WO2024254013A1 - Cellules tueuses naturelles pourvues d'un nouveau knock-in d'il-15 et leurs méthodes d'utilisation - Google Patents

Cellules tueuses naturelles pourvues d'un nouveau knock-in d'il-15 et leurs méthodes d'utilisation Download PDF

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WO2024254013A1
WO2024254013A1 PCT/US2024/032309 US2024032309W WO2024254013A1 WO 2024254013 A1 WO2024254013 A1 WO 2024254013A1 US 2024032309 W US2024032309 W US 2024032309W WO 2024254013 A1 WO2024254013 A1 WO 2024254013A1
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functional fragment
cells
cell
construct
ipscs
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Wei Li
David Zou
Anping CHEN
Hao-Ming Chang
Sourindra Maiti
Antonio Arulanandam
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Cytovia Therapeutics LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present disclosure relates generally to populations of Natural Killer (NK) cells with novel IL- 15 knock-in and methods of use thereof.
  • NK Natural Killer
  • Lymphocytes such as natural killer (NK) cells are potent anti-tumor effectors that play an important role in innate and adaptive immunity.
  • NK cells There are several activating receptors found on NK cells, including NKp30, NKp44, and NKp46, which are collectively known as Natural Cytotoxicity Receptors (NCRs), as well as NKG2D, CD16 and TRAIL.
  • NCRs Natural Cytotoxicity Receptors
  • NKp46 is an established marker for the identification of NK cells.
  • NKp46 is an NK cell specific triggering molecule found on both resting and activated NK cells. It is an important mediator in NK cell activation against numerous targets, including tumors and virally infected cells.
  • NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus.
  • NK cells are critical effectors of the early innate immune response toward transformed and virus-infected cells.
  • NK cells constitute about 10% of the lymphocytes in human peripheral blood.
  • NK cells are effector cells known as large granular lymphocytes because of their larger size and the presence of characteristic azurophilic granules in their cytoplasm.
  • NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus.
  • NK cells can be detected by specific surface markers, such as CD56 and CD45 in humans.
  • NK cells do not express T cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.
  • Stimulation of NK cells may be achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors.
  • the activation status of NK cells is regulated by a balance of intracellular signals received from an array of germ-line-encoded activating and inhibitory receptors (MacFarlane and Campbell, Curr Top Microbiol Immunol. 2006; 298:23-57).
  • NK cells encounter an abnormal cell (e.g., tumor or virus-infected cell) and activating signals predominate, the NK cells can rapidly induce apoptosis of the target cell through directed secretion of cytolytic granules containing perforin and granzymes or engagement of death domaincontaining receptors.
  • Activated NK cells can also secrete type I cytokines, such as interferon-'/, tumor necrosis factor-a and granulocyte-macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells as well as other cytokines and chemokines (Wu and Lanier, Adv Cancer Res. 2003; 90:127-56).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Production of these soluble factors by NK cells in early innate immune responses significantly influences the recruitment and function of other hematopoietic cells.
  • NK cells are central players in a regulatory crosstalk network with dendritic cells and neutrophils to promote or restrain immune responses.
  • NK cells have several characteristics which make them advantageous to use in therapeutic settings. For example, they do not require antigen priming and they are HLA agnostic, which decreases the risk of developing of Graft vs. Host Disease in allogeneic use. Nonetheless, the use of immune cells for adoptive cell therapies remains challenging and there are unmet needs for improvement. For example, NK cells are usually short-lived, and the clinical efficacy of NK cells is often compromised by their limited persistence. Therefore, there is a need to screen for optimal version(s) of IL- 15 to be knocked into NK cells to help their physical and functional persistence. There are significant opportunities that remain to harness the full potential of NK cells in adoptive immunotherapy .
  • a cell population comprising induced pluripotent stem cells (iPSCs) or NK cells wherein the iPSCs or NK cells comprise an IL- 15 construct comprising sequences encoding IL- 15 or a functional fragment thereof, wherein the IL- 15 or a functional fragment thereof is trapped in the endoplasmic reticulum.
  • the IL- 15 construct further comprises sequences encoding IL-2RP or a functional fragment thereof.
  • a cell population comprising induced pluripotent stem cells (iPSCs) or NK cells wherein the iPSCs or NK cells comprise an IL- 15 construct comprising sequences encoding (i) IL- 15 or a functional fragment thereof, and (ii) IL-2RP or a functional fragment thereof.
  • the IL- 15 construct encodes a cell membranebound form of IL- 15 (mbIL-15) or a functional fragment thereof.
  • the IL- 15 construct encodes a soluble form of IL- 15 or a functional fragment thereof.
  • the IL- 15 construct encodes an IL- 15 trapped in the endoplasmic reticulum (ER) or a functional fragment thereof.
  • the IL- 15 construct encodes a fusion protein comprising IL- 15 or a functional fragment thereof and IL-2RP or a functional fragment thereof.
  • the IL- 15 or a functional fragment thereof and IL-2RP or a functional fragment thereof are expressed as individual proteins.
  • the IL- 15 can be a membrane-bound IL- 15 or a functional fragment thereof, soluble IL- 15 or a functional fragment thereof, or an ER- trapped IL- 15 or a functional fragment thereof.
  • the above IL- 15 constructs further comprise sequences encoding IL- 15 Receptor alpha (IL-15Ra) or a functional fragment thereof.
  • the IL- 15 construct encodes a fusion protein comprising (i) IL- 15 or a functional fragment thereof, (ii) IL-2RP or a functional fragment thereof, and (iii) IL-15Ra or a functional fragment thereof.
  • the IL- 15 or a functional fragment thereof, IL-2RP or a functional fragment thereof, and IL-15Ra or a functional fragment thereof are expressed as individual proteins.
  • the IL- 15 can be a membrane-bound IL- 15 or a functional fragment thereof, soluble IL- 15 or a functional fragment thereof, or an ER-trapped IL- 15 or a functional fragment thereof.
  • the NK cells are derived from iPSCs.
  • the iPSCs or iNK cells comprise a knock-in of the IL- 15 construct into a B2M gene or a TGFPR2 gene of the iNK cells.
  • the IL- 15 construct is operably linked to a promoter which is silent or minimally active (e.g., ⁇ 10% of its full activity) in iPSC.
  • promoters that are silent or minimally active in iPSC include, but are not limited to, a NK cell-specific promoter.
  • the NK cell-specific promoter can be a NKp46 promoter.
  • the iNK cells further comprise a knockout of a cytokine inducible SH2 containing protein (CISH).
  • the iNK cells further comprise one or more knockouts of one or more NK inhibitory receptors.
  • the NK inhibitory receptor can be T-cell immunoglobulin and ITIM domain (TIGIT) receptor.
  • the iNK cells further comprise both a knockout of a cytokine inducible SH2 containing protein (CISH) and one or more knockouts of one or more NK inhibitory receptors.
  • CISH cytokine inducible SH2 containing protein
  • the NK inhibitory receptor can be T-cell immunoglobulin and ITIM domain (TIGIT) receptor.
  • the IL- 15 construct encodes a cell membrane-bound form of IL- 15 (mbIL-15) or a functional fragment thereof.
  • the IL- 15 construct encodes a soluble form of IL- 15 or a functional fragment thereof. In another embodiment, the IL- 15 construct encodes an IL- 15 trapped in the endoplasmic reticulum or a functional fragment thereof.
  • the IL- 15 construct encodes a fusion protein comprising IL- 15 or a functional fragment thereof and IL-2RP or a functional fragment thereof.
  • the IL- 15 or a functional fragment thereof and IL-2R0 or a functional fragment thereof are expressed as individual proteins.
  • the above IL- 15 constructs further comprise sequences encoding IL- 15 Receptor alpha (IL-15Ru) or a functional fragment thereof.
  • the IL- 15 construct encodes a fusion protein comprising (i) TL-15 or a functional fragment thereof, (ii) IL- 2Rp or a functional fragment thereof, and (iii) IL- 15Ra or a functional fragment thereof.
  • the IL- 15 or a functional fragment thereof, IL-2RP or a functional fragment thereof, and IL- 15 Rot or a functional fragment thereof are expressed as individual proteins.
  • the IL- 15 construct in the above method is operably linked to a promoter which is silent or minimally active (e.g., ⁇ 10% of its full activity) in iPSC.
  • a promoter which is silent or minimally active in iPSC include, but are not limited to, a NK cellspecific promoter.
  • the NK cell- specific promoter can be a NKp46 promoter.
  • the genetic editing of the above method comprises a knock-in of the IL-15 construct into a B2M gene or a TGFPR2 gene of the iPSCs.
  • the genetic editing further comprises a knockout of a cytokine inducible SH2 containing protein (CISH).
  • the genetic editing further comprises one or more knockouts of one or more NK inhibitory receptors.
  • the NK inhibitory receptor can be T-cell immunoglobulin and ITIM domain (TIGIT) receptor.
  • the genetic editing further comprises both a knockout of a cytokine inducible SH2 containing protein (CISH) and one or more knockouts of one or more NK inhibitory receptors.
  • the genetic editing comprises using a TALEN construct or a Cas9 or Casl2 enzyme.
  • the present disclosure includes cell populations disclosed herein. In another embodiment, the present disclosure comprises cell populations produced by the methods disclosed herein.
  • a pharmaceutical composition comprising the cell populations disclosed herein, or the cell populations produced by the methods disclosed herein.
  • a method of treating cancer or autoimmune diseases in a subject in need thereof comprising administering to the subject an effective amount of the pharmaceutical composition disclosed herein.
  • the cancer is a solid tumor or a hematological cancer.
  • the subject is further administered with one or more immune checkpoint inhibitors.
  • the immune checkpoint inhibitors can be inhibitors of one or more NK cell inhibitory receptors, for example, antibodies against the NK cell inhibitory receptors.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-CTLA-4 antibody, or an anti-TIGIT antibody.
  • Figure 1 shows a map of donor 107 plasmid.
  • Figure 2 shows a map of donor 107ER plasmid.
  • Figure 3 shows a map of donor 108 plasmid.
  • Figure 4 shows a map of donor 108B2M plasmid.
  • Figure 5 shows a map of donor 115 plasmid.
  • Figure 6 shows a map of a vector 1 encoding a membrane-bound IL- 15.
  • Figure 7 shows a map of a vector 2 encoding a membrane-bound IL- 15 and IL-15Ra.
  • Figure 8 shows a map of a vector 3 encoding a membrane-bound IL- 15, IL-15Ra, IL-15Rb, and a full cytoplasmic portion of the common cytokine receptor y chain.
  • Figure 9 shows a map of a vector 4 encoding a membrane-bound IL- 15, IL-15Ra, IL-15Rb with a portion of the cytoplasmic region deleted, and a full cytoplasmic portion of the common cytokine receptor y chain.
  • Figure 10 shows a map of a vector 5 encoding an ER-trapped IL- 15.
  • Figure 11 shows a map of a vector 6 encoding an IL-15 and IL-15Ra that are both trapped in the ER.
  • Figure 12 shows a map of a vector 7 encoding IL-15Ra, IL-15Rb, and a soluble IL- 15.
  • Figure 13 shows a map of a vector 8 encoding IL-15Ra and a soluble IL- 15.
  • Figure 14 shows a map of a vector 9 encoding an ER- trapped fusion protein comprising IL-15, IL-15Ra, IL-15Rb, and a full cytoplasmic portion of the common cytokine receptor y chain.
  • Figure 15 shows the IL-15-cnginccrcdiNK cells showed proliferative advantage compared to control iNK cells during ex vivo expansion. iNK cells were transduced at the beginning of the stage 3 week 2, and expanded with K562 feeders in 1:5 ratio for a total of 20 days.
  • iNK cells were harvested and measured for the viable cell number (tdT: tdTomato-transduced; control: no transduction).
  • 2-6 are iNK cells incorporating vector 2; 3-2 arc iNK cells incorporating vector 3; 4-8 arc iNK cells incorporating vector 4; 5-8 are iNK cells incorporating vector 5; 7 are iNK cells incorporating vector 7.
  • Figure 16 shows the IL-15-engineered iNK cells showed advantage in survivability in cytokine-starved condition. Following 20 days of ex vivo expansion, the IL-15-engineered iNK cells were grown in cytokine-free medium for 6 days and the viable cell number was monitored at day 0, day 2, and day 6.
  • Figure 17 shows the IL- 15 -engineered, cytokine-starved iNK cells maintain pSTAT5 activation in response to IL2 and Ki67 proliferation marker.
  • the IL- 15 -engineered iNK cells were grown in cytokine-free medium for 6 days, or in cytokine-free medium for 6 days and then pulse-treated with 100 lU/mL of IL2 for 30 minutes.
  • the % positive population of phospho-STAT5 and proliferation marker Ki-67 were analyzed at day 0 and day 6.
  • Figure 18 shows the IL-15-engineered iNK cells showed persistence in peripheral blood in vivo.
  • the IL-15-engineered iNK cells were injected into mice. The mice looked healthy post injection, and peripheral blood was withdrawn from the mice at hour 3, day 3, day 7, day 10, day 14, and day 17.
  • the persistence of the IL- 15 -engineered iNK cells was examined by flow cytometry on huCD45+ cell population. Compared to the control iNK cells, the IL-15-engineered iNK cells exhibited better survivability in vivo during the time course.
  • Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See c.g., Sambrook ct al. Molecular Cloning: A Laboratory Manual (2d cd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).
  • the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art.
  • Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • the materials, methods, and examples disclosed herein are for illustrative purpose only and are not intended to be necessarily limiting. Each literature reference or other citation referred to herein is incorporated herein by reference in its entirety.
  • a cell includes a plurality of cells, including mixtures thereof.
  • a cell population comprising differentiated cells derived from induced pluripotent stem cells (iPSCs), said differentiated cells having a Natural Killer cell phenotype.
  • iPSCs induced pluripotent stem cells
  • iNK cells Natural Killer cell phenotype
  • any suitable source of iPSC may be used to generate the iPSC-NK cells provided herein.
  • a method of producing a cell population comprising iPSC-NK cells, wherein the iPSC-NK cells express novel IL-15 constructs as disclosed herein comprising (i) genetically editing a population of induced pluripotent stem cells (iPSCs); (ii) differentiating said population of iPSCs into a population of Natural Killer (NK) cells; and (iii) expanding the population of NK cells.
  • iPSCs induced pluripotent stem cells
  • NK Natural Killer
  • iPSC may be produced by reprogramming somatic cells to induce pluripotency.
  • the reprogramming involves the activation of pluripotency genes and repression of somatic genes.
  • this process includes the expression of certain transcription factors in the somatic cells, in particular Octamer 3/4 (Oct3/4), SRY-box containing gene 2 (Sox2), Kriippel-like factor 4 (Klf4), and the protooncogene cytoplasmic Myc protein (c-Myc) (see Takahashi and Yamanaka, Cell 126:663-676).
  • the delivery of these transcription factors into the somatic cells can be accomplished by any suitable method known in the art, for example, using viral vectors, mRNA transfection, or delivery of recombinant proteins (see, e.g. Chang et al., 2019, J Korean Neurosurg Soc.62(5):493-501).
  • small molecules targeting cell signaling pathways, metabolic pathways, and epigenetic modifications may be used to induce pluripotency and reprogram somatic cells into iPSCs.
  • repression of DNA methylation, activation of Wnt signaling, activation of MAPK/ERK signaling, and induction of glycolytic metabolism have been described as mechanisms to aid in reprogramming.
  • Such small molecules include, for example, Gsk3p inhibitors, transforming growth factor P (TGFP) inhibitors, TGFR inhibitors, MEK inhibitors, AMPK inhibitors, mTOR inhibitors, VEGF inhibitors, Wnt activators, cAMP activators, retinoic acid receptor (RAR) a agonists, RAR y agonists, pyruvate dehydrogenase kinase, isozyme 1 (PDK-1) activators, HMT inhibitors, DNMT inhibitors, KDM inhibitors, HDAC inhibitors, and others.
  • Gsk3p inhibitors transforming growth factor P (TGFP) inhibitors, TGFR inhibitors, MEK inhibitors, AMPK inhibitors, mTOR inhibitors, VEGF inhibitors, Wnt activators, cAMP activators, retinoic acid receptor (RAR) a agonists, RAR y agonists, pyruvate dehydrogenase kinase, isozyme
  • iPSC lines are also available and may be used to generate the iPSC-NK cells described herein.
  • the iPSCs used to generate the iPSC-NK cells provided herein are generated, maintained and differentiated under Good Manufacturing Protocol (GMP) conditions.
  • GMP Good Manufacturing Protocol
  • iPSCs may be differentiated into NK cells using any suitable method known in the art or described herein. A description of such methods is described in, for example, Zhu, H., Kaufman, D.S. (2019). An Improved Method to Produce Clinical-Scale Natural Killer Cells from Human Pluripotent Stem Cells. In: Kaneko, S. (eds) In Vitro Differentiation of T-Cells. Methods in Molecular Biology, vol 2048. Humana, New York, NY.
  • the iNK cells may be activated for three days at high concentrations of IL-2 (100 unit/ml to 500 unit/ml), and with additional cytokines IL- 15 and IL-21 A.
  • the iPSC-NK cells provided herein may be cultured under any suitable conditions described herein or known in the art.
  • the NK cells are cultured on a feeder layer, i.e., in co-culture with another cell line. Such co-cultures can be effective in inducing proliferation in cell types that otherwise proliferate very slowly or not at all.
  • a feeder layer that is capable of inducing proliferation of iPSC-NK cells.
  • a feeder layer that is capable of activating iPSC-NK cells.
  • Examples of feeder layers that may be used for the culture of NK cells provided herein include, without limitation, K562 cells and 221 cells.
  • the feeder layer cells may be genetically modified, e.g., the feeder layer cells may be transduced with IL- 15, IL21 and/or 4-1-BB. Prior to being used in the co-culture, the feeder layer cells may be irradiated with doses sufficient to induce cell cycle arrest, such that the feeder layer cells do not proliferate in the coculture.
  • NK cell marker such as CD56 and/or CD45.
  • the iPSC-NK cells provided herein cells are genetically modified by introducing (“integrating” or “knocking in”) or deleting (“knocking out”) one or more genes.
  • knocking out or integrating genes of interest involved in NK cell exhaustion, activation, tolerance, and/or memory are thought to improve the clinical utility of the iPSC-NKs provided herein.
  • the genetic modification of the iPSC-NK cells provided herein may be achieved by any suitable method known in the art or described herein.
  • the genome of the iPSC-NK cells provided herein may be modified by introducing DNA double strand breaks, which are then repaired by the cell’s endogenous repair mechanisms, such as homologous recombination.
  • DNA double strand breaks may be introduced using targeted endonucleases, such as Zinc-finger nucleases, transcription activation-like effector-nucleases (TALENs), meganucleases, or the CRISPR/Cas system, which relies on the Cas9 endonuclease for inducing the DNA breaks and a guide RNA (gRNA) for site- specificity.
  • targeted endonucleases such as Zinc-finger nucleases, transcription activation-like effector-nucleases (TALENs), meganucleases, or the CRISPR/Cas system, which relies on the Cas9 endonuclease for inducing the DNA breaks and a guide RNA (gRNA) for site- specificity.
  • gRNA guide RNA
  • DualaseTM platform may be used to edit the iPSC cells described herein.
  • the DualaseTM is a gene editing technology which cuts DNA twice and leaves non-compatible DNA ends, which is hypothesized to lead to higher fidelity repair than non-compatible ends.
  • TALENs employ a bacterial DNA cleavage domain and specifically bind DNA via highly conserved 33-35 amino acid TALE repeats which resemble the DNA-binding domains of transcription factors.
  • the TALE repeats each bind a single base pair of DNA.
  • the specificity of TALEN DNA binding is dictated by two hypervariable residues.
  • Multiple modular TALE repeats can be linked together into a longer array with custom DNA-binding specificities. See e.g., Maeder and Gersbach, 2016, Mol Ther.24(3):430-446; Carrol, 2017, Yale J Biol Med 90:653-659.
  • Methods for designing TALEN sequences targeting a desired locus are well known in the art and described in, e.g., Cermak et al., Nucleic Acids Res.2011 Jul;39(12):e82.
  • Cas-based DNA editing systems are well known in the art. Any suitable Cas enzyme can be used to edit the iPSC-NK cells described herein, including, without limitation, Cas9 and Cas 12.
  • the polynucleotide encoding the knocked-in gene is introduced in such a way that the polynucleotide is operatively linked to a promoter.
  • the term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • control sequence refers to polynucleotide sequences which are necessary to affect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. In eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • polynucleotide as referred to herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • the iPSC-NK cells are modified by knocking in an IL- 15 construct as disclosed herein.
  • the IL- 15 construct encodes a cell membrane-bound form of IL- 15 (mbIL-15) or a functional fragment thereof.
  • the IL-15 construct encodes a soluble form of IL- 15 or a functional fragment thereof.
  • the IL- 15 construct encodes an IL- 15 trapped in the endoplasmic reticulum (ER) or a functional fragment thereof.
  • the IL- 15 construct comprises (i) IL-2R or a functional fragment thereof, and (ii) a form of IL- 15 as disclosed herein, or a functional fragment thereof.
  • the IL- 15 construct comprises (i) IL-2RP or a functional fragment thereof, (ii) IL- 15Ra or a functional fragment thereof, and (iii) a form of IL- 15 as disclosed herein, or a functional fragment thereof.
  • functional fragment is meant a fragment of a protein (e.g., IL-15 or IL- 2RP) which retains one or more desired activities of the parental protein.
  • an iPSC-NK cells provided herein expresses a polypeptide comprising the sequence of human IL-15 isoform S48AA (SEQ ID NO:1).
  • an iPSC- NK cells provided herein expresses a polypeptide comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:1.
  • an iPSC-NK cells provided herein expresses a polypeptide comprising the sequence of human IL-15 isoform S21AA (SEQ ID NO:2). In some embodiments, an iPSC-NK cells provided herein expresses a polypeptide comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO:2.
  • an iPSC-NK cells provided herein comprises a polynucleotide encoding the amino acid sequences of human IL- 15 isoform S48AA (SEQ ID NO:1).
  • an iPSC-NK cells provided herein comprises a polynucleotide encoding a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequences of SEQ ID NO:1.
  • an iPSC-NK cells provided herein comprises a polynucleotide encoding the amino acid sequences of human IL- 15 isoform S21AA (SEQ ID NO:2).
  • an iPSC-NK cells provided herein comprises a polynucleotide encoding a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequences of SEQ ID NO:2.
  • the IL-15 or functional fragment thereof may be a membrane bound form of IL- 15 (mbIL-15) or a functional fragment thereof.
  • Expression of a membrane-bound form of IL-15 may be achieved by expressing a nucleic acid encoding a soluble form of IL-15 (e.g., SEQ ID NO:41) as a fusion protein with the hinge and transmembrane domains of CD8.
  • a nucleic acid encoding a soluble form of IL-15 e.g., SEQ ID NO:41
  • An illustrative nucleic acid sequence encoding the hinge and transmembrane domains of CD8 is set forth in SEQ ID NO:46.
  • the IL- 15 or functional fragment thereof may be a form of IL- 15 that is trapped in the endoplasmic reticulum (ER). This may be achieved by, for example, expressing a polynucleotide encoding IL- 15 or a functional fragment thereof (e.g., SEQ ID NO:41) as a fusion protein with an ER retention signal.
  • ER retention signal sequence is set forth as SEQ ID NO:54.
  • the IL-15 or functional fragment thereof may be a form of IL-15 (e.g., a membrane-bound form of IL-15, a soluble form of IL-15, or an ER-trapped form of IL-15) or functional fragment thereof fused to the IL- 15 receptor alpha (IL-15Ra).
  • IL-15Ra IL- 15 receptor alpha
  • This may be accomplished by expressing the IL- 15 or functional fragment thereof as a fusion protein with IL- 15Ra.
  • An illustrative nucleic acid sequence encoding IL-15Ra is set forth in SEQ ID NO:43.
  • the iPSC-NK cells provided herein are modified by knocking in an IL-15 construct encoding IL-2RP or a functional fragment thereof, and interleukin 15 (IL-15) or a functional fragment thereof.
  • the IL- 15 can be in a soluble form, a membrane-bound form, or an ER-trapped form.
  • the iPSC-NK cells provided herein are modified by knocking in an IL- 15 construct encoding IL-2RP or a functional fragment thereof, interleukin 15 (IL- 15) or a functional fragment thereof, and IL-15Ra or a functional fragment thereof.
  • the IL- 15 can be in a soluble form, a membrane-bound form, or an ER-trapped form.
  • polypeptides and vectors comprising the gRNAs and/or the donor DNA sequences provided herein.
  • the gRNAs provided herein may be used in combination with any suitable DNA editing enzyme known in the ail or described herein, including, for example, Cas9 and Casl2.
  • the IL- 15 construct disclosed herein may be knocked into the iPSC-NK cell genome at any suitable position.
  • the IL- 15 construct comprises sequences encoding IL- 15, IL-15Ra and IL-2Rp.
  • the integration locus is the B2M locus.
  • the integration locus is the CD38 locus.
  • the integration locus is the TGFPR2 locus.
  • An illustrative gene editing strategy for a non-disruptivc knock-in of IL-15 into the B2M locus is shown in FIG. 2A of WO 2023/060136.
  • An illustrative gene editing strategy for a disruptive knock-in of IL- 15 into the TGFPR2 locus is shown in FIG. 2B of WO 2023/060136.
  • Exemplary gRNAs that may be used for the knock-in of the IL- 15 construct at the B2M locus are shown in SEQ ID NOs:3-4.
  • nucleic acid donor constructs that may be used to deliver the IL- 15 construct disclosed herein to a target site in the genome.
  • Illustrative donor sequences that may be used to insert the IL-15 construct into a cell are set forth in SEQ ID NOs:27- 38.
  • a construct that may be used to deliver the IL- 15 construct disclosed herein to a target site comprises a nucleic acid sequence encoding the desired polypeptide (e.g. IL-15 and IL-2RP, or IL-15, IL-15Ra and IL-2RP), flanked by a left homology arm (LHA) and a right homology arm (RHA).
  • LHA left homology arm
  • RHA right homology arm
  • the LHA and RHA of a given donor construct comprise nucleic acid sequences with homology to the target site (e.g., the B2M locus or the TGFPR2 locus).
  • the sequence and length of the RHA and LHA sequences may vary based on the targeted site.
  • the LHA sequence comprises a nucleic acid sequence that is homologous to the 5’ upstream sequence of the B2M gene.
  • the RHA sequence comprises a nucleic acid sequence that is homologous to exon 1 and intron 1 of B2M.
  • An illustrative LHA-RHA sequence pair that may be used for targeted insertion into the B2M locus is the pair of sequences set forth in SEQ ID NO:39 (LHA) and SEQ ID NO:45 (RHA).
  • the LHA sequence comprises a nucleic acid sequence that is homologous to intron 2 and exon 3 of TGFPR2.
  • the RHA sequence comprises a nucleic acid sequence that is homologous to exon3 and intron 3 of TGFPR2.
  • An illustrative LHA-RHA sequence pair that may be used for targeted insertion into the TGFPR2 locus is the pair of sequences set forth in SEQ ID NO:52 (LHA) and SEQ ID NO:47 (RHA).
  • the donor construct further comprises one or more of the following: spacer domains, one or more insulator domains, a CD8-hinge-transmembrane domain, a promoter, an endoplasmic reticulum (ER) retention signal sequence, a polyA sequence (e.g., a bGHpA sequence), and/or an TRES element (e.g., an IRES2 element).
  • the elements of a nucleic acid construct may be separated by spacer elements, insulators, and/or 2A sequences (e.g., a P2A sequence).
  • FIG. 1 An illustrative donor sequence (donor 107 plasmid) for inserting an IL-15 construct into the TGF0R2 locus of a cell is shown in Fig. 1: RHA(TGFpR2)-Promoter(NKp46)-CD8aSP-IL15- IL15Ra-IL2RP-ICD-bGHpA-LHA(TGFpR2) (SEQ ID NO:64).
  • FIG. 2 An illustrative donor sequence (donor 107ER plasmid) for inserting an IL-15 construct (comprising an ER-trapped IL-15) into the TGFPR2 locus of a cell is shown in Fig. 2: RHA(TGFpR2)-Promoter(NKp46)-CD8aSP-GSEKDEL-IL15-IL15Ra-IL2RP-ICD-bGHpA- LHA(TGFPR2) (SEQ ID NO:65).
  • FIG. 3 An illustrative donor sequence (donor 108 plasmid) for inserting an IL-15 construct into the TGFPR2 locus of a cell is shown in Fig. 3: RHA(TGFpR2)-Promoter(NKp46)-IL15Ra-P2A- IL2Rp-P2A-IL15-bGHpA-LHA(TGFpR2) (SEQ ID NO:66).
  • FIG. 4 An illustrative donor sequence (donor 108B2M plasmid) for inserting an IL- 15 construct into the B2M locus of a cell is shown in Fig. 4: LHA(B2M)-B2M-IRES-IL15Ra-P2A-IL2RP-P2A- IL15-bGHpA-RHA(B2M) (SEQ ID NO:67).
  • FIG. 5 An illustrative donor sequence (donor 115 plasmid) for inserting an IL- 15 construct into the B2M locus of a cell is shown in Fig. 5: LHA(B2M)-IL15(soluble)-IRES2-IL15Ra-P2A- IL2RP-P2A-RHA(B2M) (SEQ ID NO:68).
  • Figure 6 shows a map of a vector 1 comprising sequences encoding a membrane-bound IL-15.
  • the vector has the polynucleotide sequence of SEQ ID NO:55.
  • Figure 7 shows a map of a vector 2 comprising sequences encoding a membrane-bound IL- 15 and IL-15Ra.
  • the vector has the polynucleotide sequence of SEQ ID NO:56.
  • Figure 8 shows a map of a vector 3 comprising sequences encoding a membrane-bound IL- 15, IL-15Ra, IL-15Rb, and a full cytoplasmic portion of the common cytokine receptor y chain.
  • the vector has the polynucleotide sequence of SEQ ID NO:57.
  • Figure 9 shows a map of a vector 4 comprising sequences encoding a membrane-bound IL- 15, IL-15Ra, IL-15Rb with a portion of the cytoplasmic region deleted, and a full cytoplasmic portion of the common cytokine receptor y chain.
  • the vector has the polynucleotide sequence of SEQ ID NO:58.
  • Fig. 10 shows a map of a vector 5 comprising sequences encoding an
  • the vector has the polynucleotide sequence of SEQ ID NO:59.
  • Fig. 11 shows a map of a vector 6 comprising sequences encoding an
  • the vector has the polynucleotide sequence of SEQ ID NO:60.
  • Fig. 12 shows a map of a vector 7 comprising sequences encoding IL- 15Ra, IL-15Rb, and a soluble IL- 15.
  • the vector has the polynucleotide sequence of SEQ ID NO:61.
  • Fig. 13 shows a map of a vector 8 comprising sequences encoding IL-
  • the vector has the polynucleotide sequence of SEQ ID NO:62.
  • Fig. 14 shows a map of a vector 9 comprising sequences encoding an ER-trapped fusion protein comprising IL-15, IL-15Ra, IL-15Rb, and a full cytoplasmic portion of the common cytokine receptor y chain.
  • the vector has the polynucleotide sequence of SEQ ID NO:63.
  • the iPSC-NK cells comprise a knock-in of an IL- 15 construct disclosed herein into a gene of the iPSC-NK cells, wherein the IL- 15 construct is operably linked to a promoter that is not active in iPSC, but becomes active during or after iPSC differentiation.
  • the promoter can be an exogenous or endogenous promoter.
  • the promoter can be expressed in common progenitor/precursor cells during stem cell differentiation, or expressed in many different cell types after differentiation, or can be cell-type specific.
  • promoters include, but are not limited to, NKp46 that is active in NK cells; CD3y promoter that is active in T cells; CD 19 promoter for B cells; VEGFR promoter and ICAM1 promoter that arc active in endothelial cells; B2M promoter that is active in many different cells types; IL2RP promoter that is active in many immune cells; Netin promoter, Sox 2 promoter, Olig 1 promoter, Olig 2 promoter, Olig3 promoter, MBP promoter, OSP promoter, MOG promoter, or Sox 10 promoter that is active in neural cells; Sca-1 promoter, CD27 promoter, CD34 promoter, CD38 promoter, CD43 promoter, CD48 promoter, CD117 promoter, or CD 150 promoter that is active in hematopoietic progenitor cells; IL-7R promoter that is active lymphoid precursors; CD45RA promoter, IL-3R0C promoter, or thrombopoietin receptor promoter
  • the NK cells arc genetically modified by deleting or inactivating (or “knocking out”) a gene encoding TGF receptor 2 (TGF R2).
  • TGF R2 TGF receptor 2
  • such iNK cells comprising a deletion in TGFPR2 further comprise a knock-in of an IL- 15 construct as disclosed herein.
  • a gene may be inactivated, for example, by introducing a homozygous or heterozygous inactivating mutation into said gene.
  • a homozygous inactivating mutation results in complete loss of protein function and, in some cases, loss of protein expression.
  • the NK cells provided herein are genetically modified by inactivating the TGFPR2 gene, e.g., by targeting an exon of TGFPR2.
  • the TGFPR2 gene may be inactivated by introducing the IL- 15 construct as disclosed herein into the TGFPR2 locus.
  • the TGFPR2 gene may be inactivated by introducing a dominant negative form of TGFPR2 into the cell. Exemplary sequences of gRNAs that may be used to knockout TGFPR2 in the iPSC NK cells described herein are provided in WO 2023/06013.
  • the iPSC-NK cells provided herein are modified such that they are deficient in TGFPR2 signaling, e.g., by deleting the intracellular signaling domain of TGFPR2. This may be accomplished by, e.g., introducing a stop codon into a suitable position in the TGFPR2 amino acid sequence (for example, introducing a stop codon after the transmembrane domain).
  • exemplary gRNA sequences and their corresponding donor DNA sequences that may be used to knockout the TGFPR2 signaling domain by introducing a stop codon after the TGFPR2 transmembrane domain are provided in WO 2023/06013.
  • the method described herein result in a population of iPSC-NK cells wherein about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90 to about 95%, or about 95% to 100% of cells comprise an inactivating mutation in TGFBR2 and/or express detectable levels of IL- 15 or a functional fragment thereof.
  • the method described herein result in a population of iPSC-NK cells wherein at least 50%, at least 55%, at least 60%, at least 65%, at last 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more than 98% of cells comprise an inactivating mutation in TGFBR2 and/or express detectable levels of IL- 15 or a functional fragment thereof.
  • the expression level of IL- 15 or a functional fragment thereof may be determined using any suitable method known in the art or described herein, including, for example, flow cytometry, Western Blotting, Enzyme Linked Immunosorbent Assays (ELISA), quantitative real-time PCR (qPCR) and RNA sequencing.
  • the iNK cells comprise an inactivating (e.g., a frameshift) mutation in TGF0R2 and express a truncated form of TGF0R2 protein.
  • the inactivating mutation in TGFPR2 may be biallelic or mono allelic.
  • the iNK cells provided herein are resistant to the suppressive effect of TGFb signaling.
  • the expression level of NKG2D, DNAM and/or NKp30 on the surface of an iNK cell may remain comparable after treatment with TGFbl to the levels before treatment.
  • the iNK cells provided herein are able to survive without the stimulation of exogenous cytokines.
  • Cell survival may be determined by measuring cell counts or cell viability.
  • the iNK cells provided herein show higher cell killing ability than unmodified NK cells.
  • Cell killing ability may be determined by incubating the iNK (and unmodified control NK) cells with target cells and measuring the disappearance of the target cells using, e.g., a fluorescent marker.
  • the iNK cells provided herein kill target cells with an efficiency that is about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 2-3 times, about 3- 4 times, about 4-5 times, about 5-6 times, about 6-7 times, about 7-8 times, about 8-9 times or about 9-10 times higher than that of NK cells not comprising the IL- 15 construct disclosed herein.
  • the iNK cells described herein persist longer in the circulation after intravenous administration to a patient.
  • the iNK cells comprising the IL- 15 construct disclosed herein persist in the circulation of a subject about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 2-3 times, about 3-4 times, about 4-5 times, about 5-6 times, about 6-7 times, about 7-8 times, about 8-9 times or about 9-10 times longer than NK cells not comprising the IL- 15 construct.
  • the tumor microenvironment has a suppressive effect on NK cell function and inhibits NK cell function e.g., via soluble factors (e.g., cytokines), hypoxic conditions and/or low nutrient levels. See, e.g., Mclaiu ct al.. Front. Immunol.10:3038.
  • soluble factors e.g., cytokines
  • hypoxic conditions e.g., hypoxic conditions
  • low nutrient levels e.g., hypoxic conditions and/or low nutrient levels.
  • the iNK cells described herein are resistant to the suppressive effects of the tumor microenvironment.
  • the average expression level of TGF0R2 in a population of iNK cells provided herein is about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95% or more than 95% lower than the average expression level of TGF R2 in a population of unedited NK cells.
  • the expression level of TGF0R2 in the iNK cells is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% lower than the expression level of TGFBR2 in unedited NK cells.
  • iPSC-NK cells iPSC-NK cells
  • Modified iPSC cells may be cryoprcscrvcd before differentiation into iNK cells.
  • the iNK cells provided herein comprise polynucleotide sequences encoding IL- 15 and IL-2R0, and a knockout of cytokine inducible SH2 containing protein (CISH).
  • the IL- 15 can be a membrane-bound form of IL- 15, a soluble form of IL- 15, or an ER-trapped form of IL-15.
  • the IL-15 is further expressed as fusion protein with an IL-15R or a functional fragment thereof, e.g. IL-15Ra or IL-15Rb.
  • Knockout of CISH can be accomplished by genetic editing.
  • the genetic editing comprises using a TALEN construct or a Cas9 or Casl2 enzyme.
  • the iNK cells provided herein comprise polynucleotide sequences encoding IL- 15 and IL-2R0, and a knockout of a NK cell inhibitory receptor, e.g. T-cell immunoglobulin and ITIM domain (TIGIT) receptor.
  • the IL- 15 can be a membrane-bound form of IL- 15, a soluble form of IL- 15, or an ER-trapped form of IL- 15.
  • the IL-15 is further expressed as fusion protein with an IL-15R or a functional fragment thereof, e.g. IL-15Ra or IL-15Rb.
  • Knockout of TIGIT can be accomplished by genetic editing.
  • the genetic editing comprises using a TALEN construct or a Cas9 or Casl2 enzyme.
  • the iNK cells provided herein comprise polynucleotide sequences encoding IL- 15 and IL-2R0, and a knockout of CISH and a knockout of a NK cell inhibitory receptor, e.g. T-cell immunoglobulin and ITIM domain (TIGTT) receptor.
  • the IL- 15 can be a mcmbranc-bound form of IL- 15, a soluble form of IL- 15, or an ER-trapped form of IL- 15.
  • the IL- 15 is further expressed as fusion protein with an IL- 15R or a functional fragment thereof, e.g. IL-15Ra or IL-15Rb.
  • a pharmaceutical composition comprises a dose ranging from about IxlO 5 to about 5 x 10 5 iNK cells, about 5 x 10 5 to about 1 x 10 6 iNK cells, about 1 x 10 6 to about 5 x 10 6 iNK cells, about 5 x 10 6 iNK cells to about 1 x 10 7 iNK cells, about 1 x 10 7 to about 5 x 10 7 iNK cells, about 5 x 10 7 to 1 x 10 8 iNK cells, about 1 x 10 8 to about 5 x 10 8 iNK cells, about 5 x 10 8 to about 1 x 10 9 iNK cells, about 1 x 10 9 to about 5 x 10 9 iNK cells, about 5 x 10 9 to 1 x IO 10 iNK cells, about 1 x IO 10 to about 5 x IO 10 iNK cells, about 5 x 10 9 to 1 x IO 10 iNK cells, about 5 x 10 9 to 1 x IO 10 iNK cells, about 5 x
  • a pharmaceutical composition is cryopreserved.
  • a composition comprising the iPSCs or the iNK cells provided herein may be cryopreserved for about 1-3 months, about 3-6 months, about 6-9 months, or about 9-12 months.
  • a composition comprising iPSC-NK cells provided herein may be cryopreserved for more than 3 months, more than 6 months, more than 9 months, more than 12 months, more than 18 months, more than 2 years, or more than 3 years before thawing and use in a method described herein.
  • a formulation comprising the iPSCs or the iPSC-NK cells described herein may further comprise a cryoprotectant.
  • the viability of the iPSC-NK cells is at least 30%, at least 50%, or at least 70% as determined by a suitable assay known in the ail or described herein.
  • compositions typically comprise the iPSCs or the iNK cells and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but arc not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration, e.g., intravenous administration.
  • Solutions or suspensions used for intravenous administration can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl para
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • Such molecules arc suitably present in combination in amounts that are effective for the purpose intended.
  • therapeutic formulations of the invention which comprise the iPSC-NK cells of the invention, are used to treat or alleviate a symptom associated with a cancer, including solid cancers and hematological cancers.
  • cancers that may be treated with a method described herein include, without limitation, leukemias, lymphomas, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung & bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney and renal pelvis cancer, oral cavity & pharynx cancer, uterine corpus cancer, and/or melanoma. Examples of cancers are also disclosed in WO 2023/060136.
  • the present invention also provides methods of inhibiting the proliferation of tumor cells in a subject, comprising administering to the subject a population of the iPSCs or the iPSC-NK cells provided herein.
  • a therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a cancer, using standard methods.
  • Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the disease or disorder indicates that the treatment confers a clinical benefit. In some embodiments, a method provided herein results in decreased tumor proliferation in the subject.
  • the “administration” of an agent, (e.g., a population of iPSC-NK cells), to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function.
  • the population of iPSC-NK cells is administered intravenously.
  • the population of iPSC-NK cells provided herein is administered by intravenous infusion, e.g., an intravenous infusion over about 15min, about 30min, about 45min, about 60min, about 90min, about 2 hours, about 3 hours, about 4 hours, or about 5 hours, or an intravenous infusion over about 15min to about 30min, about 30min to about 45min, about 45 min to about 60min, about 60 min to about 90min, about 90min to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, or about 4 hours to about 5 hours.
  • the rate of infusion may vary with the number of cells being infused to the subject.
  • a therapeutically effective amount of the iPSC-NK cells of the invention relates generally to the amount needed to achieve a therapeutic objective. It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean “substantial”, which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
  • one or more doses of the iPSC-NK cells are administered. If two or more doses of the iPSC-NK cells are administered, the duration between the administrations should be sufficient to allow time for propagation of the cells in the individual. In specific embodiments the duration between doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more weeks.
  • “treating” or “treatment” of a disease in a subject refers to (1) inhibiting the disease or arresting its development; or (2) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.
  • iPSC-NK cells are delivered to an individual in need thereof, and the individual has been diagnosed with a cancer. Without wishing to be bound by theory, the cells then enhance the individual's immune system to attack or directly attack the respective cancer or pathogenic cells. COMBINATION THERAPIES
  • the iPSCs or the iPSC-NK cells described herein may be administered in combination with one or more other therapeutic agents.
  • the additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
  • the additional therapy may be in the form of adjuvant or neoadjuvant therapy.
  • the iPSC-NK cells described herein may be administered in combination with one or more other anti-NK inhibitory receptor agents, such as antibody, RNAi- or small molecule-based agents.
  • the anti-NK inhibitory receptor agents include, but are not limited to, checkpoint inhibitors, such as PD-1/PD-L1 inhibitors, TIGIT inhibitors, TIM-3 inhibitors, and LAG-3 inhibitors.
  • TIGIT/PVR inhibitors include, but are not limited to, Ociperlimab, BAT6005, BMS 986207, PH 804, AGEN 1777, TSRF-786C, liothyronine;
  • TIM-3 inhibitors include but are not limited to Cobolimab BMS-986258, Sabatolimab.
  • the anti-NK inhibitory receptor agents can also be inhibitors of therapeutic targets upstream of NK cell inhibitory receptor, such as Elraglusib, a selective small-molecule inhibitor of glycogen synthase kinase-3 beta that reduces expression of immune checkpoint molecules PD-1, TIGIT and LAG-3.
  • the subject can be administered nonmyeloablative lymphodepleting chemotherapy prior to administering the iNK cells provided herein.
  • the nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route.
  • the nonmyeloablative lymphodepleting chemotherapy can comprise, for example, the administration of cyclophosphamide and fludarabine.
  • An exemplary route of administering cyclophosphamide and fludarabine is intravenous.
  • any suitable dose of cyclophosphamide and fludarabine can be administered. For example, around 60 mg/kg of cyclophosphamide is administered for two days after which around 25 mg/m2 fludarabine is administered for five days.
  • the nonmyeloablative lymphodepleting immunotherapy can comprise, for example, the administration of an anti-CD52 agent or anti-CD20 agent.
  • the lymphodepleting immunotherapy is an anti-CD52 antibody.
  • the anti-CD52 antibody is alemtuzumab.
  • the lymphodepleting immunotherapy is an anti-CD20 antibody.
  • Exemplary anti-CD20 antibodies include, but are not limited to rituximab, ofatumumab, ocrelizumab, obinutuzumab, ibritumomab or iodine 131 tositumomab.
  • An exemplary route of administering anti-CD52 agent or anti-CD20 agent is intravenous. Likewise, any suitable dose of anti-CD52 agent or anti-agent can be administered.
  • immune cell growth factor that promotes the growth and activation of the immune cells is administered to the subject either concomitantly with the iNK cells provided herein or subsequently to the iNK cells provided herein.
  • the immune cell growth factor can be any suitable growth factor that promotes the growth and activation of the immune cells.
  • suitable immune cell growth factors include, but are not limited to, interleukin (IL)-2, IL-7, IL- 15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.
  • the iPSC-NK cells are not administered in combination with an interleukin.
  • the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent.
  • the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.).
  • the additional therapy is radiation therapy.
  • the additional therapy is surgery.
  • the additional therapy is a combination of radiation therapy and surgery.
  • the additional therapy is gamma irradiation.
  • the additional therapy is therapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.
  • the additional therapy may be one or more of the chemotherapeutic agents known in the art.
  • Combination therapies can include, but are not limited to, one or more anti-microbial agents (for example, antibiotics, anti-viral agents and anti-fungal agents), anti-tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (for example, azathioprine, or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory agents such as acetyls alicylic acid, ibuprofen or naproxen sodium), cytokine antagonists (for example, anti-TNF and anti-IL), anti-
  • immunosuppressive or tolerogenic agents including but not limited to calcineurin inhibitors (e.g., cyclosporin and tacrolimus); mTOR inhibitors (e.g., Rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, orB cells); chemotherapeutic agents (e.g., Methotrexate, Treosulfan, Busulfan); irradiation; or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL- 2R, IL-4, JAK kinase inhibitors) can be administered.
  • calcineurin inhibitors e.g., cyclosporin and tacrolimus
  • mTOR inhibitors e.g., Rapamycin
  • mycophenolate mofetil antibodies
  • chemotherapeutic agents e.g., Methotrexate, Treosulfan, Bus
  • Such additional pharmaceutical agents can be administered before, during, or after administration of the iNK cells provided herein, depending on the desired effect.
  • This administration of the iNK cells and the agent can be by the same route or by different routes, and either at the same site or at a different site.
  • the iPSC-NK cells provided herein may be administered before, during, or after, an additional therapeutic agent, such as an immune checkpoint inhibitor.
  • the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
  • an additional therapeutic agent such as an immune checkpoint inhibitor.
  • the anti-cancer therapy e.g., an immune checkpoint inhibitor
  • immunotherapies may be used in combination or in conjunction with methods of the embodiments described herein.
  • immunotherapeutic s generally rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Rituximab (RITUXAN®) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Examples of immunotherapies are described in WO 2023/060136.
  • the immunotherapy may be an immune checkpoint inhibitor.
  • Immune checkpoints either turn up a signal (e.g., co- stimulatory molecules) or turn down a signal.
  • Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA).
  • A2AR adenosine A2A receptor
  • B7-H3 also known as CD276
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
  • An article of manufacture or a kit comprising the iPSCs or the iPSC-NK cells described herein is also provided herein.
  • the article of manufacture or kit can further comprise a package insert comprising instructions for using the iPSC-NK to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer.
  • Suitable containers include, for example, bottles, vials, bags and syringes.
  • the container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or poly olefin), or metal alloy (such as stainless steel or hastelloy).
  • the container holding the formulation and the label on, or associated with, the container may indicate directions for use.
  • the article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent).
  • iNK cells were transduced at the beginning of the stage 3 week 2, and expanded with K562 feeders in 1:5 ratio for a total of 20 days. At day 3, day 9, day 16, and day 20 the transduced iNK cells were harvested and measured for the viable cell number (tdT: tdTomato-transduced; control: no transduction).
  • the IL- 15 -engineered iNK cells disclosed herein showed advantage in survivability in cytokine-starved condition. Following 20 days of ex vivo expansion, the IL-15-engineered iNK cells were grown in cytokinc-frcc medium for 6 days and the viable cell number was monitored at day 0, day 2, and day 6 (Fig. 16). The IL- 15 -engineered iNK cells exhibited advantage in survivability in cytokine-starved condition compared to the tdTomato-transduced iNK cells and control iNK cells (i.e., no transduction).
  • the IL-15-engineered, cytokine-starved iNK cells maintain pSTAT5 activation in response to IL2 and Ki67 proliferation marker.
  • the IL- 15- engineered iNK cells were grown in cytokine-free medium for 6 days, or in cytokine-free medium for 6 days and then pulse-treated with 100 lU/mL of IL2 for 30 minutes.
  • the % positive population of phospho-STAT5 and proliferation marker Ki-67 were analyzed at day 0 and day 6 (Fig. 17).
  • the cytokine- starved IL- 15 -engineered iNK cells maintained pSTAT5 activation by IL2 and Ki67 proliferation marker, whereas the cytokine- starved control iNK cells failed to be activated and proliferated.
  • the IL-15-engineered iNK cells disclosed herein showed persistence in peripheral blood in vivo.
  • the IL-15-engineered iNK cells were injected into mice. The mice looked healthy post injection, and peripheral blood was withdrawn from the mice at hour 3, day 3, day 7, day 10, day 14, and day 17.
  • the persistence of the IL- 15 -engineered iNK cells was examined by flow cytometry on huCD45+ cell population. Compared to the control iNK cells, the IL-15-engineered iNK cells exhibited better survivability in vivo during the time course (Fig. 18).

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Abstract

La présente divulgation concerne une population de cellules comprenant des cellules souches pluripotentes induites (iPSC) ou des cellules NK, les iPSC ou les cellules NK comprenant une construction d'IL-15 comprenant (i) des séquences codant pour IL-15 ou un fragment fonctionnel de celle-ci, l'IL-15 ou un fragment fonctionnel de celle-ci étant piégé(e) dans le réticulum endoplasmique, ou (ii) des séquences codant pour IL-15 ou un fragment fonctionnel de celle-ci et IL-2Rb ou un fragment fonctionnel de celle-ci. Dans un mode de réalisation, les cellules NK sont dérivées des iPSC (cellules iNK), et les cellules iNK peuvent être utilisées pour traiter le cancer chez un sujet.
PCT/US2024/032309 2023-06-04 2024-06-03 Cellules tueuses naturelles pourvues d'un nouveau knock-in d'il-15 et leurs méthodes d'utilisation Ceased WO2024254013A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022057851A1 (fr) * 2020-09-16 2022-03-24 Beigene, Ltd. Constructions d'interleukine 15 et procédés d'utilisation
WO2022216514A1 (fr) * 2021-04-07 2022-10-13 Century Therapeutics, Inc. Compositions et méthodes pour générer des lymphocytes t gamma-delta à partir de cellules souches pluripotentes induites
WO2022232796A1 (fr) * 2021-04-28 2022-11-03 The General Hospital Corporation Il2 attachée à son récepteur il2rbêta et protéines formant des pores en tant que plate-forme pour améliorer l'activité des cellules immunitaires
US20230044451A1 (en) * 2016-03-19 2023-02-09 Exuma Biotech Corp. Methods and compositions for the delivery of modified lymphocytes and/or retroviral particles
WO2023060136A1 (fr) * 2021-10-05 2023-04-13 Cytovia Therapeutics, Llc Cellules tueuses naturelles et leurs méthodes d'utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230044451A1 (en) * 2016-03-19 2023-02-09 Exuma Biotech Corp. Methods and compositions for the delivery of modified lymphocytes and/or retroviral particles
WO2022057851A1 (fr) * 2020-09-16 2022-03-24 Beigene, Ltd. Constructions d'interleukine 15 et procédés d'utilisation
WO2022216514A1 (fr) * 2021-04-07 2022-10-13 Century Therapeutics, Inc. Compositions et méthodes pour générer des lymphocytes t gamma-delta à partir de cellules souches pluripotentes induites
WO2022232796A1 (fr) * 2021-04-28 2022-11-03 The General Hospital Corporation Il2 attachée à son récepteur il2rbêta et protéines formant des pores en tant que plate-forme pour améliorer l'activité des cellules immunitaires
WO2023060136A1 (fr) * 2021-10-05 2023-04-13 Cytovia Therapeutics, Llc Cellules tueuses naturelles et leurs méthodes d'utilisation

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