1) one or more exogenous promoters comprising CMV, EFla, PGK, CAG, UBC, or other constitutive, inducible, temporal-, tissue-, or cell type- specific promoters; or (2) one or more endogenous promoters comprised in the selected sites comprising AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus meeting the criteria of a genome safe harbor.
  • exogenous promoters comprising CMV, EFla, PGK, CAG, UBC, or other constitutive, inducible, temporal-, tissue-, or cell type- specific promoters
  • endogenous promoters comprised in the selected sites comprising AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus
  • the genome-engineered iPSCs generated using the above method comprise one or more different exogenous polynucleotides encoding proteins comprising caspase, thymidine kinase, cytosine deaminase, B-cell CD20, ErbB2 or CD79b wherein when the genome-engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated in different safe harbor locus comprising AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1.
  • exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitory proteins such as PD1, PD-L1, and CTLA4 as well as proteins that target the CD47/signal regulatory protein alpha (SIRPa) axis .
  • the genome-engineered iPSCs generated using the method provided herein comprise in/del at one or more endogenous genes associated with targeting modality, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins suppressing engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of the iPSCs or derivative cells thereof.
  • one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of an iPSC.
  • Genome editing, or genomic editing, or genetic editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and/or replaced in the genome of a targeted cell.
  • Targeted genome editing (interchangeable with “targeted genomic editing” or “targeted genetic editing”) enables insertion, deletion, and/or substitution at pre-selected sites in the genome.
  • targeted integration referring to a process involving insertion of one or more exogenous sequences at pre-selected sites in the genome, with or without deletion of an endogenous sequence at the insertion site.
  • Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach.
  • nuclease-independent targeted editing approach homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be inserted, through the enzymatic machinery of the host cell.
  • targeted editing could be achieved with higher frequency through specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases.
  • DSBs double strand breaks
  • Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, the NHEJ often leads to random insertions or deletions (in/dels) of a small number of endogenous nucleotides.
  • NHEJ non-homologous end joining
  • the exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in a “targeted integration.”
  • HDR homology directed repair
  • DSBs Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), RNA-guided CRISPR (Clustered Regular Interspaced Short Palindromic Repeats) systems. Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases is also a promising tool for targeted integration.
  • ZFN zinc-finger nucleases
  • TALEN transcription activator-like effector nucleases
  • CRISPR Clustered Regular Interspaced Short Palindromic Repeats
  • 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 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. No.
  • a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
  • transcription activator-like effector DNA binding domain By “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” it is meant the polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA.
  • TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains.
  • TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD).
  • RVD repeat variable-diresidues
  • TALENs are described in greater detail in U.S. Patent Application No. 2011/0145940, which is herein incorporated by reference.
  • the most recognized example of a TALEN in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.
  • a targeted nuclease that finds use in the subject methods is a targeted Spoil nuclease, a polypeptide comprising a Spol 1 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.
  • 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 the present application include, but not limited to Bxbl, phiC3 1, R4, PhiBTl, and Wp/SPBc/TP901-l, 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.
  • CRISPR/Cpfl comprises two major components: (1) a CPfl endonuclease and (2) a crRNA.
  • RNP ribobnucleoprotein
  • the crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpfl to target selected sequences.
  • MAD7 is an engineered Cas12a variant originating from the bacterium Eubacterium rectale that has a preference for 5'-TTTN-3' and 5'-CTTN-3' PAM sites and does not require a tracrRNA. See, for example, PCT Publication No. 2018/236548, the disclosure of which is incorporated herein by reference.
  • DICE mediated insertion uses a pair of recombinases, for example, phiC31 and Bxbl, to provide unidirectional integration of an exogenous DNA that is tightly restricted to each enzymes’ own small attB and attP recognition sites. Because these target att sites are not naturally present in mammalian genomes, they must be first introduced into the genome, at the desired integration site. See, for example, U.S. Application Publication No. 2015/0140665, the disclosure of which is incorporated herein by reference.
  • One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genome integration.
  • the construct further comprises a pair of homologous arm 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 Cpfl expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cpfl -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 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.
  • Sites for targeted integration include, but are not limited to, genomic safe harbors, which are intragenic or extragenic regions of the human genome that, theoretically, are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism.
  • the genome safe harbor for the targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes.
  • the site for targeted integration is selected for deletion or reduced expression of an endogenous gene at the insertion site.
  • the term “deletion” with respect to expression of a gene refers to any genetic modification that abolishes the expression of the gene. Examples of “deletion” of expression of a gene include, e.g., a removal or deletion of a DNA sequence of the gene, an insertion of an exogenous polynucleotide sequence at a locus of the gene, and one or more substitutions within the gene, which abolishes the expression of the gene.
  • MHC deficient including MHC- class I deficient, or MHC-class II deficient, or both, refers to cells that either lack, or no longer maintain, or have reduced level of surface expression of a complete MHC complex comprising a MHC class I protein heterodimer and/or a MHC class II heterodimer, such that the diminished or reduced level is less than the level naturally detectable by other cells or by synthetic methods.
  • MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p21), or deletion or reducing the expression level of one or more MHC class-I associated genes including, not being limited to, beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene and Tapasin genes.
  • B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers.
  • B2M null cells are MHC-I deficient.
  • MHC class II deficiency can be achieved by functional deletion or reduction of MHC-II associated genes including, not being 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 null cells are MHC-II deficient.
  • one or more of the exogenous polynucleotides are integrated at one or more loci of genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby delete or reduce the expression of the gene(s) with the integration.
  • the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are of genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl 1, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided at least one of the one or more loci is of a MHC gene, such as a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.
  • a MHC gene such as a gene selected from the group consist
  • the one or more exogenous polynucleotides are integrated at a locus of an MHC class-I associated gene, such as a beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or Tapasin gene; and at a locus of an MHC-II associated gene, such as a RFXANK, CIITA, RFX5, RFXAP, or CIITA gene; and optionally further at a locus of a safe harbor gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes. More preferably, the one or more of the exogenous polynucleotides are integrated at the loci of CIITA, AAVS1 and B2M genes.
  • B2M beta-2 microglobulin
  • the first exogenous polynucleotide is integrated at a locus of AAVS1 gene;
  • the second exogenous polypeptide is integrated at a locus of CIITA gene;
  • the third exogenous polypeptide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides delete or reduce expression of CIITA and B2M genes.
  • the first exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 62;
  • the second exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 75;
  • the third exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 67.
  • the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62;
  • the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and
  • the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.
  • the invention relates to a cell derived from differentiation of an iPSC, a derivative cell.
  • the genomic edits introduced into the iPSC cell are retained in the derivative cell.
  • the derivative cell is a hematopoietic cell, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, B cells, antigen presenting cells (APC), monocytes and macrophages.
  • the derivative cell is an immune effector cell, such as a NK cell or a T cell.
  • the application provides a natural killer (NK) cell or a T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant and an interleukin 15 (IL- 15), wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide, such as an autoprotease peptide of a porcine tesehovirus-1 2A (P2A) peptide; and (iii) a deletion or reduced expression of an MHC class I associated gene and an MHC class II associated gene, such as an MHC class-I associated gene selected from the group consisting of a B2M gene, TAP 1 gene, TAP 2 gene and Tapasin gene, and an MHC- II associated gene selected from the group consisting of a RFXAN
  • the NK cell or T cell further comprises a third exogenous polynucleotide encoding at least one of a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G).
  • HLA-E human leukocyte antigen E
  • HLA-G human leukocyte antigen G
  • a NK cell or a T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having the amino acid sequence of SEQ ID NO: 61; (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71, an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and (iii) a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; wherein the first, second and third exogenous polynucleotides are integrated at loci of AAVS1, CIITA and B2M genes, respectively, to thereby delete or reduce
  • the first exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 62; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.
  • HPC hematopoietic progenitor cell
  • iPSC induced pluripotent stem cell
  • a CD34+ hematopoietic progenitor cell derived from an induced pluripotent stem cell (iPSC) comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope and an interleukin 15 (IL- 15), wherein the inactivated cell surface receptor and the IL- 15 are operably linked by an autoprotease peptide; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.
  • the CD34+ HPC further comprises a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and/or human leukocyte antigen G (HLA-G).
  • the CAR comprises (i) a signal peptide; (n) an extracellular domain comprising a binding domain that specifically binds the CD 19 antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain.
  • the method comprises differentiating the iPSC under conditions for cell differentiation to thereby obtain the derivative cell.
  • An iPSC of the application can be differentiated by any method known in the art. Exemplary methods are described in US8846395, US8945922, US8318491, WO2010/099539, W02012/109208, W02017/070333, WO2017/179720, W02016/010148, WO2018/048828 and WO2019/157597, each of which are herein incorporated by reference in its entirety.
  • the differentiation protocol may use feeder cells or may be feeder-free.
  • 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 iPSC derivative cells of the invention are NK cells which are prepared by a method of differentiating an iPSC cell into an NK cell by subjecting the cells to a differentiation protocol including the addition of recombinant human IL-12p70 for the final 24 hours of culture.
  • a differentiation protocol including the addition of recombinant human IL-12p70 for the final 24 hours of culture.
  • cells that are primed with IL- 12 demonstrate more rapid cell killing compared to those that are differentiated in the absence of IL-12 (FIG. 5A).
  • the cells differentiated using the IL-12 conditions demonstrate improved cancer cell growth inhibition (FIG. 5B).
  • the invention in another general aspect, relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) useful for an invention according to embodiments of the application.
  • CAR chimeric antigen receptor
  • the coding sequence of a CAR can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding CARs of the application can be altered without changing the amino acid sequences of the proteins.
  • the isolated nucleic acid encodes a CAR targeting CD 19.
  • the isolated nucleic acid encoding the CAR comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 62, preferably the polynucleotide sequence of SEQ ID NO: 62.
  • the application provides a vector comprising a polynucleotide sequence encoding a CAR useful for an invention according to embodiments of the application.
  • Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the vector is a recombinant expression vector such as a plasmid.
  • the vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication.
  • the promoter can be a constitutive, inducible, or repressible promoter.
  • a number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a CAR in the cell.
  • Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.
  • the application provides vectors for targeted integration of a CAR useful for an invention according to embodiments of the application.
  • the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding a CAR according to an embodiment of the application; and (c) a terminator/polyadenylation signal.
  • the promoter is a CAG promoter.
  • the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63.
  • Other promoters can also be used, examples of which include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.
  • the terminator/ poly adenylation signal is a SV40 signal.
  • the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64.
  • Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.
  • the polynucleotide sequence encoding a CAR comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 62.
  • the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.
  • left homology arm and right homology arm refers to a pair of nucleic acid sequences that flank an exogenous polynucleotide and facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. Sequences of the left and right arm homology arms can be designed based on the integration site of interest. In some embodiment, the left or right arm homology arm is homologous to the left or right side sequence of the integration site.
  • the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 90.
  • the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 91.
  • the vector comprises a polynucleotide sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 92, preferably the polynucleotide sequence of SEQ ID NO: 92.
  • the invention relates to an isolated nucleic acid encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application.
  • the coding sequence of an inactivated cell surface receptor can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein.
  • nucleic acid sequences encoding an inactivated cell surface receptor of the application can be altered without changing the amino acid sequences of the proteins.
  • an isolated nucleic acid encodes any inactivated cell surface receptor described herein, such as that comprises a monoclonal antibody- specific epitope, and a cytokine, such as an IL- 15 or IL-2, wherein the monoclonal antibody-specific epitope and the cytokine are operably linked by an autoprotease peptide.
  • the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by an antibody, such as ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosuma
  • an antibody
  • the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epithelial growth factor (tEGFR) variant.
  • the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab or panitumumab, preferably cetuximab.
  • the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin.
  • the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, such as an epitope specifically recognized by rituximab.
  • the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of Her 2 receptor, such as an epitope specifically recognized by trastuzumab
  • the autoprotease peptide comprises or is a porcine tesehovirus-1 2 A (P2A) peptide.
  • the truncated epithelial growth factor (tEGFR) variant consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71.
  • the monoclonal antibody-specific epitope specifically recognized by polatuzumab vedotin consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78.
  • the monoclonal antibody-specific epitope specifically recognized by rituximab consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80.
  • the monoclonal antibody-specific epitope specifically recognized by trastuzumab consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 82%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82.
  • the IL- 15 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • the autoprotease peptide has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74.
  • the isolated nucleic acid encoding the inactivated cell surface receptor comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 75, preferably the polynucleotide sequence of SEQ ID NO: 75.
  • the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79.
  • the application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application.
  • Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the vector is a recombinant expression vector such as a plasmid.
  • the vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication.
  • the promoter can be a constitutive, inducible, or repressible promoter.
  • a number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a inactivated cell surface receptor in the cell.
  • Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.
  • the application provides a vector for targeted integration of an inactivated cell surface receptor useful for an invention according to embodiments of the application.
  • the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an inactivated cell surface receptor, such as an inactivated cell surface receptor comprising a truncated epithelial growth factor (tEGFR) variant and an interleukin 15 (IL- 15), wherein the tEGFR variant and the IL- 15 are operably linked by an autoprotease peptide, such as a porcine teseho virus- 1 2A (P2A) peptide, and (c) a terminator/polyadenylation signal.
  • tEGFR truncated epithelial growth factor
  • IL- 15 interleukin 15
  • the promoter is a CAG promoter.
  • the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63.
  • Other promoters can also be used, examples of which include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.
  • the terminator/polyadenylation signal is a SV40 signal.
  • the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64.
  • Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK.
  • the polynucleotide sequence encoding an inactivated cell surface receptor comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 75.
  • the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.
  • the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 84.
  • the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 85
  • the vector comprises a polynucleotide sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 86, preferably the polynucleotide sequence of SEQ ID NO: 86.
  • the invention relates to an isolated nucleic acid encoding an HLA construct useful for an invention according to embodiments of the application.
  • the coding sequence of an HLA construct can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein.
  • nucleic acid sequences encoding an HLA construct of the application can be altered without changing the amino acid sequences of the proteins.
  • the isolated nucleic acid encodes an HLA construct comprising a signal peptide, such as an HLA-G signal peptide, operably linked to an HLA coding sequence, such as a coding sequence of a mature B2M, and/or a mature HLA-E.
  • the HLA coding sequence encodes the HLA-G and B2M, which are operably linked by a 4X GGGGS linker, and/or the B2M and HLA- E, which are operably linked by a 3X GGGGS linker.
  • the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67.
  • the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.
  • the application provides a vector comprising a polynucleotide sequence encoding a HLA construct useful for an invention according to embodiments of the application.
  • Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the vector is a recombinant expression vector such as a plasmid.
  • the vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication.
  • the promoter can be a constitutive, inducible, or repressible promoter.
  • a number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a HLA construct in the cell.
  • Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.
  • the application provides vectors for targeted integration of a HLA construct useful for an invention according to embodiments of the application.
  • the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an HLA construct; and (c) a terminator/polyadenylation signal.
  • the promoter is a CAG promoter.
  • the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63.
  • Other promoters can also be used, examples of which include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.
  • the terminator/ poly adenylation signal is a SV40 signal.
  • the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64.
  • Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK.
  • a polynucleotide sequence encoding a HLA construct comprises a signal peptide, such as a HLA-G signal peptide, a mature B2M, and a mature HLA-E, wherein the HLA-G and B2M are operably linked by a 4X GGGGS linker (SEQ ID NO: 31) and the B2M transgene and HLA-E are operably linked by a 3X GGGGS linker (SEQ ID NO: 25).
  • the HLA construct comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67.
  • the HLA construct comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.
  • the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.
  • the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 87.
  • the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 88.
  • the vector comprises a polynucleotide sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 89, preferably the polynucleotide sequence of SEQ ID NO: 89.
  • the application provides a host cell comprising a vector of the application and/or an isolated nucleic acid encoding a construct of the application.
  • Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of exogenous polynucleotides of the application.
  • the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.
  • host cells include, for example, recombinant cells containing a vector or isolated nucleic acid of the application useful for the production of a vector or construct of interest; or an engineered iPSC or derivative cell thereof containing one or more isolated nucleic acids of the application, preferably integrated at one or more chromosomal loci.
  • a host cell of an isolated nucleic acid of the application can also be an immune effector cell, such as a T cell or NK cell, comprising the one or more isolated nucleic acids of the application.
  • the immune effector cell can be obtained by differentiation of an engineered iPSC of the application. Any suitable method in the art can be used for the differentiation in view of the present disclosure.
  • the immune effector cell can also be obtained transfecting an immune effector cell with one or more isolated nucleic acids of the application.
  • the application provides a composition comprising an isolated polynucleotide of the application, a host cell and/or an iPSC or derivative cell thereof of the application.
  • the composition further comprises one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, , a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).
  • a therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, , a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive mo
  • the composition is a pharmaceutical composition comprising an isolated polynucleotide of the application, a host cell and/or an iPSC or derivative cell thereof of the application and a pharmaceutically acceptable carrier.
  • pharmaceutical composition means a product comprising an isolated polynucleotide of the application, an isolated polypeptide of the application, a host cell of the application, and/or an iPSC or derivative cell thereof of the application together with a pharmaceutically acceptable carrier.
  • Polynucleotides, polypeptides, host cells, and/or iPSCs or derivative cells thereof of the application and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.
  • the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.
  • the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition described herein or the biological activity of a composition described herein. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in a polynucleotide, polypeptide, host cell, and/or iPSC or derivative cell thereof can be used.
  • compositions of the application are known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g. 21st edition (2005), and any later editions).
  • additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents.
  • One or more pharmaceutically acceptable carrier may be used in formulating the pharmaceutical compositions of the application.
  • the application provides a method of treating a disease or a condition in a subject in need thereof.
  • the methods comprise administering to the subject in need thereof a therapeutically effective amount of cells of the application and/or a composition of the application.
  • the disease or condition is cancer.
  • the cancer can, for example, be a solid or a liquid cancer.
  • the cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a liver cancer, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, an endometrial cancer, a prostate cancer, a thyroid cancer, a glioma, a glioblastoma, and other solid tumors, and anon-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma/ disease (HD), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.
  • the cancer is a non-Hodgkin’s
  • the composition comprises a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, a host cell, and/or an iPSC or derivative cell thereof.
  • therapeutically effective amount refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject.
  • a therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a therapeutically effective amount means an amount of the cells and/or the pharmaceutical composition that modulates an immune response in a subject in need thereof.
  • a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or
  • the therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.
  • the cells of the application and/or the pharmaceutical compositions of the application can be administered in any convenient manner known to those skilled in the art.
  • the cells of the application can be administered to the subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and/or transplantation.
  • compositions comprising the cells of the application can be administered transarterially, subcutaneously, intradermaly, intratumorally, intranodally, intramedullary, intramuscularly, inrapleurally, by intravenous (i.v.) injection, or intraperitoneally.
  • the cells of the application can be administered with or without lymphodepletion of the subject.
  • compositions comprising cells of the application can be provided in sterile liquid preparations, typically isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, or the like, which are typically buffered to a selected pH.
  • the compositions can comprise carriers, for example, water, saline, phosphate buffered saline, and the like, suitable for the integrity and viability of the cells, and for administration of a cell composition.
  • Sterile injectable solutions can be prepared by incorporating cells of the application in a suitable amount of the appropriate solvent with various other ingredients, as desired.
  • Such compositions can include a pharmaceutically acceptable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like, that are suitable for use with a cell composition and for administration to a subject, such as a human.
  • Suitable buffers for providing a cell composition are well known in the art. Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the application.
  • the cells of the application and/or the pharmaceutical compositions of the application can be administered in any physiologically acceptable vehicle.
  • a cell population comprising cells of the application can comprise a purified population of cells.
  • Those skilled in the art can readily determine the cells in a cell population using various well known methods.
  • the ranges in purity in cell populations comprising genetically modified cells of the application can be from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%.
  • the cells of the application are generally administered as a dose based on cells per kilogram (cells/kg) of body weight of the subject to which the cells and/or pharmaceutical compositions comprising the cells are administered.
  • the cell doses are in the range of about 10 4 to about 10 10 cells/kg of body weight, for example, about 10 5 to about 10 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 , or about 10 5 to about 10 6 , depending on the mode and location of administration.
  • a higher dose is used than in regional administration, where the immune cells of the application are administered in the region of a tumor and/or cancer.
  • Exemplary dose ranges include, but are not limited to, 1 x 10 4 to 1 x 10 8 , 2 x 10 4 to 1 x 10 8 , 3 x 10 4 to 1 x 10 8 , 4 x 10 4 to 1 x 10 8 , 5 x 10 4 to 6 x 10 8 , 7 x 10 4 to 1 x 10 8 , 8 x 10 4 to 1 x 10 8 , 9 x 10 4 to 1 x 10 8 , 1 x 10 5 to 1 x 10 8 ,
  • the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject.
  • the terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer.
  • “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.
  • the cells of the application and/or the pharmaceutical compositions of the application can be administered in combination with one or more additional therapeutic agents.
  • the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, , a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).
  • a peptide a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, , a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).
  • iPSC Induced pluripotent stem cell
  • Gene fragments (gBlocks) encoding the transgene of interest, with the promoter, terminator, and homology arms were designed and synthesized by chemical synthesis at IDT, Inc.
  • the gBlock gene fragments were assembled into a pUC19 plasmid using the In-Fusion® Cloning HD Plus kit (Takara Bio; Shiga, Japan) according to manufacturer’s protocol.
  • Reaction products from In-Fusion Cloning, i.e. expression constructs were transformed into Stbl3 bacterial cells (Thermo Fisher; Waltham, MA) for amplification according to manufacturer’s protocol.
  • Vector (plasmid) from the amplified expression construct was purified from bacterial cell culture using the HiSpeed Plasmid Maxi Prep kit (Qiagen; Hilden, Germany) according to the manufacturer's protocol. Research grade sequencing was performed on purified plasmid DNA and evaluated by restriction digestion to confirm transgene sequence. The concentration of purified plasmid DNA was measured by absorbance. Additionally, the absorbance ratio at A260/A280 nm and A260/A230 nm were measured to evaluate residual RNA and protein levels, respectively. CIITA Targeting Plasmid
  • the CIITA targeting plasmid contains a CAG promoter (SEQ ID NO: 63), SV40 terminator / polyadenylation (SEQ ID NO: 64), and tEGFR-IL15 coding sequence.
  • the tEGFR-IL15 transgene encodes tEGFR-IL15, which contains residues 322-333 of domain 2, all of domains 3 and 4 and the transmembrane domain of the native EGFR sequence (SEQ ID NO: 71).
  • the tEGFR-IL15 transgene is followed by an in frame P2A peptide sequence (SEQ ID NO: 73) and then the full-length IL-15 sequence (SEQ ID NO: 72).
  • a schematic of the CIITA targeting transgene plasmid is shown in FIG. 1 A.
  • the AAVS1 targeting plasmid contains a CAG promoter (SEQ ID NO: 63), SV40 terminator / polyadenylation (SEQ ID NO: 64), and anti-CD19 scFv chimeric antigen receptor (CAR) sequence (SEQ ID NO: 62).
  • the encoded CAR contains the GMCSFR signal peptide connected to the FMC63 scFv followed by residues 114 to 220 of CD28 and residues 52 to 163 of CD3zeta isoform 3.
  • FIG. IB A schematic of the AAVS1 targeting transgene plasmid is shown in FIG. IB.
  • the B2M targeting plasmid contains a CAG promoter (SEQ ID NO: 63), SV40 terminator / polyadenylation (SEQ ID NO: 64), and Peptide-B2M-HLA-E coding sequence (SEQ ID NO: 67).
  • the B2M-transgene encoded protein (SEQ ID NO: 66) contains the signal peptide from HLA-G followed by the nine amino acid peptide VMAPRTLIL connected to a 4X GGGGS linker, the mature B2M sequence connected to a 3X GGGGS linker and then the mature HLA-E sequence (SEQ ID NO: 65).
  • a schematic of the B2M targeting transgene plasmid is shown in FIG. 1C.
  • Transgene insertion into the B2M (exon 2) and CIITA (exon 1) results in disruption of the coding sequences and prevents translation of the full-length sequence. Loss of expression of B2M will prevent proper MHC Class I assembly and disrupt expression. Loss of CIITA will prevent HLA II gene transcription and prevent MHC Class II expression. Insertion of the transgene into intron 1 of the AAVS1 locus does not result in any coding sequence alterations. Homology arm sequences were designed to sit on the 5’ and 3’ sides of a Cpfl genome nuclease cut site and include from 500-1200 bp of target locus-specific sequence.
  • Cell line establishment consisted of transfection, electroporation, CAR- Engineered iPSC expansion, cell sorting, and cell cloning steps.
  • Three serial rounds of transfection and electroporation were performed with the associated purified plasmid DNA and recombinant Cpfl ultra/guide RNA Ribonucleoprotein (RNP) complexes that are specific to a single target locus (either B2M, CIITA, or AAVS1).
  • Guide RNAs gRNAs
  • a vial of iPSC cells from the parental cell line was thawed into Complete Essential 8TM medium (Thermo Fisher) with Hl 152 Rho Kinase Inhibitor, pelleted, and resuspended in Complete Essential 8 medium.
  • the cell suspension was then transferred to the wells of a Vitronectin-coated, 6-well plate containing Complete Essential 8 medium with Hl 152 and incubated at 37°C, 5% CO 2 , low O 2 .
  • Cells from one well were expanded into a T-75 flask. When the flask reached 60-70% confluency, it was propagated into another T-75 flask. When this flask became 60-70% confluent, the cells were used for transfection.
  • Hl 152 was added to a T-75 flask containing iPSC cells and the cells were incubated at 37°C, 5% CO 2 , low O 2 .
  • RNP Ribonucleoprotein
  • IDT Alt-R® CRISPR-Cpfl crRNA
  • IDT Alt-R® Cpfl Ultra Nuclease
  • Electroporated cells were then added to the wells of a pre-warmed, Vitronectin-coated, 24-well plate containing Complete Essential 8 medium and NU7026 and were incubated at 37°C, 5% CO 2 , low O 2 .
  • Cells were cultured for a minimum of 10 days in Complete Essential 8 medium on Vitronectin-coated plates for homology directed repair to occur. Once cells on the 24- well plate were 60-70% confluent, they were dissociated and propagated into one well of a 6-well plate. After reaching confluency, one well of a 6-well plate was propagated into a T-75 flask. Cells were maintained in culture for the minimum 10-day duration, after which the culture was analyzed for the presence of inserted transgenes and/or absence of deleted endogenous genes by flow cytometry. Cells were then subjected to flow cytometry sorting to isolate the modified population.
  • Sorting after each round of engineering included markers from the previous rounds and may require multiple rounds of sorting to sufficiently enrich the population for all the respective markers. Sorting was performed on a MacsQuant Tyto cell sorter (Miltenyi Biotech; Bergisch Gladbach, Germany) using fluorescently labeled antibodies to human HLA-E, human EGFR and a fusion protein of human CD19-Fc.
  • single cell clones were isolated by limited dilution cloning.
  • Cells were washed once with DPBS and dissociated from the plate.
  • Cells were resuspended in Complete Essential 8 medium, filtered through a 70-pm cell strainer, counted, and diluted to a final density of 1000 cells/mL in Complete Essential 8 medium.
  • the cells were then transferred in 200- ⁇ L aliquots to 9 mL StemFit® (Amsbio; Abington, United Kingdom) with 1 mL CloneRTM supplement (StemCell Technologies; Vancouver, Canada), plated at 100 ⁇ L/well, and rested for 24 hours.
  • Hematopoietic Progenitor Cell Differentiation iPSC cells thawed from cryopreserved cell banks were grown on plates coated with Vitronectin in E8 medium supplemented with Hl 152 Rho Kinase Inhibitor. The iPSC cells were passaged twice through dissociation with TrypLETM (Thermo Fisher), and re-seeding on to Vitronectin plates with E8 media + Hl 152. After two passages through dissociation TrypLE treatment, the iPSC cells were once more treated with TrypLE and the cells were resuspended in an optimized concentration in HDM-I media plus Hl 152.
  • TrypLETM Thermo Fisher
  • HDM media contains IMDM medium, Ham’s F12 medium, CTS B27 minus Vitamin A supplement, Non Essential Amino Acids, Ascorbic Acid Mg 2-phosphate, Monothioglycerol, and Heparin.
  • HDM-I media contains HDM + CHIR99021 GSK3 inhibitor, FGF2, and VEGF. The resuspended cells were then seeded into the appropriate vessels depending on scale. The next day (DI), 80% of the medium was replaced with Fresh HDM-I medium. At days 2, 3, and 4, 80% of the medium was removed and replaced with HDM-II medium (HDM media + BMP4, FGF2, and VEGF). At day 5, HPCs may start to appear in the cultures, budding off of the cell aggregates.
  • HPCs were harvested 2 days later, but no earlier than day 8. Starting at day 5; every day 80% of the medium was removed, and any HPCs in the removed media are collected by centrifugation and the cells resuspended in HDM-III (HDM +BMP4, SCF, TPO, FLT3L, and IL3) and added back to the culture. HPCs were then harvested at day 8 or day 9 (depending on day of initial appearance of the HPCs) Natural Killer INK) Cell and T Cell Differentiation and Activation
  • the HPCs were differentiated to generate NK or T cells.
  • Cells were thawed, washed, and seeded into retronectin/DLL4-coated G-Rex bioreactors. Notch signaling, specific cytokines, and growth factors were used for differentiation into lymphoid lineage and subsequent NK or T cell maturation and activation.
  • the culture was concentrated and washed, formulated using a defined cryopreservation medium, and filled into AT vials using an Ml filling station. Vials were visually inspected, cryopreserved in a controlled rate freezer, and stored in the vapor phase of a LN2 freezer.
  • NK and T cells can also be differentiated using feeder cells. Briefly, K562 myelogenous leukemia cells engineered to express class I molecules, CD64, 4-1BBL and transmembrane are cultured with the HCPs for a sufficient time to promote differentiation of NK or T cells.
  • CAR iNK or CAR/IL-15 iNK cells were cultured in media alone or co-cultured with K562 myelogenous leukemia cells (ATCC) at a 1 : 1 effector to target ratio.
  • Supernatants were collected after incubating for 24, 28,72 or 96 hours and assayed for IL- 15 concentration using an MSD immunoassay (Cat #K151URK-4) according to manufacturer’s protocol (Meso Scale Diagnostic; Rockville, MD).
  • MSD immunoassay Cat #K151URK-4
  • iNK cells engineered to express the IL- 15 transgene demonstrated superior IL- 15 release into the culture media (FIG. 3 A).
  • CAR iNK or CAR/IL- 15 iNK cells (10E6 cells) were injected intravenously into immunodeficient NSGTM mice (The Jackson Laboratory; Bar Harbor, ME) on Day 0. On Day 20 post-injection, blood and lungs were analyzed for the presence of human CD45 + CD56 + cells (infused iNK cells) using Fluorescence-activated Cell Sorting (FACS). Only when infused cells carried the human IL-15 transgene were they detectable after 20 days (FIG. 3B).
  • mice were intravenously infused with 1x10 7 CAG-CAR or CAG-CAR/IL-15 cells on study Day 1.
  • Half of the mice were supplemented with exogenous recombinant human IL-15 (1 pg/mouse daily, intraperitoneal) for the duration of the study.
  • lungs were harvested and processed for flow cytometry analysis. Samples were stained with Fixable LiveDead NearIR (Thermo fisher), anti- huCD45, anti-huCD56.
  • iNK cells were defined as CD56/CD45 double positive cells and recorded as a percentage of the live cell population (FIG. 3C).
  • CAR/IL-15 iNK cells were cultured at a 1 : 1 effector to target ratio (E:T) with Irradiated Reh cells (2Gy) for 3-4 days. Results showed that CAR/IL-15-iNK cells perform serial killing for seven rounds before exhausting (FIG. 4A). At the end of the bulk culture no Reh target cells were detectable.
  • CAR/IL-15 iNK cells were counted on the ViCell Blue to track expansion and allow for setting up subsequent bulk culture and Incucyte assays. Incucyte based killing assays were set up in parallel to each bulk culture, using the cells harvested from the prior bulk culture as the effector population with multiple E:Ts 5: 1, 1 : 1, 1 :5.
  • the CAR/IL-15-iNK cells were compared to CAR-iNK cells not expressing IL-15.
  • the CAR/IL-15-iNK cells showed superior proliferation compared to CAR-iNK cells (FIG. 4B).
  • the CAR/IL-15-iNK cells also showed superior serial killing of Raji cells compared to CAR-iNK cells (FIG. 4C).
  • Interleukin- 12 is a cytokine that stimulates the production of interferon-gamma (IFN- ⁇ ) and tumor necrosis factor-alpha (TNF- ⁇ ) from T cells and natural killer (NK) cells.
  • IFN- ⁇ interferon-gamma
  • TNF- ⁇ tumor necrosis factor-alpha
  • iNK cells were differentiated in the standard protocol (no IL- 12) or with inclusion of 10 ng/ml recombinant human IL-12p70 (PeproTech; Rocky Hill, NJ) for the final 24 hours of culture.
  • iNK cells were used in an Incucyte killing assay to determine efficacy for killing the Raji CD 19+ B cell leukemia cell line (ATCC; Manassas, VA). Cells that were primed with IL-12 demonstrated more rapid killing of Raji cells compared to those that were differentiated in the absence of IL-12 (FIG. 5A).
  • IL-12 primed iNK cells were further tested for effects on tumorigenesis in vivo.
  • Luciferase-labeled Burkitt’s lymphoma cell line Raji was intravenously (iv) implanted in female NSGTM mice on study Day 0.
  • Mice were intravenously infused with 1x10 7 unprimed or IL12-primed CAG-CAR-IL15 iNK cells on study days 1, 4, and 7.
  • Mice were supplemented with intraperitoneal recombinant human IL-2 (100,000 IU, PeproTech #200-02) three times weekly for the duration of the study, beginning on day 1. An untreated group served as the control.
  • CAR/IL15 iNK cells were engineered to express tEGFR as an elimination feature, intended to operate as the target of antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) through dosing with Cetuximab, an EGFR inhibitor.
  • iNK cells with or without EGFR expressed from a transgene were co-cultured with human PBMCs. Increasing doses of Cetuximab were added to facilitate ADCC and cells were incubated for 3 hours. Control cells were treated with human IgGl. Results are shown in FIGs. 6A-6B. Only EGFR-expressing iNK cells showed dose-dependent cell death (Annexin V staining) in the ADCC assay. This data demonstrates that the CAR/IL15/tEGFR iNK cells can be efficiently eliminated using Cetuximab.
  • Example 6 MHC Class I and Class II Deletion
  • the plasmid constructs described in the present application are designed to target integration of exogenous polypeptides useful for the invention of the application and simultaneously delete or reduce expression of MHC class I and class II genes.
  • Genomic engineering of IPSCs using the B2M and CIITA targeting plasmids is done as described above.
  • After differentiation to an NK cell confirmation of MHC class I and II expression is confirmed using flow cytometry using antibodies specific for HLA I (alpha chain) and HLA II (alpha or beta chain).
  • Non-classical HLA Expression iNK cells of application are engineered to further express non-classical HLA proteins, HLA-E or HLA-G. Expression is confirmed at all stages by flow cytometry using antibodies specific for HLA-E or HLA-G.
  • iNK clone iNK1248-iPSC611 and primary peripheral blood NK (PB-NK) cells from 3 PBMC donors were assessed for the ability to kill K562 cells using the Incucyte live imaging platform.
  • the Incucyte platform allows for real-time quantification of fluorescently labeled target cells, depletion of which serves as a measurement of target lysis.
  • CML cell K562 cell line The chronic myelogenous leukemia (CML) cell K562 cell line was obtained from ATCC. K562 cells were transduced with NucLight Red lentivirus following the standard Sartorius protocol. Transduced cells were selected and cultured in IMDM culture media containing lug/mL of puromycin. Cells were cultured spitting every 2-3 days keeping cell density between le5 cells/mL and le6 cells/mL.
  • CML chronic myelogenous leukemia
  • PBMCs from 3 donors were thawed in a 37C and centrifuged for 3min at 300G. Supernatant was aspirated and cells were resuspended in RPMI + 10%FBS > lOng/mL fl- 15 and rested overnight. NK cells were isolated from rested PBMCs using CD56 MicroBeads, human (Miltenyi, part 130-050-401) according to manufacturers recommended protocol.
  • CAR-iNK clones and PB-NK donors were plated at 100K cells per well in 96 well U-bottom plate (BD falcon 353077). All wash steps were carried out by centrifugation at 300xG for 3min and flicking supernatants into the sink. Cells were washed 2x in PBS and stained with 100ul of a 1 :1000 dilution (in PBS) of LIVE/DEADTM Fixable Near-IR viability dye (thermo Fisher) for 15min at room temperature (RT). Cells were washed 2x in BD FACS stain buffer BSA (BD).
  • TrustainFcX human Fc receptor block
  • BD FACS stain buffer BSA BD
  • a staining cocktail was made by diluting the mAbs for CD16, CD4, CD19, CD45, CD3, CD56, CD14 and 1 : 100 in BD FACS Stain buffer.
  • Cells were stained with 50ul of staining cocktail and incubated for 30min at 4C protected from light.
  • Cells were washed 2x using BD FACS Stain buffer fixed in 100ul of BD Stabilizing fixative. All samples were run with the same voltage on the BD Symphony A3 Lite. Flow cytometry data was analyzed using FlowJo 10.7.2.
  • NKCM assay media is made up of 500 mL of IMDM and 500 mL Ham’s F-12 Nutrient Mix as base media.
  • Base media is supplemented with 2%, CTS B- 27 Supplement, XenoFree, w/o Vitamin A, 1% MEM Non-Essential Amino Acids Solution, 250 ⁇ M Ascorbic acid Mg 2-phosphate, 100 ⁇ M Mono-Thio-Glycerol, 1% GlutaMax and 2 mM Nicotinamide.
  • Results iNK1248-iPSC61 land PB-NK cells showed ability to kill K562 cells at effector to target ratios of 20: 1, 10: 1 and 1 : 1.
  • the Incucyte based assay measured the loss of Nuclight Red K562 cells over time with effector to target ratios of (A) 20: 1, (B) 10: 1, and (C) 1 : 1.
  • Normalized target cell count as a percentage of target cell only count for four iNK1248-iPSC611 and the average of 3 PB-NKs. With respect to purity, the PB-NK cells were between 87% and 96% CD45+ / CD56+ post isolation.
  • iNK1248-iPSC611 was 98.8% CD45+ / CD56+.
  • PB-NK cells were between 20.15% and 0.195% CD3+ and between 19.1% and 0.075% CD3+ / CD56+.
  • iNK1248-iPSC611 was 0.08% Cd3+ and 0.048% CD3+ CD56+ (FIG. 8).
  • CAR-iNK clone iNK1248-iPSC611
  • the Incucyte live cell imaging platform was used to demonstrate the cytolytic activity of iNK1248-iPSC611 at multiple effector to target ratios (E:T) through real-time quantification of fluorescently labeled target cells, depletion of which serves as a measurement of efficacy of effector target cell killing.
  • Isogenic pairs of Reh and NALM6 cell lines, naively expressing CD 19 or CD 19 knock out (KO) were used to demonstrate CAR mediated lysis of CD 19+ target cells.
  • Reh and NALM6 cells were obtained from ATCC.
  • Cell lines were transduced with Incucyte NucLightRed Lentivirus Reagent (EF1 ⁇ Promoter, Puromycin Selection) according to manufacturer’s protocol at MOI of 3, in cell culture media with 8 pg/mL Polybrene.
  • NLR-transduced cells were selected and cell lines were cultured in RPMI with 10% FBS and 1 pg/mL Puromycin.
  • Reh-CD19KO and NALM6-CD19KO were generated using the Lonza CRISPR- Cas-9 system on parental Reh and NALM6 cells (previously NucLight Red transduced) according to manufacturer’s protocol for Amaxa 4-D Nucleofector.
  • Target sequence for custom CD19KO crRNA used was: GCTGTGCTGCAGTGCCTCAA.
  • CD 19+ cells were removed using Human CD 19 Positive Selection Kit II according to manufacturer’s protocol, and CD 19 expression was assayed via flow cytometry. Cell lines were cultured in RPMI- 10% FBS, 1 pg/mL puromycin. Table 5.
  • FIG. 9 shows the results of an Incucyte based assay measuring the loss of Nuclight Red target cells over time with four effector to target ratios. Normalized target cell count in Reh and Reh-CD19KO co-cultured with iNK1248-iPSC611 at E:T ratios of (A) 10: 1, (B) 5: 1, (C) 1 : 1, and (D) 1 :5 as a percentage of target cell only counts.
  • FIG. 9 shows the results of an Incucyte based assay measuring the loss of Nuclight Red target cells over time with four effector to target ratios. Normalized target cell count in Reh and Reh-CD19KO co-cultured with iNK1248-iPSC611 at E:T ratios of (A) 10: 1, (B) 5: 1, (C) 1 : 1, and (D) 1 :5 as a percentage of target cell only counts.
  • FIG. 9 shows the results of an Incucyte based assay measuring the loss of
  • FIG. 10 shows the results of an Incucyte based assay measuring the loss of Nuclight Red target cells over time with four effector to target ratios.
  • the single cell iNK clone iNK1248-iPSC611 was engineered to secrete the NK homeostatic cytokine IL-15.
  • IL-2 10nM-0nM
  • the in-vitro persistence of iNK1248-iPSC611 was compared to that of a bulk non engineered “wild type” (WT) iNK iNK1487-iPSC005 cell and the NK cell leukemia line KHYG-1. Every 3-4 days cells were harvested counted on a ViCell Blue and re-seeded in fresh media. Cumulative fold expansion was calculated using viable cell counts collected from the ViCell Blu.
  • 1.5x10 6 of iNK1248-iPSC611, WT iNK1487-iPSC005 or KHYG-1 immortalized NK cells were added to individual wells of a 24 well plate at 0.5e6/mL in a total of 3 mL of NKCM containing six different concentrations of IL2.
  • 1.5x10 6 KHYG-1 immortalized NK cells were also added to individual wells of a 24 well plate at 0.5e6/mL in a total of 3 mL of RPMI + 10% HI FBS + IX Pen Strep containing six different concentrations of IL2. Both plates were transferred to an incubator set at 37 °C with 5% CO2. Every 72 or 96 hours, cells were harvested and transferred into individual 15 mL conical tubes.
  • the cells were centrifuged again at 300g for 10 minutes. Supernatants were aspirated and cells were resuspended in NKCM assay media or RPMI assay media containing the corresponding concentration of IL2 at 0.5e6 cells/mL. Cells were replated at 0.5e6 cells/mL in 3 mL per well. If cells were resuspended at 0.5e6 cells/mL in a volume less than 3 mL, the total volume was plated. If resuspension volume fell below 200 uL, the cell line was not re-plated. At the end of 14 days, the assay was terminated and the cells were discarded.
  • Cells were cultured in basal NKCM for 14 days at 37 °C with 5% CO2. Every 3-4 days, all conditions were harvested, counted on the ViCell Blu, resuspended at 0.5e6/mL in appropriate media and then replated. After 21 days, cumulative fold change was calculated.
  • the single cell clone iNK1248-IPSC611 persisted in-vitro longer than the WT iNK1487-iPSC005 in the absence of exogenous IL-2 indicating that the IL- 15 transgene is functional and exhibits the intended mode of action, namely enhanced persistence.
  • the IL-15 released by iNK1248-IPSC611 is adequate to support homeostatic survival of the cells but not sufficient to cause mitogenic expansion.
  • Cells were cultured in NKCM containing one of six IL2 concentrations: lOnM (FIG. 12A), 3nM (FIG. 12B), InM (FIG. 12C), 0.3nM (FIG. 12D), 0.1nM (FIG. 12E), 0nM (FIG. 12F) for 21 days at 37 °C with 5% CO2.
  • Exogenous IL-2 support increased the persistence of both iNK1248- iPSC611 and WT iNK1487-iPSC005 indicating that additional homeostatic cytokine is required to enable limited mitogenic expansion of iNK1248-IPSC611.
  • ADCC Antibody-dependent cellular cytotoxicity
  • NK natural killer
  • neutrophils neutrophils
  • macrophages macrophages
  • eosinophils recognition of bound immunoglobulin via their Fc receptors, particularly CD 16 (Fc ⁇ RIII).
  • Cetuximab is a chimeric mouse-human antibody targeted against the extracellular domain of epidermal growth factor receptor (EGFR). It has been demonstrated to mediate ADCC against EGFR-expressing tumor cell lines via its human IgGl Fc region (Kurai, 2007)
  • iPSC-derived NK (iNK) development candidate 611 e.g., therapeutic iNK
  • iNK iPSC-derived NK
  • Cetuximab compared with an isotype control antibody when cultured with interleukin (IL)-2 activated peripheral blood mononuclear cells (PBMC).
  • IL interleukin
  • PBMC Primary Effector Cell Isolation & Culture
  • PBMC Peripheral mononuclear blood cells
  • ADCC Assays iNK cells were labeled with 2.5 mM CTV (Life Technologies) and 2.5x10 4 cells plated/well as targets in a 96 well flat bottom plate (Coming) in triplicate. Cetuximab (Selleckchem) or a human IgGl isotype control (Invivogen) were pre-incubated with therapeutic iNK targets at concentrations of 10 pg/mL-10 mg/mL for 30’ prior to addition of effector cells.
  • IL-2 activated effector PBMCs were added at an effectortarget (E:T) ratio of 25: 1 in triplicate wells/condition and cultures incubated for 16 hours in a 5% CO2, 37% C° incubator.
  • Dead cells were identified by flow cytometry using LIVE/DEADTM Fixable Near-IR Dead Cell Stain (ThermoFisher) according to manufacturer’s protocol. Samples were acquired on a Symphony A3 (BD Biosciences) and analyzed on FlowJo version 10.7.1 software.
  • PE molecules can be converted per cell to antibodies per cell.
  • Quantibrite beads were gated on by FSC-A vs SSC-A. Subsequently the PE fluorescence was visualized as a histogram and gates were drawn for each of the 4 distinct peaks. Geometric mean fluorescence was exported for each PE peak and used for ABC calculations.
  • Lymphocytes were gated on based on forward scatter area (FSC-A) and side scatter area (SSC-A). Singlets were excluded based on forward scatter area (FSC-A) vs forward scatter height (FSC-H) gate. Gates were drawn on CTV + therapeutic iNK targets or CTV effector cells, and a subsequent gate drawn on CTV + therapeutic iNK cells that labeled positive for LIVE/DEAD Fixable Near-IR. As shown in FIG. 13, cells were gated on lymphocytes, followed by exclusion of doublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finally on LIVE/DEADTM Near-IR+ to determine % of dead therapeutic iNK targets.
  • FSC-A forward scatter area
  • SSC-A side scatter area
  • FSC-H forward scatter height
  • CTV CellTrace Violet
  • NIR Near-IR.
  • Percent specific cell lysis for ADCC assays was calculated as in (Kim, 2007)Error! Reference source not found, using the following equation where LIVE/DEAD NIR + CTV + targets are considered dead iNK and the percent of spontaneous iNK cell death is determined by iNK cells cultured without addition of effector cells (0: 1 E:T):
  • FIG. 14 shows EGFR PE levels on therapeutic iNK stained with EGFR (black histogram) compared with unstained therapeutic iNK (gray histogram) or an unedited WT iNK (dashed line). EGFR expression was observed on therapeutic iNK cells by flow cytometry with values of 7,341 ABC. This level of EGFR was sufficient to observe ADCC activity mediated by Cetuximab with an EC50 of 2.0 ng/mL in co-cultures of iNK with IL-2 activated PBMC.
  • FIG. 15 shows the percent specific cell lysis of therapeutic iNK cells mediated by Cetuximab (black triangles) compared with human IgGl isotype control (open triangles).
  • IL-2 activated PBMC were co-cultured with therapeutic iNK at a 25: 1 E:T ratio for 16 hours and percent specific cell death of iNK determined. Each data point is a mean of triplicate wells, error bars ⁇ standard deviation. Addition of human IgGl isotype control did not mediate ADCC of therapeutic iNK targets, although some background killing was observed at the highest concentration of antibody.
  • Example 12 Antibody and Complement Evasion Using B2M Knockout Allogeneic cell therapy products derived from induced pluripotent stem cells (iPSC) have the potential to be used as an off-the-shelf treatment for many diseases but may generate a vigorous immune response by the host due to incompatibilities in human leukocyte antigen (HLA) genes.
  • HLA human leukocyte antigen
  • some patients may have pre-existing antibodies (Ab) to these polymorphic proteins (1, 2). If Abs to HLA Class I molecules do exist, there is the potential for complement-mediated cytotoxicity (CDC) of the effector cells.
  • a strategy to eliminate binding by Abs to HLA Class I molecules is by deletion of beta-2 microglobulin (b2M), which encodes a subunit common to HLA Class I protein and is required for cell surface expression.
  • b2M beta-2 microglobulin
  • the CDC assay is a simple method to measure how well an Ab induces the killing of cells in the presence of complement proteins (3).
  • Plasma, as well as serum contains the full spectrum of complement proteins, which is referred to as the complement cascade.
  • these molecules are labile and as such, collected serum samples must be quickly frozen before use in CDC assays.
  • rabbit complement can be used as a reagent in assays to substitute for human complement.
  • iNK cells were tested to demonstrate the sensitivity of wild-type (WT) HLA Class I expressing iNK cells and protection of B2M knock out (KO) Clone 611 iNK cells from CDC.
  • iNK cells WT 005 and Clone 611 were diluted to 4x10e6/mL in RPMI-1640 basal media.
  • iNK cells were seeded at 200K cells/well in a polypropylene, U-well, 96- well plate (50 uL/well). Samples were seeded in triplicate. Abs were diluted in RPMI- 1640 at 40 ug/mL and dispensed at 50 uL/well (10 ug/mL final). Baby rabbit complement (BRC) was thawed just prior to use, then diluted 1 :5 in RPMI-1640 and dispensed at 100 uL/well (10% BRC final).
  • BRC Baby rabbit complement
  • Elimination of B2M from iNK cells protects from complement-mediated cytotoxicity in the presence of Abs to HLA-ABC molecules and complement.
  • both freshly thawed WT 005 and Clone 611 iNK cells were found to maintain high viability when cultured for 1 hr in RPMI-1640 alone or with the isotype Ab plus BRC.
  • only the WT 005 iNK cells were killed in the presence of the HLA- ABC Ab plus BRC with no effect on clone 611 iNK cells.
  • Clone 611 iNK cells were still sensitive to complement-mediated killing, we included an Ab to CD52. Addition of the anti-CD52 Ab plus BRC resulted in the killing of both iNK cells.
  • Example 13 Comparison of CTL Activation and iNK Cell Lysis Between ⁇ 2M- Deficient, iPSC-Derived NK Cells and ⁇ 2M-Expressing, Wild-Type iNK Cells
  • Allogeneic cell therapy products derived from induced pluripotent stem cells have the potential to be used as an off-the-shelf treatment for many diseases, but may generate a vigorous immune response by the host due to incompatibilities in human leukocyte antigen (HLA) genes (Lanza, et al. Nat Rev Immunol. 2019 Dec;19(12):723- 733).
  • HLA human leukocyte antigen
  • direct lysis of mismatched class I HLA-bearing cells occurs via activation of host CD8+ T cells that interact with the class I HLA molecules (Felix, et al. Nat Rev Immunol. 2007 Dec;7(12):942-53).
  • Activation of host CD8 T cells is thwarted by deletion of beta-2 microglobulin ( ⁇ 2M), which encodes a subunit common to all class I HLA genes and is required for their surface expression (Krangel, et al. Cell. 1979 Dec;18(4):979-91; and Zijlstra, et al. Nature. 1989 Nov 23;342(6248):435-8).
  • ⁇ 2M beta-2 microglobulin
  • iPSC-derived NK which are genetically edited to be ⁇ 2M-deficient (KO) are cultured with CD8+ cytotoxic lymphocytes (CTL) derived from peripheral blood mononuclear cells (PBMC) from multiple donors to determine whether they induce CTL activation and lysis of iNK cells compared with wild-type iNK which express ⁇ 2M.
  • CTL cytotoxic lymphocytes
  • CTL Effector Cytotoxic Lymphocytes
  • PBMC peripheral mononuclear blood cells
  • PBMC peripheral mononuclear blood cells
  • CTLs with specific reactivity to the parental iPSC line were generated. Briefly, T cells were isolated from 5x10 7 PBMC with Human T cell Isolation kit (StemCell Technologies) according to manufacturer’s instructions and primed three times by co-culture with parental iPSC-derived iNK cells in media with IL2, followed by another round of T cell isolation, and then expanded with Immunocult anti- CD2/CD3/CD28 stimulation reagent (StemCell Technologies) in media with IL2, IL7 and IL15. Expanded cells were cryopreserved in CS-10 (StemCell Technologies) buffer at 107 cells/ml.
  • iNK cells were labeled with 5 ⁇ M CTV (Life Technologies) according to manufacturer’s instructions and 5x10 4 cells plated/well as targets in a 96-well U-bottom plate (Falcon) in duplicate. Cryopreserved CTLs were thawed and added at an effectortarget (E:T) ratio of 5: 1 in triplicate wells/condition and cultures incubated for 48 hours in a 5% CO2, 37% Co incubator.
  • E:T effectortarget
  • Lymphocytes and quantitation beads were gated based on forward scatter height (FSC-H) and side scatter area (SSC-A). Singlets were excluded based on forward scatter area (FSC-A) vs forward scatter height (FSC-H) gate. Live cells were gated as negative for LIVE/DEAD NIR staining. Based on CTV and TCR ⁇ , T cells (TCR ⁇ positive, CTV negative) and iNK cells (CTV positive and TCR ⁇ negative) were gated. Within the T cell gate, CD8 positive and CD4 negative cells were selected. Within the CD8+ T cell population, expression of CD25 was assessed, the CD25-positive gate determined to capture minimal positive background events among T cells cultured alone without targets (FIG. 17). As shown in FIG.
  • the live iNK number for each well was normalized by dividing the acquired CTV+ gate event count by the event count from the quantitation bead gate. Average of duplicate wells for each donor condition was used for calculated values. Specific lysis of iNK cells by CTL was determined by the following calculation:
  • iNKc is the normalized CTV+ event count in the given iNK:CTL co- culture condition
  • iNK a is the normalized CTV+ event count in the corresponding control iNK alone condition.
  • FIG. 19A 64-84% of CTL were activated by the parental wild-type iNK, while 1-3% were activated by ⁇ 2MKO iNK, and 0.5-5% activated without target cells present (FIG. 19B).
  • Allogeneic cell therapy products derived from induced pluripotent stem cells have the potential to be used as an off-the-shelf treatment for many diseases, but may generate a vigorous immune response by the host due to incompatibilities in human leukocyte antigen (HLA) genes.
  • a strategy to eliminate activation of host CD8 T cells is by deletion of beta-2 microglobulin ( ⁇ 2M), which encodes a subunit common to class I major histocompatibility complex (MHC) and is required for surface expression of MHC class I (Krangel, et al. Cell. 1979 Dec;18(4):979-91; and Zijlstra, et al. Nature. 1989 Nov 23;342(6248):435-8).
  • ⁇ 2M beta-2 microglobulin
  • HLA-E is a minimally polymorphic ligand which presents peptides derived from signal sequences of other HLA class I molecules and binds the inhibitory NK receptor complex CD94/NKG2A (Braud, et al. Nature 349, 329-331 (1991); Miller, et al. J Immunol. 2003 Aug 1; 171(3): 1369-75).
  • iPSC-derived NK which are edited to be ⁇ 2M -/- but express HLA-E (e.g., therapeutic iNK) are cultured with peripheral blood mononuclear cells (PBMC) to determine whether they are less susceptible to killing by PBMC compared with iNK which lack ⁇ 2M and do not express HLA-E.
  • PBMC peripheral blood mononuclear cells
  • PBMC Peripheral mononuclear blood cells
  • iNK cells were labeled with 2.5 ⁇ M CTV (Life Technologies) according to manufacturer’s instructions and 2.5x10 4 cells plated/well as targets in a 96 well flat bottom plate (Coming) in triplicate.
  • Cryopreserved PBMCs were thawed and added at an effectortarget (E:T) ratio of 25: 1 in triplicate wells/condition and cultures incubated for 72 hours in a 5% CO2, 37% Co incubator.
  • Dead cells were identified by flow cytometry using LIVE/DEADTM Fixable Near-IR Dead Cell Stain (ThermoFisher) according to manufacturer’s protocol. Samples were acquired on a Symphony A3 (BD Biosciences) and analyzed on FlowJo version 10.7.1 software.
  • iNK cells were labeled with mouse IgGl PE isotype control (BioLegend) or HLA-E PE (BioLegend) for 15’ at RT in the dark, washed with Cell Staining Buffer (BioLegend), and fixed for 10’ at RT in the dark with Fixation Buffer (BioLegend).
  • a single tube of BD Quantibrite beads (BD Biosciences) was reconstituted with 500 mL PBS per manufacturer’s protocol. Labeled iNK cells and a BD Quantibrite PE tube were acquired on a Symphony A3 (BD Biosciences) using the same voltages and settings, and all samples were analyzed on FlowJo version 10.7.1 software.
  • PE molecules can be converted per cell to antibodies per cell.
  • Quantibrite beads were gated on by FSC- A vs SSC-A. Subsequently the PE fluorescence was visualized as a histogram and gates were drawn for each of the 4 distinct peaks. Geometric mean fluorescence was exported for each PE peak and used for ABC calculations.
  • NK cell phenotyping cells were transferred to a 96 well round bottom plate (Falcon), washed in lx PBS pH 7.2 (Life Technologies) and resuspended in PBS containing LIVE/DEADTM Fixable Near-IR Dead Cell Stain (ThermoFisher) according to manufacturer’s protocol. Non-specific binding to Fc receptors (FcR) was blocked using Human TruStain FcX Fc receptor blocking solution (BioLegend) prior to addition of antibodies. Cells were incubated with antibodies against CD3, CD56, and CD16 for 20’ at RT and washed three times with Cell Staining Buffer (BioLegend) before fixation with Fixation Buffer (BioLegend). Samples were collected on a Symphony A3 (BD Biosciences) and all FCS files analyzed on FlowJo version 10.7.1 software.
  • Lymphocytes were gated on based on forward scatter area (FSC-A) and side scatter area (SSC-A). Singlets were excluded based on forward scatter area (FSC-A) vs forward scatter height (FSC-H) gate. Gates were drawn on CTV + iNK targets or CTV effector cells, and a subsequent gate drawn on CTV + iNK cells that labeled positive for LIVE/DEAD Fixable Near-IR (FIG. 20). Cells were gated on lymphocytes, followed by exclusion of doublets, followed by gating on CellTrace Violet (CTV)+ iNK, and finally on LIVE/DEADTM Near-IR+ to determine % of dead iNK targets.
  • FSC-A forward scatter area
  • SSC-A side scatter area
  • FSC-H forward scatter height
  • CTV CellTrace Violet
  • NIR Near-IR.
  • Cell death for allo-evasion assays was calculated by determining the mean percent of LIVE/DEAD NIR + CTV + targets (dead iNK) for each iNK group and dividing by the mean percent of LIVE/DEAD NIR+CTV+ WT iNK targets. Results are presented as “Cell death relative to WT iNK”.
  • HLA-E expression was measured on edited iNK cells from line 004 by flow cytometry with a value of 3.625 ABC. Expression of HLA-E on therapeutic iNK cells was sufficient to observe a reduction in cell death when cultured with PBMC compared with HLA-E negative, ⁇ 2M KO iNK.
  • HLA-E binds the heterodimer CD94/NKG2A, an inhibitory receptor which is expressed on NK cells. Because CD94 can also pair with NKG2C to form an activating receptor, it was not assessed here.
  • Frequency of NKG2A-expressing NK cells within a PBMC milieu was measured on two donors. Cryopreserved PBMC were thawed and stained for cells expressing NK cell markers (CD3'CD56 + CD16 +/ ‘) and frequencies of NKG2A-expressing NK cells assessed. In donor 1, 63.7% of NK cells expressed NKG2A while donor 2 contained 44.1% NKG2A + NK cells. (FIG. 22). PBMC samples were gated on viable lymphocytes, followed by a gate on CD3-CD56+ cells (“NK cells”). Frequencies of NKG2A-expressing NK cells were then determined based on an FMO.
  • iNK cells lacking surface HLA (b2M KO, white bars) exhibited an approximate 2.25 and 1.5-fold increase in cell death relative to WT (black bars) in PBMC co-cultures with donors 1 and 2, respectively.
  • Therapeutic iNK cells which express HLA-E, reduced cell death to the level of WT iNK (gray bars) (FIG. 23 and Table 7).
  • Freshly thawed PBMC were co- cultured with therapeutic iNK at a 25: 1 E:T ratio in the presence of 10 ng/mL IL- 15 for 72 hours and cell death of edited iNK relative to WT determined as described in methods. Each data point is a mean of triplicate wells.
  • Example 15 In Vivo Evaluation of Anti-tumor Efficacy of iNK Cells The purpose of this study is to evaluate the in vivo anti-tumor efficacy of cryopreserved iPSC611 CD19iNK cells. A secondary purpose of this study is to evaluate single-dose 7-day persistence of cryopreserved iPSC611 CD19iNK.
  • mice femalae NSG (NOD.Cg-Prkdc scid II2rg tmlWjl SzJ) mice (Jackson
  • mice were 7-9 weeks of age, and initial body weight was an average of 23 grams. Animals were acclimated for one week prior to any experimental procedures being performed.
  • NALM6-Fluc-Puro (ALL) tumor cells (Imanis Life Sciences, CL151) were maintained in RPMI 1640 medium with 10 mM HEPES, 2.5 pg/mL Puromycin, and 10% (v/v) HI FBS. Each mouse received 1x10 5 NALM6-Fluc-Puro cells in serum-free RPMI 1640 medium in a total volume of 0.2 mL.
  • the tumor cell implant day was designated as Study Day 0.
  • mice On Days 1, 8, and 15 following i.v. NALM6-Fluc-Puro tumor cell implantation, mice were intravenously injected with 10x10 6 or 15x10 6 cryopreserved iPSC611 therapeutic iNK cells thawed and resuspended in Lactated Ringer’ s/5% Human Serum Albumin (Groups 2, 3), in a volume of 0.2 mL. Group 1 remained as an untreated control (Table 8, Efficacy Study Design). Table 8. Efficacy Study Design
  • mice received intraperitoneal recombinant human IL-2 (PeproTech® 200-02) on Days 1, 2, 4, 7, 8, 10, 12, 15, 17, 19, 21, 23, 25, and 28, at a dose of 100,000 international units (IU) per mouse in 0.2 mL. Briefly, lyophilized rhIL-2 (1 mg) is centrifuged at 2000 g for 1 minute, resuspended and solubilized in 1 mL 100 mM acetic acid, then mixed with 4 mL 0.1% BSA in PBS. 1 mL aliquots are frozen at -80°C until use, at which point the aliquot is thawed at ambient temperature and mixed with 3 mL PBS for a final concentration of 500,000 lU/mL.
  • PeproTech® 200-02 All mice received intraperitoneal recombinant human IL-2 (PeproTech® 200-02) on Days 1, 2, 4, 7, 8, 10, 12, 15, 17, 19, 21, 23, 25, and 28, at a dose of 100,000 international units (IU) per mouse in
  • mice were injected i.p. with 150 mg/kg D-Luciferin (VivoGloTM Luciferin, PromegaTM), anesthetized via 2.5-3.5% vaporized isoflurane in oxygen, and imaged on automatic exposure ventrally and dorsally 20 minutes post- luciferin injection.
  • Total whole-body bioluminescence is calculated by adding the average radiance of ventral and dorsal images.
  • mice were provided with HydroGel® ad libitum on the day of treatment (Days 1, 8, and 15).
  • mice were intravenously implanted with NALM6-Fluc-Puro cells as described previously, on Day 0.
  • Group 1 remained as an untreated control Table 9, Persistence Study Design). All animals received recombinant human IL-2, dosed as described previously, on Days 1, 3, 5, and 7.
  • mice on study plus one naive age-matched mouse were humanely euthanized and sampled.
  • Whole blood was collected via cardiac puncture into lithium heparin-coated tubes (BD 365965).
  • Lungs were flushed with PBS through the right ventricle in situ, trimmed, and placed into 2.4 mL lx Buffer S (Miltenyi Biotech GmbH, 130-095-927) on wet ice until processing. Cervical lymph nodes were harvested and placed into 2.4 mL lx Buffer S on wet ice until processing.
  • Blood was processed by transferring to a 96 well 2mL deep well plate containing 1.5mL of PBS. The plate was centrifuged for 5 min at 300g and supernatant was decanted. The cell pellets were resuspended in 750 ⁇ L of ACK lysis solution and incubated at room temp for 5 min, at which point 750 ⁇ L of PBS was added to each well. The plate was centrifuged for centrifuged for 5 min at 300g and supernatant was decanted. ACK lysis was repeated 2X as described above. After completion of ACK lysis the resulting Cell pellets were resuspended in 150pl of PBS and transferred to a 96well U bottom plate for FACS staining and analysis.
  • Tissues were processed using the Miltenyi Biotech GmbH Lung Dissociation Kit. Briefly, lx Buffer S was prepared by mixing 1 mL 20x Buffer S with 19 mL sterile water. Enzyme D was reconstituted with 3 mL lx Buffer S, using gentle inversion every minute until solubilized. Enzyme A was reconstituted with 1 mL lx Buffer S, using gentle inversion every minute until solubilized. Tissues were individually collected into lx Buffer S in gentleMACS C Tubes. Immediately prior to processing, 100 ⁇ L of Enzyme D and 15 ⁇ L of Enzyme A were added to each tube.
  • Tubes were placed on the gentleMACS Dissociator on program “m_lung_01.” The tubes were then placed in incubation at 37C on the MACSmix Tube Rotator for 30 minutes, followed by further mechanical dissociation using the gentleMACS Dissociator on program “m_lung_02.” Samples were then filtered through a MACS SmartStrainer (70 pm) placed on a 50 mL tube and washed with 10 mL PBS. The suspension was centrifuged at 300xg for 10 minutes, supernatant aspirated, and cell pellet resuspended in PBS at 10x10 6 cells/mL for plating, staining, and FACS analysis.
  • Cell suspensions from blood, lung and cervical lymph nodes were plated at approximately le6 cells per well in a 96-well U-bottom plate (BD falcon 353077). All wash steps carried out by centrifugation at 300xG for 3min and flicking supernatants into the sink. Cells were washed 2x in PBS and stained with 50pl of a 1 : 1000 dilution (in PBS) of LIVE/DEADTM Fixable Near-IR viability dye (thermo Fisher) for 15min at room temperature (RT). 50pl of Fc Receptor Blocker (Innovex NB309) was added to each well and incubated for 20 minutes at 4°C. Cells were washed 2x in BD FACS stain buffer BSA (BD).
  • BD FACS stain buffer BSA
  • a staining cocktail were made by diluting the mAbs for CD45 and CD56 1 :20 in BD FACS Stain buffer. Cells were stained with 50 ⁇ l of staining cocktail and incubated for 30min at 4C protected from light. Cells were washed 2x using BD FACS Stain buffer fixed in 100 ⁇ l of BD Stabilizing fixative. All samples were run with the same voltage on the BD Symphony A3 Lite collecting all events. Flow cytometry data was analyzed using FlowJo 10.7.2. iNK cells were defined as live singlets that were CD45+ and CD56+ and represented as #iNK cells per 100K live lymphocytes. The lower limit of detection (LLOD) was defined as maximum + 1 standard deviation (SD) of the control group that received no iNK treatment. Samples above the LLOD were plotted in graph pad Prism.
  • SD standard deviation
  • Body weights are graphically represented as percent change in mean group body weight, using the formula: where ‘W’ represents mean body weight of the treated group on a particular day, and ‘Wo’ represents mean body weight of the same treated group at initiation of treatment.
  • TGI Percent tumor growth inhibition
  • results are plotted as the percentage survival against days post-tumor implantation.
  • Adverse clinical signs indicating excessive tumor burden (such as ruffled/ matted fur, hunched posture, inactivity, or hind limb weakness) are used as a surrogate endpoint for death.
  • Median survival is determined utilizing Kaplan Meier survival analysis.
  • Tumor bioluminescent data, body weight, survival, and persistence were graphically represented and statistically analyzed utilizing GraphPad Prism software (Version 9.0.1).
  • Statistical significance for tumor bioluminescence was evaluated using an ordinary two-way analysis of variance (ANOVA) and Tukey multiple comparisons, with a 95% confidence interval. Differences between groups were considered significant when the probability value (p) was ⁇ 0.05.
  • Statistical significance for probability of survival was evaluated using a Mantel-Cox test with a Gehan-Breslow-Wilcoxon test.
  • Statistical significance for persistence was evaluated using an ordinary one-way analysis of variance (ANOVA) and Tukey multiple comparisons, with a 95% confidence interval. Differences between groups were considered significant when the probability value was ⁇ 0.05.
  • Cryogenic iPSC611 cells were well-tolerated as determined by body weight and clinical observations. iPSC611 demonstrated significant anti -tumor efficacy at both dose levels. Enhanced increased life span was observed in mice treated with iPSC611 cells. Cryogenic iPSC611 had limited in vivo persistence one week post-injection.
  • Group mean body weight changes of NALM6-Fluc-Puro tumor-bearing mice treated with iPSC611 cells or tumor alone control are graphically represented in FIG. 24 ( Mean percent body weight change of untreated mice ( ⁇ ), or mice treated intravenously with iPSC611 at 10x10 6 ( ⁇ ) and 15x10 6 ( ⁇ ) (cryogenic) cells). Means are plotted where ⁇ 50% of the treatment group are present. Arrows represent dosing days. No significant body weight loss (>10% loss from the start of treatment) was observed in any group receiving iPSC611 cells or in the tumor alone control.
  • %ILS Percent Increased Life Span
  • iPSC611 FACS analysis of lungs and blood indicated limited persistence of cryogenic iPSC611 (FIG. 27). Mice were left untreated, or received a single intravenous dose of iPSC611 at 15x10 6 cryogenic cells. One-week post-injection, lungs and blood were harvested for FACS analysis. Number of iNK per 100,000 lymphocytes is plotted for individual mice (o), and average per group represented by bars. iNK were detected in lungs and blood of two of the five mice injected with iPSC611, one week post-injection. Example 16. In Vivo Evaluation of Elimination of iNK Cells
  • the purpose of this study is to evaluate the in vivo elimination of cryopreserved iPSC611 CD19iNK cells, using Erbitux (cetuximab).
  • mice Female NSG (NOD.Cg-Prkdc scid II2rg tmlWjl SzJ) mice (Jackson Labs, Bar Harbor, Maine, USA), were used. At study initiation, mice were 10-12 weeks of age, and initial body weight was an average of 24.3 grams. Animals were acclimated for one week prior to any experimental procedures being performed.
  • mice were intravenously injected with 15x10 6 cryopreserved iPSC611 cells thawed and resuspended in Lactated Ringer’ s/5% Human Serum Albumin (Groups 2, 3), in a volume of 0.2 mL. Group 1 remained as an untreated control.
  • mice in Groups 1, 2, and 3 received intraperitoneal recombinant human IL-2 (PeproTech® 200-02) on Days 1 and 3, at a dose of 100,000 international units (IU) per mouse in 0.2 mL. Briefly, lyophilized rhIL-2 (1 mg) is centrifuged at 2000 g for 1 minute, resuspended and solubilized in 1 mL 100 mM acetic acid, then mixed with 4 mL 0.1% BSA in PBS. 1 mL aliquots are frozen at -80°C until use, at which point the aliquot is thawed at ambient temperature and mixed with 3 mL PBS for a final concentration of 500,000 lU/mL. On Days 2 and 3, mice received intraperitoneal antibody therapy. Group 2 was dosed with 20 mL/kg PBS, IP. Group 3 was dosed with 40 mg/kg cetuximab in a volume of 20 mL/kg, IP.
  • mice on study were humanely euthanized and sampled. Blood was collected via cardiac puncture into Lithium Heparin coated tubes (BD Microtainer 365965). Lungs were flushed with PBS through the right ventricle in situ, trimmed, and placed into PBS+2% FBS on wet ice until processing.
  • Lithium Heparin coated tubes BD Microtainer 365965
  • Blood was processed through 2 rounds of ACK lysis following the following protocol. Blood was transferred to a 2 ml deep well plate and tubes rinsed with 1 ml of PBS. Deep well was centrifuged for 3 min at 300xG. The supernatant was removed and 1 mL of ACK added to each well. The plate was incubated for 2 minutes and then 1 mL of PBS was added to stop osmotic lysis. The plate was centrifuged for 3 minutes at 300xG and supernatant was removed. ACK lysis was repeated 1-2 more times as needed. Samples were resuspended in 200 ⁇ L of BD FACS stain buffer and transferred to a 96 well U-bottom plate for staining.
  • Lungs were processed to a single-cell suspension using mechanical dissociation and gentle enzymatic digestion. Briefly, lung tissue was transferred into a dish without medium, and minced into a homogenous paste ( ⁇ 1 mm in size) using a razor blade or scalpel. Minced tissue was transferred to 2 mL digestion medium containing 10% Collagenase/Hyaluronidase, 15% DNase I Solution (1 mg/mL), and 75% RPMI 1640 Medium, and incubated at 37°C for 20 minutes on a shaking platform. The tissue was then passed through a 70 pm nylon mesh strainer over a 50mL conical tube using the rubber end of a syringe plunger to obtain a cell suspension.
  • the suspension was passed through a new 70 pm nylon mesh strainer over a 50 mL conical tube to filter, and rinsed with 10 mL RPMI.
  • the cell suspension was transferred into a 15 mL conical tube and centrifuge at 500xG for 10 minutes at room temperature with the brake on low. Supernatant was removed and discarded.
  • Cells were resuspended in 10 mL of PBS and counted, adjusted to 10x10 6 cells/mL, and underwent one ACK lysis step before plating, staining, and FACS analysis.
  • Cell suspensions from lung were plated at approximately le6 cells per well in a 96-well U-bottom plate (BD falcon 353077). All wash steps carried out by centrifugation at 300xG for 3 minutes and flicking supernatants into the sink. Cells were washed 2x in PBS and stained with 50pl of a 1 :1000 dilution (in PBS) of LIVE/DEADTM Fixable Near- IR viability dye (thermo Fisher) for 15 minutes at room temperature (RT). 50pl of Fc Receptor Blocker (Innovex NB309) was added to each well and incubated for 20 minutes at 4°C. Cells were washed 2x in BD FACS stain buffer BSA (BD).
  • BD FACS stain buffer BSA
  • a staining cocktail were made by diluting the mAbs for CD45 and CD56 1 :20 in BD FACS Stain buffer. Cells were stained with 50pl of staining cocktail and incubated for 30 minutes at 4°C protected from light. Cells were washed 2x using BD FACS Stain buffer fixed in lOOpl of BD Stabilizing fixative. All samples were run with the same voltage on the BD Symphony A3 Lite collecting all events. Flow cytometry data was analyzed using FlowJo 10.7.2. iNK cells were defined as live singlets that were CD45+ and CD56+ and represented as #iNK cells per 100K live lymphocytes. The lower limit of detection (LLOD) was defined as maximum + 1 standard deviation (SD) of the control group that received no iNK treatment. Samples above the LLOD were plotted in graph pad Prism.
  • SD standard deviation
  • Body weights are graphically represented as percent change in mean group body weight, using the formula: where ‘W’ represents mean body weight of the treated group on a particular day, and ‘Wo’ represents mean body weight of the same treated group at initiation of treatment.
  • Body weight and persistence were graphically represented and statistically analyzed utilizing GraphPad Prism software (Version 9.0.1). Statistical significance for elimination was evaluated using an unpaired one-tailed t-test with Welch’ s correction, with a 95% confidence interval. Differences between groups were considered significant when the probability value was ⁇ 0.05.
  • mice were significantly reduced in the lungs and blood of mice that received cetuximab treatment.
  • Group mean body weight changes of mice are graphically represented in FIG. 28 (mean percent body weight change of mice treated intravenously with iPSC611 at 15x10 6 cells receiving IP PBS ( ⁇ ), or cetuximab at 40 mg/kg ( ⁇ )). No significant body weight loss (>10% loss from the start of treatment) was observed in any group receiving iPSC611 cells and antibody.

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WO2025094915A1 (ja) * 2023-10-30 2025-05-08 武田薬品工業株式会社 細胞傷害性ｔ細胞の製造方法
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MX2023006395A (es) 2023-06-15
US20230382999A1 (en) 2023-11-30
US12269888B2 (en) 2025-04-08
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