WO2023143475A1 - Méthodes et compositions pour immunothérapies cellulaires - Google Patents

Méthodes et compositions pour immunothérapies cellulaires Download PDF

Info

Publication number
WO2023143475A1
WO2023143475A1 PCT/CN2023/073482 CN2023073482W WO2023143475A1 WO 2023143475 A1 WO2023143475 A1 WO 2023143475A1 CN 2023073482 W CN2023073482 W CN 2023073482W WO 2023143475 A1 WO2023143475 A1 WO 2023143475A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
population
composition
vitro method
Prior art date
Application number
PCT/CN2023/073482
Other languages
English (en)
Inventor
Luhan Yang
Yangbin Gao
Juanjuan ZHAO
Yanan YUE
Yang Yang
Xingyue WANG
Tiantian ZHU
Original Assignee
Hangzhou Qihan Biotechnology Co., Ltd.
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 Hangzhou Qihan Biotechnology Co., Ltd. filed Critical Hangzhou Qihan Biotechnology Co., Ltd.
Publication of WO2023143475A1 publication Critical patent/WO2023143475A1/fr

Links

Classifications

    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • A61K35/48—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • Cancer e.g., neoplasm, tumor
  • cancer is a leading cause of death worldwide, accounting for about 10 million deaths annually. Cancer continues to bring increasing health, economic, and emotional burden on individuals, families, communities, and countries. Increase understanding of cancer biology (e.g., specifically cancer immune biology) and genetic engineering has encouraged development of adoptive cell therapies (e.g., cellular immunotherapy) , with a goal to treat or control a number of different cancers.
  • adoptive cell therapies e.g., cellular immunotherapy
  • the present disclosure provides methods and systems for generating immune cells (e.g., engineered immune cells) or progenitors thereof. Some aspects of the present disclosure provide generation of immune cells exhibiting specific cytolytic and regulatory functions. Some aspects of the present disclosure provide generating engineered immune cells (e.g., engineered natural killer (NK) cells) and methods of use thereof for treatment of cancer, such as, e.g., hematologic malignancies or solid tumors.
  • engineered immune cells e.g., engineered natural killer (NK) cells
  • NK natural killer
  • the present disclosure provides an in vitro method of generating stem cell-derived mesodermal cells, the method comprising: contacting a population of stem cells with a composition comprising an activator of Wnt/ ⁇ -catenin signaling pathway (activator) , wherein the activator is sufficient to transform the population of stem cells into a population of mesodermal cells.
  • activator Wnt/ ⁇ -catenin signaling pathway
  • the contacting occurs in a medium that is substantially free of a transforming growth factor ⁇ 1 (TGF ⁇ 1) for about 2 days.
  • TGF ⁇ 1 transforming growth factor ⁇ 1
  • the contacting occurs in a medium that is substantially free of a basic fibroblast growth factor (bFGF) for about 2 days.
  • bFGF basic fibroblast growth factor
  • the population of mesodermal cells exhibits a greater propensity to differentiate into immune cells, as compared to a control population of mesodermal cells that have been transformed from stem cells in a medium comprising TGF ⁇ 1 and/or bFGF.
  • the activator is a small molecule.
  • the activator is an inhibitor of GSK-3 ⁇ / ⁇ .
  • the activator is CHIR.
  • the population of mesodermal cells is characterized by expressing a greater level of a mesodermal marker selected from the group consisting of Kinase Insert Domain Receptor (KDR) , Eomesodermin (EOMES) , Mix Paired-Like Homeobox (MIXL1) , and CD235a, as compared to that of the population of stem cells.
  • KDR Kinase Insert Domain Receptor
  • EOMES Eomesodermin
  • MIXL1 Mix Paired-Like Homeobox
  • CD235a CD235a
  • the population of mesodermal cells does not exhibit enhanced hemogenic endothelium (HE) potential as compared to the population of stem cells.
  • HE hemogenic endothelium
  • the population of stem cells is contacted by the composition for less than or equal to about 5 days.
  • the population of stem cells is contacted by the composition for less than or equal to about 3 days.
  • the population of stem cells comprises pluripotent stem cells.
  • the present disclosure provides an in vitro method of generating mesodermal cells having hemogenic endothelium (HE) potential, the method comprising: contacting a population of mesodermal cells with a composition comprising (i) a bone morphogenetic protein (BMP) , (ii) a vascular endothelial growth factor (VEGF) , and (iii) an inhibitor of activin receptor-like kinase (ALK) receptor signaling pathway, to transform the population of mesodermal cells into a population of mesodermal cells having HE potential.
  • BMP bone morphogenetic protein
  • VEGF vascular endothelial growth factor
  • ALK activin receptor-like kinase
  • an amount of the BMP is less than 30 ng/ml.
  • the amount of the BMP is less than 25 ng/ml.
  • the amount of the BMP in the composition is 20 ng/ml.
  • a mass of the BMP in the composition is less than 50%of a mass of the VEGF in the composition.
  • the BMP comprises BMP4.
  • the inhibitor is a small molecule.
  • the ALK receptor is selected from the group consisting of ALK4 (TGF ⁇ receptor 1) , ALK4 (Activin receptor type-1B) , and ALK7 (Activin A receptor) .
  • the ALK receptor is ALK4.
  • the inhibitor is SB431542.
  • the composition further comprises a fibroblast growth factor (FGF) .
  • FGF fibroblast growth factor
  • the FGF comprises bFGF.
  • a mass of the BMP in the composition is less than 50%of a mass of the FGF in the composition.
  • the population of mesodermal cells prior to the contacting is KDR+ and CD235a+
  • the population of mesodermal cells having definitive HE potential is KDR+ and CD235a-.
  • the population of stem cells is contacted by the composition for less than or equal to about 10 days.
  • the population of stem cells is contacted by the composition for less than or equal to about 5 days.
  • the present disclosure provides an in vitro method of generating hemogenic endothelial (HE) cells, the method comprising: contacting a population of mesodermal cells with a composition comprising (i) interleukin-7 (IL-7) , and (ii) at least one member selected from the group consisting of bone morphogenetic protein (BMP) and vascular endothelial growth factor (VEGF) , to transform the population of mesodermal cells into a population of HE cells.
  • a composition comprising (i) interleukin-7 (IL-7) , and (ii) at least one member selected from the group consisting of bone morphogenetic protein (BMP) and vascular endothelial growth factor (VEGF)
  • a mass of the IL-7 in the composition is less than or equal to a mass of the at least one member.
  • the at least one member comprises the BMP.
  • the at least one member comprises the VEGF.
  • the at least one member comprises the BMP and the VEGF.
  • a mass of the VEGF in the composition is less than or equal to a mass of the BMP in the composition. In some embodiment, the mass of the VEGF in the composition is less than the mass of the BMP in the composition.
  • the BMP comprises BMP4.
  • the composition further comprises an additional interleukin that is not IL-7.
  • a mass of the additional interleukin in the composition is less than or equal to a mass of the IL-7 in the composition.
  • the mass of the additional interleukin in the composition is less than the mass of the IL-7 in the composition.
  • the additional interleukin comprises IL-3 and/or IL-15.
  • the composition further comprises a FMS-like tyrosine kinase receptor ligand (FLTL) .
  • FLTL FMS-like tyrosine kinase receptor ligand
  • a mass of the FLT receptor ligand in the composition is less than or equal to a mass of the IL-7 in the composition.
  • the mass of the FLT receptor ligand in the composition is less than the mass of the IL-7 in the composition.
  • the FLT receptor ligand comprises FLT3L.
  • the population of mesodermal cells prior to the contacting, exhibit HE potential.
  • the population of HE cells is characterized by expressing a greater level of CD34, as compared to that of the population of mesodermal cells.
  • the population of mesodermal cells is contacted by the composition for less than or equal to about 10 days.
  • the population of mesodermal cells is contacted by the composition for less than or equal to about 5 days.
  • the present disclosure provides an in vitro method of generating a population of natural killer (NK) cells from a population of stem cells, the method comprising contacting the population of stem cells or progenies thereof to a plurality of compositions in a sequential manner, to generate the population NK cells, wherein the plurality of compositions comprises: a first composition comprising an activator of Wnt/ ⁇ -catenin signaling pathway; a second composition comprising one or more members selected from the group consisting of bone morphogenetic protein (BMP) , a vascular endothelial growth factor, an inhibitor of activin receptor-like kinase (ALK) receptor signaling pathway, and a FGF; a third composition comprising an interleukin-7 (IL-7) , an IL-7, an IL-3, a BMP, a VEGF, and a FMS-like tyrosine kinase receptor ligand (FLTL) ; and a fourth composition comprising an IL-7 and/or
  • the population of stem cells or progenies thereof are subjected to each composition of the plurality of compositions in a medium that is substantially free of the other of the plurality of compositions.
  • one or more members of (i) the population of mesodermal cells, (ii) the population of mesodermal cells having HE potential, (iii) the population of HE cells, (iv) the population of NK cells, and/or (v) a progeny thereof is usable to treat a subject in need thereof.
  • the subject has or is suspected of having a disease.
  • the disease is cancer or tumor.
  • FIGs. 1A-1C illustrate validation of induced pluripotent stem cell (iPSC) pluripotency.
  • FIG. 1A show bright field photograph of iPSC before passaging for differentiation.
  • FIG. 1B shows flow cytometry of iPSCs for expression of SSEA4 and TRA-1-81.
  • FIG. 1C shows data quantification of flow cytometry of iPSCs for expression of SSEA4 and TRA-1-81.
  • FIGs. 2A-2B illustrate validation of hemogenic endothelium cells differentiated from iPSCs.
  • FIG. 2A shows flow cytometry of hemogenic endothelium cells differentiated from iPSCs for expression of CD31 and CD34 at day 7 of differentiation.
  • FIG. 2B shows data quantification of flow cytometry of hemogenic endothelium cells differentiated from iPSCs for expression of CD31 and CD34 at day 7 of differentiation.
  • FIGs. 3A-3D illustrate validation of natural killer (NK) cell differentiation at day 21.
  • FIG. 3A shows flow cytometry of NK cells for expression of CD56 at day 21 of differentiation.
  • FIG. 3B shows flow cytometry of NK cells for expression of NKp30 at day 21 of differentiation.
  • FIG. 3C shows flow cytometry of NK cells for expression of NKG2A at day 21 of differentiation.
  • FIG. 3D shows data quantification of flow cytometry of NK cells for expression of CD56, NKp30, and NKG2A at day 21 of differentiation.
  • FIGs. 4A-4D illustrate validation of NK cell differentiation at day 30.
  • FIG. 4A shows flow cytometry of NK cells for expression of CD56 at day 30 of differentiation.
  • FIG. 4B shows flow cytometry of NK cells for expression of NKp30 at day 30 of differentiation.
  • FIG. 4C shows flow cytometry of NK cells for expression of NKp44 at day 30 of differentiation.
  • FIG. 4D shows data quantification of flow cytometry of NK cells for expression of CD56, NKp30, and NKp44 at day 30 of differentiation.
  • FIGs. 5A-5D illustrate validation of NK cell differentiation at day 37.
  • FIG. 5A shows flow cytometry of NK cells for expression of CD56 at day 37 of differentiation.
  • FIG. 5B shows flow cytometry of NK cells for expression of NKp30 at day 37 of differentiation.
  • FIG. 5C shows flow cytometry of NK cells for expression of NKp44 at day 37 of differentiation.
  • FIG. 5D shows data quantification of flow cytometry of NK cells for expression of CD56, NKp30, and NKp44 at day 30 of differentiation.
  • FIG. 6 illustrates NK cell yield from one iPSC at day 30 and day 37.
  • FIG. 7 illustrates NK cell yield from one iPSC using conventional method versus method disclosed herein.
  • the term “at least, ” “greater than, ” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values the term “at least, ” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.
  • achimeric transmembrane receptor includes a plurality of chimeric transmembrane receptors.
  • the term “about” or “approximately” generally mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1%of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • a cell generally refers to a biological cell.
  • a cell can be the basic structural, functional and/or biological unit of a living organism.
  • a cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g.
  • algal cell e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like
  • seaweeds e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like
  • seaweeds e.g.
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g. fruit fly, cnidarian, echinoderm, nematode, etc.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a cell is not originating from a natural organism (e.g. a cell can be a synthetically made, sometimes termed an artificial cell) .
  • differentiated generally refers to a process by which an unspecialized (“uncommitted” ) or less specialized cell acquires the features of a specialized cell such as, e.g., an immune 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” generally 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 generally refers to the ability of a cell to form all lineages of the body or soma (i.e., 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 can be a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell) , 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) .
  • iPSCs induced pluripotent stem cells
  • differentiated cells e.g., differentiated adult, neonatal, or fetal cells
  • iPSCs reprogrammed stem cells
  • the iPSCs produced do not refer to cells as they are found in nature.
  • iPSCs can be engineered to differentiation directly into committed cells (e.g., natural killer (NK) cells) .
  • NK natural killer
  • iPSCs can be engineered to differentiate first into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) ) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
  • tissue-specific stem cells e.g., hematopoietic stem cells (HSCs)
  • HSCs hematopoietic stem cells
  • ESCs generally refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm.
  • ESCs can be engineered to differentiation directly into committed cells (e.g., NK cells) .
  • ESCs can be engineered to differentiate first into tissue-specific stem cells (e.g., HSCs) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
  • isolated stem cells generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs) , etc. ) that are isolated from a multicellular organism.
  • HSCs can be isolated from a mammal’s body, such as a human body.
  • an embryonic stem cells can be isolated from an embryo.
  • isolated generally refers to a cell or a population of cells, which has been separated from its original environment.
  • a new environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist.
  • An isolated cell can be a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample.
  • the term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated form a cell culture or cell suspension. Therefore, an isolated cell is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
  • hematopoietic stem and progenitor cells generally refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into NK cells) and include, multipotent hematopoietic stem cells (hematoblasts) , myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors.
  • hematoblasts multipotent hematopoietic stem cells
  • HSCs Hematopoietic stem and progenitor cells
  • myeloid monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages T cells, B cells, NK cells
  • HSCs can be CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T cells, NK cells and B cells.
  • immune cell generally refers to a differentiated hematopoietic cell.
  • Non-limiting examples of an immune cell can include an NK cell, a T cell, a monocyte, an innate lymphocyte, a tumor-infiltrating lymphocyte, a macrophage, a granulocyte, etc.
  • NK cell or “Natural Killer cell” generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3) .
  • NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceR ⁇ , and EAT-2.
  • isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30 (e.g., NKP30A and/or NKP30B isoforms) , NKp40, NKp46, NKp44, activating and inhibitory KIRs, NKG2A and/or DNAM-1.
  • NKp30 e.g., NKP30A and/or NKP30B isoforms
  • NKp40, NKp46, NKp44 activating and inhibitory KIRs, NKG2A and/or DNAM-1.
  • NK cells can exhibit expression of activating NK receptor such as CD100 (SEMA4D) , CD16 (FcgRIIIA) , CD160 (BY55) , CD244 (2B4, SLAMF4) , CD27, CD94–NKG2C, CD94–NKG2E, CD94-NKG2H, CD96, CRTAM, DAP12, DNAM1 (CD226) , KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, Ly49, NCR, NKG2D (KLRK1, CD314) , NKp30 (NCR3) , NKp44 (NCR2) , NKp46 (NCR1) , NKp80 (KLRF1, CLEC5C) , NTB-A (SLAMF6) , PSGL1, SLAMF7 (CRACC, CS1, CD319) , CD161 (NKR-P1A, NK1.1)
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • a nucleotide can comprise a synthetic nucleotide.
  • a nucleotide can comprise a synthetic nucleotide analog.
  • Nucleotides can be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) .
  • nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives can include, for example, [ ⁇ S] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots.
  • Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM) , 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4- (4′dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) .
  • FAM 5-carboxyfluorescein
  • JE 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein
  • fluorescently labeled nucleotides can include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] ddTTP available from Perkin Elmer, Foster City, Calif.
  • Chromosome Labeled Nucleotides BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.
  • Nucleotides can also be labeled or marked by chemical modification.
  • a chemically-modified single nucleotide can be biotin-dNTP.
  • biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP) , biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP) , and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP) .
  • polynucleotide oligonucleotide, ” or “nucleic acid, ” as used interchangeably herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form.
  • a polynucleotide can be exogenous or endogenous to a cell.
  • a polynucleotide can exist in a cell-free environment.
  • a polynucleotide can be a gene or fragment thereof.
  • a polynucleotide can be DNA.
  • a polynucleotide can be RNA.
  • a polynucleotide can have any three dimensional structure, and can perform any function, known or unknown.
  • a polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase) . If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g.
  • rhodamine or flurescein linked to the sugar thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • the term “gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript.
  • genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5′and 3′ends.
  • the term encompasses the transcribed sequences, including 5′and 3′untranslated regions (5′-UTR and 3′-UTR) , exons and introns.
  • the transcribed region will contain “open reading frames” that encode polypeptides.
  • a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region” ) necessary for encoding a polypeptide.
  • genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes.
  • rRNA ribosomal RNA genes
  • tRNA transfer RNA
  • the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters.
  • a gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism.
  • a gene can refer to an “exogenous gene” or a non-native gene.
  • a non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer.
  • a non-native gene can also refer to a gene not in its natural location in the genome of an organism.
  • a non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .
  • expression generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides can be collectively referred to as “gene product. ” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.
  • Up-regulated, with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state.
  • Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time.
  • stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell.
  • a selection advantage may be a resistance towards a certain toxin that is presented to the cell.
  • amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains) .
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component.
  • amino acid and amino acids, ” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues.
  • Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid.
  • Amino acid analogues can refer to amino acid derivatives.
  • amino acid includes both D-amino acids and L-amino acids.
  • derivative, ” “variant, ” or “fragment, ” as used herein with reference to a polypeptide generally refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary) , activity (e.g., enzymatic activity) and/or function.
  • Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions) , truncations, modifications, or combinations thereof compared to a wild type polypeptide.
  • engineered, ” “chimeric, ” or “recombinant, ” as used herein with respect to a polypeptide molecule generally refers to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the polypeptide molecule, as well as cells or organisms which express the polypeptide molecule.
  • Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion.
  • an engineered or recombinant polynucleotide e.g.,
  • gene editing moiety generally refers to a moiety which can edit a nucleic acid sequence, whether exogenous or endogenous to a cell comprising the nucleic acid sequence.
  • a gene editing moiety regulates expression of a gene by editing a nucleic acid sequence.
  • a gene editing moiety can regulate expression of a gene by editing genomic DNA sequence.
  • a gene editing moiety can regulate expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can, in some cases, alter the underlying template for gene expression.
  • a gene editing moiety can be capable of regulating expression or activity of a gene by specifically binding to a target sequence operatively coupled to the gene (or a target sequence within the gene) , and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA.
  • a gene editing moiety can recruit or comprise at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA.
  • a gene editing moiety can itself bind to DNA and regulate transcription by physical obstruction, for example preventing proteins such as RNA polymerase and other associated proteins from assembling on a DNA template.
  • a gene editing moiety can regulate expression of a gene at the translation level, for example, by regulating the production of protein from mRNA template.
  • a gene editing moiety can regulate gene expression by affecting the stability of an mRNA transcript.
  • antibody generally refers to a proteinaceous binding molecule with immunoglobulin-like functions.
  • the term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) , as well as derivatives, variants, and fragments thereof.
  • Antibodies include, but are not limited to, immunoglobulins (Ig's ) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc. ) .
  • a derivative, variant or fragment thereof can refer to a functional derivative or fragment which retains the binding specificity (e.g., complete and/or partial) of the corresponding antibody.
  • Antigen-binding fragments include Fab, Fab′, F (ab′) 2, variable fragment (Fv) , single chain variable fragment (scFv) , minibodies, diabodies, and single-domain antibodies ( “sdAb” or “nanobodies” or “camelids” ) .
  • the term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies) .
  • Non-limiting examples of the antibodies can include, but are not limited to, monospecific or monovalent antibodies (e.g., CH-Fc/VL-Fc, Fc/Fab-Fc, HC-FC/LC-Fc, etc. ) , IgG-like formats with appendages of scFv and/or scFab (e.g., scFv-Fc, scFv-Fc/scFv-Fc, Fab-Fc, Fab-Fc/Fab-Fc, Fab-Fc/scFab-Fc, Fv-/Fv-CrossMab IgG, Fab-CrossMab IgG, etc.
  • monospecific or monovalent antibodies e.g., CH-Fc/VL-Fc, Fc/Fab-Fc, HC-FC/LC-Fc, etc.
  • IgG-like formats with appendages of scFv and/or scFab e.g
  • IgG formats with correct LC associations e.g., IgG, CrossMab IgG, common LC-IgG, Ortho-Fab IgG, Four-in-One Cross-Mab IgG, etc. .
  • chimeric polypeptide receptor generally refers to a non-natural polypeptide receptor comprising one or more antigen binding moieties, each antigen binding moiety capable of binding to a specific antigen.
  • a chimeric polypeptide receptor can be monospecific (i.e., capable of binding to one type of specific antigen) .
  • a chimeric polypeptide receptor can be multi-specific (i.e., capable of binding to two or more different types of specific antigens) .
  • a chimeric polypeptide receptor can be monovalent (i.e., comprising a single antigen binding moiety) .
  • a chimeric polypeptide receptor can be multivalent (i.e., comprising a plurality of antigen binding moieties) .
  • a chimeric polypeptide receptor can comprise a T-cell receptor (TCR) fusion protein (TFP) or a chimeric antigen receptor (CAR) .
  • TCR T-cell receptor
  • TFP T-cell receptor
  • an antigen binding domain generally refers to a construct exhibiting preferential binding to a specific target antigen.
  • An antigen binding domain can be a polypeptide construct, such as an antibody, modification thereof, fragment thereof, or a combination thereof.
  • the antigen binding domain can be any antibody as disclosed herein, or a functional variant thereof.
  • Non-limiting examples of an antigen binding domain can include a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy-chain-only antibody (VNAR) , Ig NAR, a chimeric antibody, a recombinant antibody, or antibody fragment thereof.
  • Non-limiting examples of antibody fragment include Fab, Fab′, F (ab) ′2, F(ab) ′3, Fv, single chain antigen binding fragment (scFv) , (scFv) 2, disulfide stabilized Fv (dsFv) , minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb, Nanobody) , recombinant heavy-chain-only antibody (VHH) , and other antibody fragments that maintain the binding specificity of the whole antibody.
  • safety switch generally refers to an engineered polypeptide construct designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. When expressed in a cell, the safety switch can induce death of the host cell, thereby inactivating activity of the cell in a host (e.g., in a subject’s body) .
  • the safety switch can be a suicide moiety.
  • the cell can be programmed to express the suicide moiety at certain stage of its life-cycle (e.g., time-programmed) . In some cases, expression of the suicide moiety in a cell can be conditional or inducible.
  • conditional regulation (e.g., expression) of a suicide moiety can include control through a small molecule-mediated post-translational activation and tissue-specific and/or temporal transcriptional regulation.
  • the safety switch can be an inducible suicide moiety.
  • a safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and/or antibody-mediated depletion.
  • a safety switch can be activated by an exogenous molecule (e.g., a drug or a prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., engineered NK cell as disclosed herein) .
  • an exogenous molecule e.g., a drug or a prodrug
  • apoptosis and/or cell death of a cell e.g., engineered NK cell as disclosed herein
  • hypo-immunity regulator generally refers to a polypeptide construct in a cell, wherein either enhanced expression (e.g., via knock-in of a heterologous gene) or reduced expression (e.g., via knock-out or knock-down of an endogenous gene) of the hypo-immunity regulator in the cell can help the cell to reduce or avoid immune response (e.g., immune attack, such as adaptive immune rejection) from a host’s body upon administration to the host’s body.
  • immune response e.g., immune attack, such as adaptive immune rejection
  • cells e.g., engineered NK cells as disclosed herein
  • the hypo-immunity regulator can be modified to exhibit either enhanced expression or reduced expression of the hypo-immunity regulator, such that the cells can evade the host immune attack upon second or further infusion of the cells into the host (i.e., recipient) .
  • the cells (i) would not be rejected by the host’s immune system and/or (ii) would be rejected at a slower rate by the host’s immune system as compared with a control cell without the enhanced expression or reduced expression of the hypo-immunity regulator.
  • a cell exhibiting the enhanced expression or reduced expression of the hypo-immunity regulator can be referred to as exhibiting “hypo-immunity” or being “immune-privileged. ”
  • immune response generally refers to T cell mediated and/or B cell mediated immune responses from a host’s immune system to an object (e.g., a foreign object) .
  • An example of an immune response include T cell responses, e.g., cytokine production and cellular cytotoxicity.
  • an immune response can be indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, such as macrophages.
  • the term “enhanced expression, ” “increased expression, ” or “upregulated expression” generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of expression can be substantially zero (or null) or higher than zero.
  • the moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain.
  • the moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain.
  • a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.
  • the term “enhanced activity, ” “increased activity, ” or “upregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of activity can be substantially zero (or null) or higher than zero.
  • the moiety of interest can comprise a polypeptide construct of the host strain.
  • the moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain.
  • a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.
  • reduced expression, ” “decreased expression, ” or “downregulated expression” generally refers to a production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is below a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of expression is higher than zero.
  • the moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain.
  • the moiety of interest can be knocked-out or knocked-down in the host strain.
  • reduced expression of the moiety of interest can include a complete inhibition of such expression in the host strain.
  • reduced activity, ” “decreased activity, ” or “downregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is below a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) .
  • the normal level of activity is higher than zero.
  • the moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain.
  • the moiety of interest can be knocked-out or knocked-down in the host strain.
  • reduced activity of the moiety of interest can include a complete inhibition of such activity in the host strain.
  • subject generally refers to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • treatment generally refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a treatment can comprise administering a system or cell population disclosed herein.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • an effective amount or “therapeutically effective amount” generally refers to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and/or NK cells) comprising a system of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof.
  • lymphocytes e.g., T lymphocytes and/or NK cells
  • therapeutically effective generally refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.
  • Differentiated cells produced from stem cells can be produced through multiple methods. Some protocols may use embryonic fibroblast feeder layers in the culture system. However, the use of feeder systems can result in pathogen transmission and viral infection of the stem cells. As a result, the eventual differentiated cells may be unusable in a clinical or research setting. The use of feeder-free culture systems with small molecules and growth factors can generate differentiated cells that are suitable to clinical applications.
  • the present disclosure describes methods for regulating cell fate, e.g., differentiation of a population of cells into a more differentiated state.
  • a population of stem cells can be differentiated into immune cells (e.g., NK cells, T cells, etc. ) via one or more steps.
  • immune cells e.g., NK cells, T cells, etc.
  • each step of the one or more steps can comprise unique compositions designed for the cell fate regulation.
  • the immunes cells generated by the methods of the present disclosure can be utilized for immunotherapy, e.g., to treat or ameliorate diseases, such as cancer or tumor.
  • the engineered immune cells that are prepared as disclosed herein can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) .
  • the engineered immune cells can be autologous to the subject.
  • the engineered immune cells can be allogeneic to the subject.
  • the present disclosure also describes compositions for regulating cell fate in accordance with the methods herein
  • the present disclosure provides a method (e.g., an in vitro or ex vivo method) of generating stem cell-derived mesodermal cells.
  • the method can comprise contacting a population of stem cells with a composition comprising an activator of Wnt/ ⁇ -catenin signaling pathway (activator) .
  • activator can be sufficient to transform the population of stem cells into a population of mesodermal cells.
  • _the contacting can occur in a medium that is substantially free of a transforming growth factor (TGF) , e.g., TGF ⁇ 1.
  • TGF transforming growth factor
  • the medium can be substantially free of at least 1, at least 2, at least 3, at least 4, at least 5, or more specific sub-types of TGF.
  • TGF can include TGF ⁇ 1, TGF ⁇ 1, TGF ⁇ 2, or TGF ⁇ 3.
  • the contacting can occur in the medium as described herein for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, or more.
  • the contacting can occur in the medium as described herein for at most about 5 days, at most about 4 days, at most about 3 days, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 20 hours, at most about 16 hours, at most about 12 hours, at most about 8 hours, at most about 6 hours, at most about 4 hours, at most about 2 hours, at most about 1 hour, or less.
  • the medium that is substantially free of TGF can have a concentration of TGF that is less than about 100 picograms per milliliter (pg/ml) , less than about 50 pg/ml, less than about 20 pg/ml, less than about 10 pg/ml, less than about 5 pg/ml, less than about 2 pg/ml, less than about 1 pg/ml, less than about 0.5 pg/ml, less than about 0.2 pg/ml, less than about 0.1 pg/ml, less than about 0.05 pg/ml, less than about 0.02 pg/ml, less than about 0.01 pg/ml, or less.
  • stem cells can be engineered into stem cell-derived mesodermal cells in a medium that comprises one or more types of TGF, e.g., TGF ⁇ 1.
  • the contacting can occur in a medium that is substantially free of a fibroblast growth factor (FGF) , e.g., bFGF.
  • FGF fibroblast growth factor
  • the medium can be substantially free of at least 1, at least 2, at least 3, at least 4, at least 5, or more specific sub-types of FGF.
  • FGF can include FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23.
  • the contacting can occur in the medium as described herein for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, or more.
  • the contacting can occur in the medium as described herein for at most about 5 days, at most about 4 days, at most about 3 days, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 20 hours, at most about 16 hours, at most about 12 hours, at most about 8 hours, at most about 6 hours, at most about 4 hours, at most about 2 hours, at most about 1 hour, or less.
  • the medium that is substantially free of FGF can have a concentration of FGF that is less than about 1,000 picograms per milliliter (pg/ml)
  • the concentration of FGF in the medium can be between about 1 pg/ml and about 999 pg/ml, between about 1 pg/ml and about 100 pg/ml, between about 100 pg/ml and about 200 pg/ml, between about 200 pg/ml and about 300 pg/ml, between about 300 pg/ml and about 400 pg/ml, between about 400 pg/ml and about 500 pg/ml, between about 500 pg/ml and about 600 pg/ml, between about 600 pg/ml and about 700 pg/ml, between about 700 pg/ml and about 800 pg/ml, between about 800 pg/ml and about
  • a concentration of the FGF in the medium can be less than about 1,000 pg/ml, less than about 500 pg/ml, less than about 200 pg/ml, less than about 100 pg/ml, less than about 50 pg/ml, less than about 20 pg/ml, less than about 10 pg/ml, less than about 5 pg/ml, less than about 2 pg/ml, less than about 1 pg/ml, less than about 0.5 pg/ml, less than about 0.2 pg/ml, less than about 0.1 pg/ml, less than about 0.05 pg/ml, less than about 0.02 pg/ml, less than about 0.01 pg/ml, or less.
  • stem cells can be engineered into stem cell-derived mesodermal cells in a medium that comprises one or more types of FGF, e.g., bFGF.
  • FGF e.g., bFGF
  • the population of mesodermal cells can exhibit a greater propensity to differentiate into immune cells (e.g., differentiation into T cells, NK cells, NKT cells, etc. from, for example, hematopoietic stem cells derived from the mesodermal cells as described herein) , as compared to a control population of mesodermal cells that have been transformed from stem cells in a control medium.
  • immune cells e.g., differentiation into T cells, NK cells, NKT cells, etc. from, for example, hematopoietic stem cells derived from the mesodermal cells as described herein
  • the population of mesodermal cells as prepared by the subject method as described herein can exhibit at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 200%, or more greater propensity to differentiate into immune cells compared to the control population.
  • the population of mesodermal cells can exhibit at most about 200%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less greater propensity to differentiate into immune cells compared to the control population.
  • the propensity to differentiate into immune cells can be ascertained by differentiation efficiency (e.g., differentiation into NK cells) , proliferative capacity of the resulting immune cells, cytotoxicity of the resulting immune cells, or cytokine release from the resulting immune cells.
  • differentiation efficiency e.g., differentiation into NK cells
  • proliferative capacity of the resulting immune cells e.g., proliferative capacity of the resulting immune cells
  • cytotoxicity of the resulting immune cells cytokine release from the resulting immune cells.
  • Number of resulting immune cells or functional activity of resulting immune cells can be ascertained by a number of methods, including, but not limited to, flow cytometry, intracellular cytokine staining, ELISpot, ELISA, proliferation assays, and cytotoxicity assays.
  • control medium as disclosed herein can comprise TGF (e.g., TGF ⁇ 1) and/or FGF (e.g., bFGF) .
  • control medium for the control population of mesodermal cells can comprise TGF, such as TGF ⁇ 1.
  • control medium for the control population of mesodermal cells can comprise FGF, such as bFGF.
  • control medium for the control population of mesodermal cells can comprise TGF and FGF.
  • the control medium can be substantially free of the activator.
  • the control medium can have an amount of the activator that is less than the subject method as described herein.
  • the activator as provided herein can be a small molecule, a nucleotide, a polynucleotide, an amino acid, a polypeptide (e.g., a small peptide of a protein, such as a cytokine, growth factor, antibody, a receptor or a fragment thereof, etc. ) , or combinations thereof. In some cases, the activator can be a small molecule.
  • the activator can have a molecular weight (e.g., number average molecular weight) of less than or equal to about 10000 Daltons, less than or equal to about 5000 Daltons, less than or equal to about 4000 Daltons, less than or equal to about 3000 Daltons, less than or equal to about 2500 Daltons, less than or equal to about 2000 Daltons, less than or equal to about 1500 Daltons, less than or equal to about 1000 Daltons, less than or equal to about 900 Daltons, less than or equal to about 800 Daltons, less than or equal to about 700 Daltons, less than or equal to about 600 Daltons, less than or equal to about 500 Daltons, less than or equal to about 400 Daltons, less than or equal to about 300 Daltons, or less than or equal to about 200 Daltons.
  • a molecular weight e.g., number average molecular weight
  • the activator can have a molecular weight (e.g., number average molecular weight) or less than or equal to about 10000 grams per mole, less than or equal to about 5000 grams per mole, less than or equal to about 4000 grams per mole, less than or equal to about 3000 grams per mole, less than or equal to about 2500 grams per mole, less than or equal to about 2000 grams per mole, less than or equal to about 1500 grams per mole, less than or equal to about 1000 grams per mole, less than or equal to about 900 grams per mole, less than or equal to about 800 grams per mole, less than or equal to about 700 grams per mole, less than or equal to about 600 grams per mole, less than or equal to about 500 grams per mole, less than or equal to about 400 grams per mole, less than or equal to about 300 grams per mole, or less than or equal to about 200 grams per mole.
  • a molecular weight e.g., number average molecular weight
  • the activator can interact with one or more components of the stem cells, such as, for example, extracellular proteins, transmembrane proteins (e.g., receptors) , intracellular proteins, intercellular polynucleotides, intracellular small molecules (e.g., lipids) , etc.
  • extracellular proteins e.g., extracellular proteins
  • transmembrane proteins e.g., receptors
  • intracellular proteins e.g., intercellular polynucleotides
  • intracellular small molecules e.g., lipids
  • the activator can be a regulator of a pathway (e.g., an intracellular signaling pathway) involving Wnt/ ⁇ -catenin, e.g., an activator of Wnt/ ⁇ -catenin.
  • the regulator can activate function or activity of one or more proteins involved in such pathway (e.g., a protein that effects activation of the Wnt/ ⁇ -catenin signaling pathway, such as Dvl) .
  • the activator can be a polypeptide.
  • the polypeptide can be a Wnt protein.
  • Wnt proteins can include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16.
  • the activator can be lithium chloride.
  • the activator can be a regulator of a pathway (e.g., an intracellular signaling pathway) involving GSK, e.g., an inhibitor of GSK, such as GSK-3 ⁇ / ⁇ .
  • the regulator can inhibit function or activity of one or more proteins involved in such pathway, such as GSK.
  • GSK inhibitors can include CHIR-99021, CHIR-98014, LY2090314, BIO, TWS119, Tideglusib, or SB216763.
  • the regulator can activate function or activity of one or more proteins involved in such pathway, such as Akt.
  • Akt can include SC79.
  • the activator can be a polypeptide.
  • the activator can a cytokine, such as a ⁇ -chain receptor-related cytokine.
  • a cytokine such as a ⁇ -chain receptor-related cytokine.
  • ⁇ -chain receptor-related cytokines can be I-2, IL-7, IL-15, or IL-21.
  • the composition comprising an activator can have a concentration of the activator between about 1 and about 20 micromolar ( ⁇ M) , such as about 10 ⁇ M.
  • concentration of the activator can be at least about 1 ⁇ M, at least about 2 ⁇ M, at least about 4 ⁇ M, at least about 6 ⁇ M, at least about 8 ⁇ M, at least about 10 ⁇ M, at least about 12 ⁇ M, at least about 14 ⁇ M, at least about 16 ⁇ M, at least about 18 ⁇ M, or at least about 20 ⁇ M.
  • the concentration of the activator can be at most about 20 ⁇ M, at most about 18 ⁇ M, at most about 16 ⁇ M, at most about 14 ⁇ M, at most about 12 ⁇ M, at most about 10 ⁇ M, at most about 8 ⁇ M, at most about 6 ⁇ M, at most about 4 ⁇ M, at most about 2 ⁇ M, or at most about 1 ⁇ M.
  • the composition comprising an activator can have a concentration of the activator between about 0.5 micrograms per milliliter ( ⁇ g/ml) and about 10 ⁇ g/ml, e.g., about 5 ⁇ g/ml.
  • the concentration of the activator can be at least about 0.5 ⁇ g/ml, at least about 1 ⁇ g/ml, at least about 1.5 ⁇ g/ml, at least about 2 ⁇ g/ml, at least about 2.5 ⁇ g/ml, at least about 3 ⁇ g/ml, at least about 3.5 ⁇ g/ml, at least about 4 ⁇ g/ml, at least about 4.5 ⁇ g/ml, at least about 5 ⁇ g/ml, at least about 5.5 ⁇ g/ml, at least about 6 ⁇ g/ml, at least about 6.5 ⁇ g/ml, at least about 7 ⁇ g/ml, at least about 7.5 ⁇ g/ml, at least about 8 ⁇ g/ml, at least about 8.5 ⁇ g/ml, at least about 9 ⁇ g/ml, at least about 9.5 ⁇ g/ml, at least about 10 ⁇ g/ml, or more.
  • the concentration of the activator can be at most about 10 ⁇ g/ml, at most about 9.5 ⁇ g/ml, at most about 9 ⁇ g/ml, at most about 8.5 ⁇ g/ml, at most about 8 ⁇ g/ml, at most about 7.5 ⁇ g/ml, at most about 7 ⁇ g/ml, at most about 6.5 ⁇ g/ml, at most about 6 ⁇ g/ml, at most about 5.5 ⁇ g/ml, at most about 5 ⁇ g/ml, at most about 4.5 ⁇ g/ml, at most about 4 ⁇ g/ml, at most about 3.5 ⁇ g/ml, at most about 3 ⁇ g/ml, at most about 2.5 ⁇ g/ml, at most about 2 ⁇ g/ml, at most about 1.5 ⁇ g/ml, at most about 1 ⁇ g/ml, at most about 0.5 ⁇ g/ml, or less.
  • the composition comprising an activator can have a concentration of the activator between about 0.5 nanogram per milliliter (ng/ml) and about 10 ng/ml, e.g., about 5,000 ng/ml.
  • the concentration of the activator can be at least about 0.5 ng/ml, at least about 1 ng/ml, at least about 1.5 ng/ml, at least about 2 ng/ml, at least about 2.5 ng/ml, at least about 3 ng/ml, at least about 3.5 ng/ml, at least about 4 ng/ml, at least about 4.5 ng/ml, at least about 5 ng/ml, at least about 5.5 ng/ml, at least about 6 ng/ml, at least about 6.5 ng/ml, at least about 7 ng/ml, at least about 7.5 ng/ml, at least about 8 ng/ml, at least about 8.5 ng/ml, at least about 9
  • the concentration of the activator can be at most about 10 ng/ml, at most about 9.5 ng/ml, at most about 9 ng/ml, at most about 8.5 ng/ml, at most about 8 ng/ml, at most about 7.5 ng/ml, at most about 7 ng/ml, at most about 6.5 ng/ml, at most about 6 ng/ml, at most about 5.5 ng/ml, at most about 5 ng/ml, at most about 4.5 ng/ml, at most about 4 ng/ml, at most about 3.5 ng/ml, at most about 3 ng/ml, at most about 2.5 ng/ml, at most about 2 ng/ml, at most about 1.5 ng/ml, at most about 1 ng/ml, at most about 0.5 ng/ml, or less.
  • the composition comprising an activator can have a concentration of the activator between 0.5 picogram per milliliter (pg/ml) and about 10 pg/ml.
  • the concentration of the activator can be at least about 0.5 pg/ml, at least about 1 pg/ml, at least about 1.5 pg/ml, at least about 2 pg/ml, at least about 2.5 pg/ml, at least about 3 pg/ml, at least about 3.5 pg/ml, at least about 4 pg/ml, at least about 4.5 pg/ml, at least about 5 pg/ml, at least about 5.5 pg/ml, at least about 6 pg/ml, at least about 6.5 pg/ml, at least about 7 pg/ml, at least about 7.5 pg/ml, at least about 8 pg/ml, at least about 8.5 pg/ml, at least
  • the concentration of the activator can be at most about 10 pg/ml, at most about 9.5 pg/ml, at most about 9 pg/ml, at most about 8.5 pg/ml, at most about 8 pg/ml, at most about 7.5 pg/ml, at most about 7 pg/ml, at most about 6.5 pg/ml, at most about 6 pg/ml, at most about 5.5 pg/ml, at most about 5 pg/ml, at most about 4.5 pg/ml, at most about 4 pg/ml, at most about 3.5 pg/ml, at most about 3 pg/ml, at most about 2.5 pg/ml, at most about 2 pg/ml, at most about 1.5 pg/ml, at most about 1 pg/ml, at most about 0.5 pg/ml, or less.
  • the activator can be CHIR, such as CHIR-99021 or CHIR-98014.
  • the population of mesodermal cells can be characterized by expressing a greater level of a mesodermal marker compared to that of the population of stem cells.
  • the mesodermal marker can be selected from the group consisting of Kinase Insert Domain Receptor (KDR) , Eomesodermin (EOMES) , Mix Paired-Like Homebox (MIXL1) , and CD235a.
  • the population of mesodermal cells can express a greater level of KDR and CD235a as compared to that of the population of stem cells.
  • the population of mesodermal cells can express a greater level of mesodermal markers compared to the population of stem cells of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 200%.
  • the population of mesodermal cells can express a greater level of mesodermal markers compared to the population of stem cells of at most about 200%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less.
  • the population of mesodermal cells can express at least one of (e.g., at least 1 of, at least 2 of, at least 3 of, or all 4 of) KDR, EOMES, MIXL1, and CD235a.
  • the population of mesodermal cells can express KDR.
  • the population of mesodermal cells can express KDR and EOMES.
  • the population of mesodermal cells can express KDR and MIXL1.
  • the population of mesodermal cells can express KDR and CD235a.
  • the population of mesodermal cells can express KDR, EOMES, and MIXL1.
  • the population of mesodermal cells can express KDR, EOMES, and CD235a.
  • the population of mesodermal cells can express KDR, MIXL1, and CD235a. In some cases, the population of mesodermal cells can express EOMES. In some cases, the population of mesodermal cells can express EOMES and MIXL1. In some cases, the mesodermal cells can express EOMES and CD235a. In some cases, the mesodermal cells can express EOMES, MIXL1, and CD235a. In some cases, the mesodermal cells can express MIXL1. In some cases, the mesodermal cells can express MIXL1 and CD235a. In some cases, the mesodermal cells can express CD235a.
  • the population of mesodermal cells can be characterized by expressing a lower level of a stem cell marker, as compared to that of the population of stem cells.
  • the stem cell marker can be selected from the group consisting of 5TA, ABCG2, Activin RIB (ALK-4) , Activin RIIB, Alkaline Phosphatase (ALPL) , B18R, E-Cadherin, Cbx2, CD9, CD30 (TNFRSF8) , CD117 (c-kit) , CDX2, CDH1, Cripto, DNMT3B, DPPA2, DPPA4, DPPA5 (ESG1) , EpCAM (TROP1) , ERR- ⁇ (NR3B2) , ERVMER34-1, ESGP, F-box protein 15 (FBXO15) , FGF-4, FGF-5, FoxD3, GBX2, GCNF (NR6A1) , GDF-3, Integrin ⁇ 6 (
  • Expression or activity level of a protein of interest can be ascertained by a number of methods, including, but not limited to phosphorylation (e.g., that of the protein of interest or a downstream signaling protein thereof) via Western blotting, and/or expression assay via Western blotting or polymerase chain reaction (PCR) assays.
  • phosphorylation e.g., that of the protein of interest or a downstream signaling protein thereof
  • PCR polymerase chain reaction
  • the population of mesodermal cells may not exhibit hemogenic endothelium (HE) potential, e.g., the population of mesodermal cells may not exhibit sufficient HE potential to effect differentiation into hemogenic endothelial lineage.
  • the population of mesodermal cells may not exhibit enhanced hemogenic endothelium (HE) potential as compared to the population of stem cells.
  • the population of mesodermal cells that does not exhibit enhanced HE potential may be KDR+ CD235a+.
  • the population of mesodermal cells that does not exhibit enhanced HE potential may be KDR+.
  • the population of mesodermal cells that does not exhibit enhanced HE potential may be CD235a+.
  • the population of stem cells can be contacted by the composition (e.g., cultured or immersed in the composition comprising a medium) as disclosed herein for less than or equal to about 5 days.
  • the population of stem cells can be contacted by the composition for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, or more.
  • the population of stem cells can be contacted by the composition for at most about 5 days, at most about 4 days, at most about 3 days, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 20 hours, at most about 16 hours, at most about 12 hours, at most about 8 hours, at most about 6 hours, at most about 4 hours, at most about 2 hours, at most about 1 hour, or less.
  • the population of stem cells can be contacted by the composition for less than or equal to 3 days. In some cases, the population of stem cells can be contacted by the composition for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 48 hours, at least about 52 hours, at least about 56 hours, at least about 60 hours, at least about 64 hours, at least about 68 hours, at least about 72 hours, or more.
  • the population of stem cells can be contacted by the composition for at most about 72 hours, at most about 68 hours, at most about 64 hours, at most about 60 hours, at most about 56 hours, at most about 52 hours, at most about 48 hours, at most about 44 hours, at most about 40 hours, at most about 36 hours, at most about 32 hours, at most about 28 hours, at most about 24 hours, at most about 22 hours, at most about 20 hours, at most about 18 hours, at most about 16 hours, at most about 14 hours, at most about 12 hours, at most about 11 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about 2 hours, at most about 1 hour, or less.
  • the population of stem cells comprises pluripotent stem cells.
  • the pluripotent stem cells can be induced pluripotent stem cells (iPSC) , germ cell-derived stem cells, embryonal carcinoma cells (ECCs) , embryonic stem cells (ESC) , embryonic stem cells made by somatic cell nuclear transfer (ntESC) , or parthenogenesis embryonic stem cells (pESC) .
  • iPSC induced pluripotent stem cells
  • ECCs embryonal carcinoma cells
  • ESC embryonic stem cells
  • ntESC embryonic stem cells made by somatic cell nuclear transfer
  • pESC parthenogenesis embryonic stem cells
  • the population of stem cells were initially terminally differentiated somatic cells that can be reverted to pluripotency.
  • the somatic cells are reverted to pluripotency by chemical reprogramming.
  • the somatic cells are reverted to pluripotency by genetic reprogramming.
  • the present disclosure provides a population of stem cell-derived mesodermal cells that can be generated by any one of the methods described herein.
  • the present disclosure provides one or more compositions for implementing any one of the methods described herein, e.g., for generating a population of stem cell-derived mesodermal cells.
  • the present disclosure provides a method (e.g., an in vitro or ex vivo method) of generating mesodermal cells that can have definitive hemogenic endothelium (HE) potential.
  • the method can comprise contacting a population of mesodermal cells with a composition comprising (i) a bone morphogenetic protein (BMP) , (ii) a vascular endothelial growth factor (VEGF) , and (iii) an inhibitor of activin receptor-like kinase (ALK) receptor signaling pathway.
  • BMP bone morphogenetic protein
  • VEGF vascular endothelial growth factor
  • ALK activin receptor-like kinase
  • an amount of the BMP in the composition can be less than about 30 ng/ml. In some cases, the amount of the BMP in the composition can be at least about 1 ng/ml, at least about 2 ng/ml, at least about 3 ng/ml, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 7 ng/ml, at least about 8 ng/ml, at least about 9 ng/ml, at least about 10 ng/ml, at least about 11 ng/ml, at least about 12 ng/ml, at least about 13 ng/ml, at least about 14 ng/ml, at least about 15 ng/ml, at least about 16 ng/ml, at least about 17 ng/ml, at least about 18 ng/ml, at least about 19 ng/ml, at least about 20 ng/ml, at least about 21 ng/ml, at least about 22
  • the amount of the BMP in the composition can be at most about 30 ng/ml, at most about 28 ng/ml, at most about 27 ng/ml, at most about 26 ng/ml, at most about 25 ng/ml, at most about 24 ng/ml, at most about 23 ng/ml, at most about 22 ng/ml, at most about 21 ng/ml, at most about 20 ng/ml, at most about 19 ng/ml, at most about 18 ng/ml, at most about 17 ng/ml, at most about 16 ng/ml, at most about 15 ng/ml, at most about 14 ng/ml, at most about 13 ng/ml, at most about 12 ng/ml, at most about 11 ng/ml, at most about 10 ng/ml, at most about 9 ng/ml, at most about 8 ng/ml, at most about 7 ng/ml, at most about 6 ng/ml,
  • the amount of the BMP in the composition can be less than about 20 ng/ml. In some cases, the amount of the BMP in the composition can be at least about 1 ng/ml, at least about 2 ng/ml, at least about 3 ng/ml, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 7 ng/ml, at least about 8 ng/ml, at least about 9 ng/ml, at least about 10 ng/ml, at least about 11 ng/ml, at least about 12 ng/ml, at least about 13 ng/ml, at least about 14 ng/ml, at least about 15 ng/ml, at least about 16 ng/ml, at least about 17 ng/ml, at least about 18 ng/ml, at least about 19 ng/ml, or more.
  • the amount of the BMP in the composition can be at most about 20 ng/ml, at most about 19 ng/ml, at most about 18 ng/ml, at most about 17 ng/ml, at most about 16 ng/ml, at most about 15 ng/ml, at most about 14 ng/ml, at most about 13 ng/ml, at most about 12 ng/ml, at most about 11 ng/ml, at most about 10 ng/ml, at most about 9 ng/ml, at most about 8 ng/ml, at most about 7 ng/ml, at most about 6 ng/ml, at most about 5 ng/ml, at most about 4 ng/ml, at most about 3 ng/ml, at most about 2 ng/ml, at most about 1 ng/ml, or less.
  • the amount of the BMP can be less than about 15 ng/ml. In some cases, the amount of the BMP can be at least 1 ng/ml, at least about 2 ng/ml, at least about 3 ng/ml, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 7 ng/ml, at least about 8 ng/ml, at least about 9 ng/ml, at least about 10 ng/ml, at least about 11 ng/ml, at least about 12 ng/ml, at least about 13 ng/ml, at least about 14 ng/ml, or more.
  • the amount of the BMP can be at most about 14 ng/ml, at most about 13 ng/ml, at most about 12 ng/ml, at most about 11 ng/ml, at most about 10 ng/ml, at most about 9 ng/ml, at most about 8 ng/ml, at most about 7 ng/ml, at most about 6 ng/ml, at most about 5 ng/ml, at most about 4 ng/ml, at most about 3 ng/ml, at most about 2 ng/ml, at most about 1 ng/ml, or less.
  • the amount of BMP in the composition may be more than about 30 ng/ml, e.g., more than about 250 ng/ml.
  • a mass of the BMP in the composition can be less than 50 %of a mass of the VEGF in the composition. In some cases, the mass of the BMP in the composition can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%or more of the mass of the VEGF in the composition.
  • the mass of the BMP in the composition can be at most about 50%, at most about 49%, at most about 48%, at most about 47%, at most about 46%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less of the mass of the VEGF in the composition.
  • the mass of the BMP in the composition can be greater than or equal to 50%of the mass of the VEGF in the composition.
  • the mass of the BMP in the composition can be at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, or more of the mass of the VEGF in the composition.
  • the mass of the BMP in the composition can be at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 54%, at most about 53%, at most about 52%, at most about 51%, or less of the mass of the VEGF in the composition.
  • the mass of the BMP in the composition can be equal to the mass of the VEGF in the composition.
  • a composition medium can have at least 1, at least 2, at least 3, at least 4, at least 5, or more specific sub-types of BMP.
  • BMP can include BMP1, BMP1b, BMP2, BMP2A, BMP2B, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, and BMP8B.
  • the BMP comprises BMP4.
  • the composition medium can have at least 1, at least 2, at least 3, at least 4, at least 5, or more specific sub-types of VEGF.
  • VEGF can include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF.
  • the composition medium may be substantially free of BMP or VEGF. In some cases, the composition medium may be substantially free of BMP. In some cases, the composition medium may be substantially free of VEGF. In some cases, the composition medium may be substantially free of BMP and VEGF.
  • the inhibitor can be a regulator of a pathway (e.g., an intracellular signaling pathway) involving Activin receptor-like kinases (ALK) , e.g., an inhibitor of ALK.
  • ALK inhibitors can include Bridatinib, Ceritinib, Lorlatinib, Alectinib, or Repotrectinib.
  • the regulator can inhibit function or activity of one or more proteins involved in such pathway, such as Phosphoinositide 3-kinase (PI3K) .
  • PI3K Phosphoinositide 3-kinase
  • Non-limiting examples of PI3K inhibitors can include 3-Methyladenine, LY294002, Dactolisib, Wortmannin, Alpelisib, TGX-221, AS-252424, or Idelalisib.
  • the regulator can inhibit function or activity of one or more proteins involved in such pathway, such as Janus tyrosine kinase (JAK) .
  • JAK inhibitors can include Ruxolitinib, Tofacitinib, AZD1480, momelotinib, Fedratinib, Itacitinib, or Decernotinib.
  • the regulator can inhibit function or activity of one or more proteins involved in such pathway, such as Extracellular signal-regulated kinases (ERK) .
  • ERK inhibitors can include SCH772984, Ulixertinib, LY3214996, or Ravoxertinib.
  • the regulator can activate function or activity of one or more proteins involved in such pathway, such as Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) .
  • SHP-1 activators can include nintedanib.
  • the inhibitor may be a small molecule. In some embodiments, the inhibitor may target an ALK receptor. In some cases, the ALK receptor can be ALK1, ALK2, ALK3, ALK4, ALK5, or ALK7.
  • ALK1 inhibitors can include ALK1-Fc or PF-03446962.
  • Non-limiting examples of ALK2 inhibitors can include Dalantercept (ACE-041) , DMH1, or K02288.
  • Non-limiting examples of ALK3 inhibitors can include LDN-212854, LDN-193189, DMH2, or VU0465350.
  • Non-limiting examples of ALK4 inhibitors can include SB-431542 or SB-505124.
  • Non-limiting examples of ALK5 inhibitors can include SB-431542, EW-7197, SB-505124, LY-2157299, or GW6604.
  • Non-limiting examples of ALK7 inhibitors can include SB-431542 or SB-505124.
  • the ALK receptor is selected from the group consisting of ALK4 (TGF ⁇ receptor 1) , ALK4 (Activin receptor type-1B) , and ALK7 (Activin A receptor) . In some cases, the ALK receptor is ALK4.
  • the ALK receptor selected can be ALK1, ALK2, ALK3, ALK5, or ALK7.
  • the ALK receptor is ALK1.
  • the ALK receptor is ALK2.
  • the ALK receptor is ALK3.
  • the ALK receptor is ALK5.
  • the ALK receptor is ALK7.
  • an amount of the ALK inhibitor in the composition can be between about 1 micromolar ( ⁇ M) and about 50 ⁇ M. In some cases, the amount of the ALK inhibitor in the composition can be at least about 1 ⁇ M, at least about 2 ⁇ M, at least about 3 ⁇ M, at least about 4 ⁇ M, at least about 5 ⁇ M, at least about 6 ⁇ M, at least about 7 ⁇ M, at least about 8 ⁇ M, at least about 9 ⁇ M, at least about 10 ⁇ M, at least about 11 ⁇ M, at least about 12 ⁇ M, at least about 13 ⁇ M, at least about 14 ⁇ M, at least about 15 ⁇ M, at least about 16 ⁇ M, at least about 17 ⁇ M, at least about 18 ⁇ M, at least about 19 ⁇ M, at least about 20 ⁇ M, at least about 25 ⁇ M, at least about 30 ⁇ M, at least about 35 ⁇ M, at least about 40 ⁇ M, at least about 45 ⁇ M, at least about 50 ⁇ M.
  • the amount of the ALK inhibitor in the composition is at most about 50 ⁇ M, at most about 45 ⁇ M, at most about 40 ⁇ M, at most about 35 ⁇ M, at most about 30 ⁇ M, at most about 25 ⁇ M, at most about 20 ⁇ M, at most about 19 ⁇ M, at most about 18 ⁇ M, at most about 17 ⁇ M, at most about 16 ⁇ M, at most about 15 ⁇ M, at most about 14 ⁇ M, at most about 13 ⁇ M, at most about 12 ⁇ M, at most about 11 ⁇ M, at most about 10 ⁇ M, at most about 9 ⁇ M, at most about 8 ⁇ M, at most about 7 ⁇ M, at most about 6 ⁇ M, at most about 5 ⁇ M, at most about 4 ⁇ M, at most about 3 ⁇ M, at most about 2 ⁇ M, at most about 1 ⁇ M, or less.
  • the ALK inhibitor is SB-431542.
  • an amount of the ALK inhibitor in the composition can be at most about 45 ⁇
  • the ALK inhibitor can be SB-505124.
  • the ALK inhibitor can be a polypeptide, such as SHP-1.
  • the composition may be substantially free of ALK inhibitor.
  • the composition comprising the ALK inhibitor can yield enhanced NK differentiation (e.g., from stem cells, such as iPSCs) as compared to that comprising the ALK inhibitor and one or more additional components such as, but not limited to, Activin A and/or BMP (e.g., BMP-4) .
  • NK differentiation e.g., from stem cells, such as iPSCs
  • additional components such as, but not limited to, Activin A and/or BMP (e.g., BMP-4) .
  • the composition may further comprise a fibroblast growth factor (FGF) , such as bFGF (basic fibroblast growth factor) .
  • FGF fibroblast growth factor
  • the composition can have at least 1, at least 2, at least 3, at least 4, at least 5, or more specific sub-types of FGF.
  • FGF can include FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23.
  • an amount of the FGF in the composition can be between about 1 ng/ml and about 200 ng/ml. In some cases, the amount of FGF in the composition can at least about 1 ng/ml, at least about 10 ng/ml, at least about 20 ng/ml, at least about 30 ng/ml, at least about 40 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, at least about 100 ng/ml, at least about 110 ng/ml, at least about 120 ng/ml, at least about 130 ng/ml, at least about 140 ng/ml, at least about 150 ng/ml, at least about 160 ng/ml, at least about 170 ng/ml, at least about 180 ng/ml, at least about 190 ng/ml, at least about 200 ng/
  • the amount of FGF in the composition can be at most about 200 ng/ml, at most about 190 ng/ml, at most about 180 ng/ml, at most about 170 ng/ml, at most about 160 ng/ml, at most about 150 ng/ml, at most about 140 ng/ml, at most about 130 ng/ml, at most about 120 ng/ml, at most about 110 ng/ml, at most about 100 ng/ml, at most about 90 ng/ml, at most about 80 ng/ml, at most about 70 ng/ml, at most about 60 ng/ml, at most about 50 ng/ml, at most about 40 ng/ml, at most about 30 ng/ml, at most about 20 ng/ml, at most about 10 ng/ml, at most about 1 ng/ml, or less.
  • the FGF comprises bFGF.
  • a mass of the BMP in the composition is less than 50 %of a mass of the FGF in the composition.
  • the mass of the BMP in the composition can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, or more of the mass of the FGF in the composition.
  • the mass of the BMP in the composition can be at most about 49%, at most about 48%, at most about 47%, at most about 46%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less of the mass of the FGF in the composition.
  • the mass of the BMP in the composition can be greater than or equal to 50%of the mass of the FGF in the composition.
  • the mass of the BMP in the composition can be at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, or more of the mass of the FGF in the composition.
  • the mass of the BMP in the composition can be at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 54%, at most about 53%, at most about 52%, at most about 51%, at most about 50%, or less of the mass of the FGF in the composition.
  • the mass of the BMP in the composition can be equal to the mass of the FGF in the composition.
  • the composition may be substantially free of FGF.
  • the population of mesodermal cells prior to contacting may be KDR+ and CD235a+, and the population of mesodermal cells having the definitive HE potential may be KDR+ and CD235a-.
  • the population of mesodermal cells prior to contacting may be KDR+.
  • the population of mesodermal cells prior to contacting may be CD235a+.
  • the population of mesodermal cells having the definitive HE potential may be KDR+.
  • the population of mesodermal cells having the definitive HE potential may be CD235a-.
  • the population of mesodermal cells may be KDR-and CD235a-.
  • the population of mesodermal cells having definitive HE potential may be KDR-and CD235a+.
  • the population of mesodermal cells can be contacted by the composition for less than or equal to about 10 days. In some cases, the population of mesodermal cells can be contacted by the composition for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, at least about 120 hours, at least about 132 hours, at least about 144 hours, at least about 156 hours, at least about 168 hours, at least about 180 hours, at least about 192 hours, at least about 204 hours, at least about 216 hours, at least about 228 hours, at least about 240 hours, or more.
  • the population of mesodermal cells can be contacted by the composition for at most about 240 hours, at most about 228 hours, at most about 216 hours, at most about 204 hours, at most about 192 hours, at most about 180 hours, at most about 168 hours, at most about 156 hours, at most about 144 hours, at most about 132 hours, at most about 120 hours, at most about 108 hours, at most about 96 hours, at most about 84 hours, at most about 72 hours, at most about 60 hours, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 12 hours, or less.
  • the population of mesodermal cells can be contacted for less than or equal to about 5 days.
  • the population of mesodermal cells can be contacted by the composition for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, or more.
  • the population of mesodermal cells can be contacted by the composition for at most about 5 days, at most about 4 days, at most about 3 days, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 20 hours, at most about 16 hours, at most about 12 hours, at most about 8 hours, at most about 6 hours, at most about 4 hours, at most about 2 hours, at most about 1 hour, or less.
  • the present disclosure provides a population of mesodermal cells having definitive hemogenic endothelium (HE) potential that can be generated by any one of the methods described herein.
  • HE hemogenic endothelium
  • the present disclosure provides one or more compositions for implementing any one of the methods described herein, e.g., for generating a population of mesodermal cells having definitive HE potential.
  • the present disclosure provides a method (e.g., an in vitro or ex vivo method) of generating hemogenic endothelial (HE) cells.
  • the method can comprise contacting a population of mesodermal cells with a composition comprising (i) interleukin-7 (IL-7) and (ii) at least one member selected from the group consisting of bone morphogenetic protein (BMP) and vascular endothelial growth factor (VEGF) .
  • BMP bone morphogenetic protein
  • VEGF vascular endothelial growth factor
  • an amount of IL-7 in the composition may be between around 1 ng/ml and about 50 ng/ml. In some cases, the amount of IL-7 in the composition may be at least about 1 ng/ml, at least about 2 ng/ml, at least about 3 ng/ml, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 8 ng/ml, at least about 10 ng/ml, at least about 12 ng/ml, at least about 14 ng/ml, at least about 16 ng/ml, at least about 18 ng/ml, at least about 20 ng/ml, at least about 22 ng/ml, at least about 24 ng/ml, at least about 26 ng/ml, at least about 28 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/
  • the amount of IL-7 in the composition may be at most about 50 ng/ml, at most about 45 ng/ml, at most about 40 ng/ml, at most about 35 ng/ml, at most about 30 ng/ml, at most about 28 ng/ml, at most about 26 ng/ml, at most about 24 ng/ml, at most about 22 ng/ml, at most about 20 ng/ml, at most about 18 ng/ml, at most about 16 ng/ml, at most about 14 ng/ml, at most about 12 ng/ml, at most about 10 ng/ml, at most about 8 ng/ml, at most about 6 ng/ml, at most about 5 ng/ml, at most about 4 ng/ml, at most about 3 ng/ml, at most about 2 ng/ml, at most about 1 ng/ml, or less.
  • a mass of the IL-7 in the composition can be less than or equal to a mass of at least one member.
  • the at least one member may be the BMP.
  • the at least one member may be the VEGF.
  • the at least one member may be the BMP and the VEGF.
  • a mass of the VEGF in the composition is less than or equal to a mass of BMP in the composition. In some cases, the mass of the VEGF in the composition can be less than the mass of the BMP in the composition.
  • reducing the amount of VEGF in the medium e.g., to 5 ng/ml, can reduce the growth of endothelial cells.
  • reducing the amount of VEGF in the medium e.g., to 5 ng/ml, can increase the growth of immune cells (e.g., NK cells) .
  • a composition medium can have at least 1, at least 2, at least 3, at least 4, at least 5, or more specific sub-types of BMP.
  • BMP can include BMP1, BMP1b, BMP2, BMP2A, BMP2B, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, and BMP8B.
  • an amount of BMP in the composition can be between about 1 ng/ml and about 100 ng/ml.
  • the amount of BMP in the composition be at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 55 ng/ml, at least about 60 ng/ml, at least about 65 ng/ml, at least about 70 ng/ml, at least about 75 ng/ml, at least about 80 ng/ml, at least about 85 ng/ml, at least about 90 ng/ml, at least about 95 ng/ml, at least about 100 ng/ml, or more.
  • the amount of BMP in the composition can be at most about 100 ng/ml, at most about 95 ng/ml, at most about 90 ng/ml, at most about 85 ng/ml, at most about 80 ng/ml, at most about 75 ng/ml, at most about 70 ng/ml, at most about 65 ng/ml, at most about 60 ng/ml, at most about 55 ng/ml, at most about 50 ng/ml, at most about 45 ng/ml, at most about 40 ng/ml, at most about 35 ng/ml, at most about 30 ng/ml, at most about 25 ng/ml, at most about 20 ng/ml, at most about 15 ng/ml, at most about 10 ng/ml, at most about 5 ng/ml, at most about 1 ng/ml, or less.
  • the BMP can be BMP4.
  • the composition medium can have at least 1, at least 2, at least 3, at least 4, at least 5, or more specific sub-types of VEGF.
  • VEGF can include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF.
  • an amount of VEGF in the composition can be between about 1 ng/ml and about 20 ng/ml.
  • the amount of VEGF in the composition can be at least about 1 ng/ml, at least about 2 ng/ml, at least about 4 ng/ml, at least about 6 ng/ml, at least about 8 ng/ml, at least about 10 ng/ml, at least about 12 ng/ml, at least about 14 ng/ml, at least about 16 ng/ml, at least about 18 ng/ml, at least about 20 ng/ml, or more.
  • the amount of VEGF in the composition can be at most about 20 ng/ml, at most about 18 ng/ml, at most about 16 ng/ml, at most about 14 ng/ml, at most about 12 ng/ml, at most about 10 ng/ml, at most about 8 ng/ml, at most about 6 ng/ml, at most about 4 ng/ml, at most about 2 ng/ml, at most about 1 ng/ml, or less.
  • the mass of the VEGF in the composition can be about 99%less than or equal to the mass of BMP in the composition. In some cases, the mass of the VEGF in the composition can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more than or equal to the mass of BMP in the composition.
  • the mass of the VEGF in the composition can be at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than or equal to the mass of BMP in the composition.
  • the mass of the VEGF in the composition can be about 99%less than the mass of BMP in the composition. In some cases, the mass of the VEGF in the composition can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more the mass of BMP in the composition.
  • the mass of the VEGF in the composition can be at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than the mass of BMP in the composition.
  • the composition may be substantially free of IL-7, VEGF, or BMP.
  • the composition may be substantially free of IL-7, VEGF, and BMP.
  • the composition may be substantially free of IL-7 and VEGF.
  • the composition may be substantially free of IL-7 and BMP.
  • the composition may be substantially free of IL-7.
  • the composition may be substantially free of BMP.
  • the composition may be substantially free of BMP and VEGF.
  • the composition may be substantially free of VEGF.
  • the composition can further comprise an additional interleukin that is not IL-7.
  • the additional interleukin can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.
  • a mass of the additional interleukin in the composition can be less than or equal to a mass of the IL-7 in the composition. In some examples, the mass of the additional interleukin in the composition can be less than the mass of the IL-7 in the composition. In some embodiments, the mass of the additional interleukin in the composition can be about 99%less than or equal to the mass of IL-7 in the composition.
  • the mass of the additional interleukin in the composition can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more than or equal to the mass of IL-7 in the composition.
  • the mass of the additional interleukin in the composition can be at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than or equal to the mass of IL-7 in the composition.
  • the mass of the additional interleukin in the composition can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more the mass of IL-7 in the composition.
  • the mass of the additional interleukin in the composition can be at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than the mass of IL-7 in the composition.
  • the additional interleukin can be IL-3 and/or IL-15. In some cases, the additional interleukin can be IL-3. In some cases, the additional interleukin can be IL-15.In some cases, the additional interleukin can be IL-3 and IL-15. In some cases, an amount of IL-3 in the composition can be between about 1 ng/ml and about 50 ng/ml.
  • the amount of IL-3 in the composition can be at least about 1 ng/ml, at least about 2 ng/ml, at least about 3 ng/ml, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 7 ng/ml, at least about 8 ng/ml, at least about 9 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, or more.
  • the amount of IL-3 in the composition can be at most about 50 ng/ml, at most about 45 ng/ml, at most about 40 ng/ml, at most about 35 ng/ml, at most about 30 ng/ml, at most about 25 ng/ml, at most about 20 ng/ml, at most about 15 ng/ml, at most about 10 ng/ml, at most about 9 ng/ml, at most about 8 ng/ml, at most about 7 ng/ml, at most about 6 ng/ml, at most about 5 ng/ml, at most about 4 ng/ml, at most about 3 ng/ml, at most about 2 ng/ml, at most about 1 ng/ml, or less.
  • an amount of IL-15 in the composition can be between about 1 ng/ml and about 100 ng/ml. In some cases, the amount of IL-15 in the composition can be at least about 1 ng/ml, at least about 2 ng/ml, at least about 4 ng/ml, at least about 6 ng/ml, at least about 8 ng/ml, at least about 10 ng/ml, at least about 12 ng/ml, at least about 14 ng/ml, at least about 16 ng/ml, at least about 18 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 55 ng/ml, at least about 60 ng/ml, at least about 65 ng/ml, at least about 70 ng/m
  • the amount of IL-15 in the composition can be at most about 100 ng/ml, at most about 95ng/ml, at most about 90 ng/ml, at most about 85 ng/ml, at most about 80 ng/ml, at most about 75 ng/ml, at most about 70 ng/ml, at most about 65 ng/ml, at most about 60 ng/ml, at most about 55 ng/ml, at most about 50 ng/ml, at most about 45 ng/ml, at most about 40 ng/ml, at most about 35 ng/ml, at most about 30 ng/ml, at most about 25 ng/ml, at most about 20 ng/ml, at most about 18 ng/ml, at most about 16 ng/ml, at most about 14 ng/ml, at most about 12 ng/ml, at most about 10 ng/ml, at most about 8 ng/ml, at most about 6 ng/ml, at most about 100
  • the composition may be substantially free of IL-3 and/or IL-15. In some cases, the composition may be substantially free of IL-3. In some cases, the composition may be substantially free of IL-15. In some cases, the composition may be substantially free of IL-3 and IL-15.
  • the composition can further comprise a FMS-like tyrosine kinase receptor ligand (FLTL) .
  • FLTL FMS-like tyrosine kinase receptor ligand
  • Non-limiting examples of FLTL can include FLTL-1, FLTL-2, FLTL-3, or FLTL-4.
  • a mass of the FLTL in the composition is less than or equal to a mass of the IL-7 in the composition.
  • the mass of the FLTL in the composition is less than the mass of the IL-7 in the composition.
  • the FLTL can comprise FLTL3.
  • the mass of the FLTL in the composition can be about 99%less than or equal to the mass of IL-7 in the composition. In some cases, the mass of the FLTL in the composition can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more than or equal to the mass of IL-7 in the composition.
  • the mass of the FLT in the composition can be at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than or equal to the mass of IL-7 in the composition.
  • the mass of the FLTL in the composition can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more the mass of IL-7 in the composition.
  • the mass of the FLTL in the composition can be at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, or less than the mass of IL-7 in the composition.
  • an amount of FLTL in the composition can be between about 1 ng/ml and about 100 ng/ml. In some cases, the amount of FLTL in the composition can be at least about 1 ng/ml, at least about 2 ng/ml, at least about 3 ng/ml, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 7 ng/ml, at least about 8 ng/ml, at least about 9 ng/ml, at least about 10 ng/ml, at least about 11 ng/ml at least about 12 ng/ml, at least about 13 ng/ml, at least about 14 ng/ml, at least about 15 ng/ml, at least about 16 ng/ml, at least about 17 ng/ml, at least about 18 ng/ml, at least about 19 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml,
  • the amount of FLTL in the composition can be at most about 100 ng/ml, at most about 95 ng/ml, at most about 90 ng/ml, at most about 85 ng/ml, at most about 80 ng/ml, at most about 75 ng/ml, at most about 70 ng/ml, at most about 65 ng/ml, at most about 60 ng/ml, at most about 55 ng/ml, at most about 50 ng/ml, at most about 45 ng/ml, at most about 40 ng/ml, at most about 35 ng/ml, at most about 30 ng/ml, at most about 25 ng/ml, at most about 20 ng/ml, at most about 19 ng/ml, at most about 18 ng/ml, at most about 17 ng/ml, at most about 16 ng/ml, at most about 15 ng/ml, at most about 14 ng/ml, at most about 13 ng/ml, at
  • the composition may have FLTL (e.g., FLTL3) in the initial period (e.g., during the first 48 hours or more) of differentiation method disclosed herein.
  • the composition may be substantially free of FLTL during the initial period of differentiation method (e.g., during the first 48 hours or more) .
  • the mesodermal cells prior to contacting, can exhibit HE potential.
  • the resulting cells after contacting, can be hemogenic endothelial (HE) cells.
  • the population of HE cells can be characterized by expressing a greater level of CD34, as compared to that of the population of mesodermal cells.
  • the population of HE cells can have greater percentage of CD34 expression compared to the population of mesodermal cells.
  • the increased percentage of CD34 expression can be between about 1%and about 200%.
  • the increased percentage of CD34 expression can be at least about 1%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or more.
  • the increased percentage of CD34 expression can be at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 1%, or less.
  • the population of mesoderm cells may have a higher expression of CD34 prior to the contacting as compared to the population of HE cells after the contacting.
  • the population of HE cells may be CD34-negative.
  • the population of mesodermal cells can be contacted by the composition for less than or equal to about 10 days. In some cases, the population of mesodermal cells can be contacted by the composition for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, at least about 120 hours, at least about 132 hours, at least about 144 hours, at least about 156 hours, at least about 168 hours, at least about 180 hours, at least about 192 hours, at least about 204 hours, at least about 216 hours, at least about 228 hours, at least about 240 hours, or more.
  • the population of mesodermal cells can be contacted by the composition for at most about 240 hours, at most about 228 hours, at most about 216 hours, at most about 204 hours, at most about 192 hours, at most about 180 hours, at most about 168 hours, at most about 156 hours, at most about 144 hours, at most about 132 hours, at most about 120 hours, at most about 108 hours, at most about 96 hours, at most about 84 hours, at most about 72 hours, at most about 60 hours, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 12 hours, or less.
  • the population of mesodermal cells can be contacted for less than or equal to about 5 days. In some cases, the population of mesodermal cells can be contacted by the composition for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, or more.
  • the population of mesodermal cells can be contacted by the composition for at most about 5 days, at most about 4 days, at most about 3 days, at most about 48 hours, at most about 36 hours, at most about 24 hours, at most about 20 hours, at most about 16 hours, at most about 12 hours, at most about 8 hours, at most about 6 hours, at most about 4 hours, at most about 2 hours, at most about 1 hour, or less.
  • the present disclosure provides a population of hemogenic endothelial cells that can be generated by any one of the methods described herein.
  • the present disclosure provides a method (e.g., an in vitro or ex vivo method) that can generate a population of NK cells from a population of stem cells.
  • the method can comprise contacting the population of stem cells or progenies thereof to a plurality of compositions in a sequential manner, to generate the population of NK cells.
  • the plurality of compositions can comprise: a first composition comprising an activator of Wnt/ ⁇ -catenin signaling pathway; a second composition comprising one or more members selected from the group consisting of a bone morphogenetic protein (BMP) , a vascular endothelial growth factor (VEGF) , an inhibitor of activin receptor-like kinase (ALK) receptor signaling pathway, and a fibroblast growth factor (FGF) ; a third composition comprising an interleukin-7 (IL-7) , an IL-15, an IL-3, a BMP, a VEGF, and a FMS-like tyrosine kinase (FLTL) ; and a fourth composition comprising an IL-7 and/or a FLTL.
  • BMP bone morphogenetic protein
  • VEGF vascular endothelial growth factor
  • ALK activin receptor-like kinase
  • FGF fibroblast growth factor
  • IL-7 interleukin-7
  • the population of stem cells or progenies thereof can be subjected to each composition of the plurality of compositions in a medium that is substantially free of the other of the plurality of compositions.
  • the present disclosure provides a population of NK cells that can be generated by any one of the methods described herein.
  • Additional aspects of the present disclosure provide methods and compositions for engineering any one of the cells disclosed herein (e.g., stem cells, mesodermal cells, hemogenic endothelial cells, hematopoietic stem cells, NK cells, T cells, etc. ) , e.g., to exhibit one or more desired characteristics (e.g., one or more engineered or heterologous characteristics) .
  • the one or more desired characteristics can be introduced to the cell prior to, during, or subsequent to one or more of the following steps: (1) generating stem cell-derived mesodermal cells, (2) generating mesodermal cells with HE potential, (3) generating hemogenic endothelial cells, and (4) generating immune cells (e.g., NK cells, T cells, etc.
  • the desired characteristics can be mediated via a heterologous polypeptide and/or a heterologous polynucleotide.
  • a heterologous polypeptide may comprise a heterologous receptor and/or a cytokine, to effect a desired characteristic (e.g., activation of a desired cellular signaling cascade) in the cell or a progeny thereof.
  • a heterologous polypeptide and/or a heterologous polynucleotide may comprise a gene editing moiety (e.g., an endonuclease complex such as Cas/gRNA complex, a small interfering RNA (siRNA) , etc. ) that is capable of regulating expression and/or activity level of a target gene (e.g., CD16) in the cell.
  • a gene editing moiety e.g., an endonuclease complex such as Cas/gRNA complex, a small interfering RNA (siRNA) , etc.
  • the gene editing moiety may effect (i) enhanced expression or activity level of a target gene, or (ii) reduced expression or activity of a target gene.
  • a cell during any one of the steps (1) through (4) can be contacted with the heterologous polypeptide and/or the heterologous polynucleotide (e.g., via transfection or transduction) , such that the cell or a progeny thereof can exhibit the one or more desired characteristics accordingly.
  • the heterologous polynucleotide can be integrated into the genome of the cell (e.g., the resulting NK cells) .
  • the heterologous polynucleotide may not and need not be integrated into the genome of the cell.
  • Non-limiting examples of the one or more desired characteristics of the cell can comprise one or more members select from the group comprising: enhanced CD16 signaling; a chimeric polypeptide receptor; reduced expression and/or activity level of endogenous CD38; reduced expression and/or activity level of an immune checkpoint inhibitor; reduced expression and/or activity level of a hypo-immunity regulator; a safety switch; a heterologous cytokine (e.g., interleukin (IL) ) and/or a heterologous receptor thereof; and/or reduced activity of endogenous cytokine signaling.
  • IL interleukin
  • the one or more desired characteristics can comprise enhanced CD16 signaling (e.g., constitutively activated signaling of CD16) .
  • the enhanced CD16 signaling can be achieved by having non-cleavable CD16 variant in the subject cell.
  • CD16 e.g., CD16a
  • immune cells e.g., NK cells
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the binding between CD16 and the monomeric IgG can induce cleavage of the CD16 protein at a cleavage site near the transmembrane domain, to regulates the cell surface density of CD16 upon immune cell activation.
  • the endogenous CD16 of the cell can be modified to enhance its signaling.
  • an enhanced signaling variant of CD16 can be artificially introduced to the cell (e.g., via transfection or transduction of a heterologous polynucleotide as disclosed herein) .
  • endogenous CD16 gene of the cell as disclosed herein can be genetically modified in its ectodomain (e.g., F176V) via action of a gene editing moiety as disclosed herein, such that the modified CD16 exhibits higher binding affinity to its target (e.g., monomeric IgG) as compared to a natural CD16.
  • a heterologous gene encoding such modified CD16 can be introduced to the cell.
  • the cell’s endogenous gene encoding CD16 can be genetically modified via action of a gene editing moiety as disclosed herein, such that the modified CD16 is non-cleavable and can induce enhanced CD16 signaling.
  • the cleavage site e.g., position 195-198 in the membrane-proximal region (position 189-212) of CD16 can be modified or eliminated (e.g., CD16 S197P variant as a non-cleavable CD16 variant) .
  • a heterologous gene encoding such modified CD16 can be introduced to the cell.
  • a heterologous gene encoding a heterologous CD16 variant that (i) exhibits higher binding affinity to its target (e.g., monomeric IgG) and (ii) is non-cleavable can be introduced to the cell (i.e., hnCD16) .
  • the heterologous CD16 variant can be a modified CD16 comprising, for example, F176V and S197P, as disclosed herein.
  • the heterologous CD variant can be a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.
  • a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.
  • the enhanced CD16 signaling of the cell e.g., the engineered NK cell
  • the cell e.g., the engineered NK cell
  • the enhanced CD16 signaling of the cell can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., SHP-1) via Western blotting or (ii) expression of a downstream gene (e.g., CD25, IFN-gamma, TNF, etc. ) via Western blotting or PCR techniques.
  • a downstream signaling protein e.g., SHP-1
  • a downstream gene e.g., CD25, IFN-gamma, TNF, etc.
  • the one or more desired characteristics can comprise a chimeric polypeptide receptor (e.g., a chimeric antigen receptor (CAR) , an engineered T cell receptor (TCR) , etc. ) comprising an antigen binding moiety capable of binding to an antigen.
  • a chimeric polypeptide receptor e.g., a chimeric antigen receptor (CAR) , an engineered T cell receptor (TCR) , etc.
  • the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include BCMA, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and/or gp10.
  • the one or more desired characteristics can comprise reduced expression and/or activity level of endogenous CD38 as compared to a control cell.
  • a control cell may be used to treat a subject who has or is suspected of having white blood cell cancer, such as multiple myeloma (MM) .
  • MM multiple myeloma
  • the one or more desired characteristics can comprise enhanced expression and/or activity level of the activating NK receptor.
  • the activating NK receptor can comprise NKG2D, NKP30 (e.g., NKP30A and/or NKP30B isoforms) , NKP44, NKP46, a modification thereof, a functional variant thereof, and/or a combination thereof.
  • the activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKG2D or a modification thereof.
  • the activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKP30 or a modification thereof.
  • the activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKP44 or a modification thereof.
  • the activating NK receptor can comprise at least a portion of (e.g., some of the entirety of) NKP46 or a modification thereof.
  • the activating NK receptor may not comprise NKG2C.
  • the activating NK receptor may comprise NKG2C.
  • the activating NK receptor can be an endogenous activating NK receptor.
  • the activating NK receptor can be a heterologous receptor (e.g., encoded by a heterologous polynucleotide sequence that is introduced to the cell as disclosed herein) .
  • the one or more desired characteristics can comprise reduced expression and/or activity level of an immune checkpoint inhibitor (e.g., PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, 2B4, etc. ) .
  • an immune checkpoint inhibitor e.g., PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, 2B4, etc.
  • the one or more desired characteristics can comprise reduced expression and/or activity level of a hypo-immunity regulator (e.g., B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, etc) .
  • a hypo-immunity regulator e.g., B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR
  • the one or more desired characteristics can comprise a safety switch capable of effecting death of the cell (e.g., upon a stimulus, such as exposure to an activating moiety, such as light or antibody) .
  • the cell can comprise a gene encoding the safety switch (e.g., integrated into the genome of the immune cell) , via action of the gene editing moiety, as disclosed herein.
  • a prodrug can be introduced to the cell (e.g., administered to a subject comprising the cell) in the event of an adverse event or when the adaptive immunotherapy is no longer necessary, and the prodrug can be activated by the safety switch molecule to kill the subject immune cell.
  • the safety switch can comprise one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, and functional variants thereof.
  • the safety switch can be activated via an activator (e.g., a small molecule or a protein, such as an antibody) for post-translational, temporal, and/or site-specific regulation of death (or depletion) of the subject cell.
  • an activator e.g., a small molecule or a protein, such as an antibody
  • Non-limiting examples of a safety switch and its activator can include Caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV) ; and cytosine deaminase (CD) and 5-fluorocytosine (5-FC) .
  • Caspase 9 or caspase 3 or 7
  • AP1903 thymidine kinase
  • GCV ganciclovir
  • CD cytosine deaminase
  • 5-FC 5-fluorocytosine
  • modified epidermal growth factor receptor (EGFR) containing epitope recognized by an antibody e.g., anti-EGFR Ab, such as cetuximab
  • an antibody e.g., anti-EGFR Ab, such as cetuximab
  • the cells e.g., the engineered NK cells
  • the cells can comprise a safety switch protein selected from the group consisting of caspase 9 (caspase 3 or 7) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.3.
  • the one or more desired characteristics can comprise a heterologous cytokine (e.g., interleukin (IL) ) and/or a heterologous receptor thereof for enhanced cytokine signaling as compared to the control cell.
  • a heterologous cytokine e.g., interleukin (IL)
  • IL interleukin
  • the heterologous cytokine (e.g., the heterologous IL) and/or the heterologous receptor thereof, as disclosed herein, can be of the same species as that of the cell (e.g., the engineered NK cell) .
  • both the heterologous cytokine (and/or the heterologous receptor thereof) and the cell can be of human origin.
  • the heterologous cytokine (and/or the heterologous receptor thereof) can be of a different species than that of the cell.
  • a heterologous cytokine e.g., the heterologous IL and/or the heterologous receptor thereof, as disclosed herein, can be introduced to the cell (e.g., engineered NK cell) by contacting a heterologous polynucleotide encoding the heterologous cytokine and/or the heterologous receptor thereof to the cell.
  • the heterologous polynucleotide can be integrated into the cell’s chromosome (e.g., nuclear chromosome) .
  • the heterologous polynucleotide may not and need not be integrated into the chromosome of the cell.
  • a mRNA encoding a heterologous cytokine can be introduced (or inserted into) the cell.
  • the cytokine as disclosed herein can be IL.
  • An IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of ILs.
  • An IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of ILs.
  • the IL can be a single type of IL.
  • Non-limiting examples of the IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36.
  • the IL can comprise one or more members selected from the group consisting of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and functional modifications thereof.
  • the cell e.g., an engineered NK cell
  • the cell as disclosed herein can comprise at least a portion of heterologous variant of any one of the IL as disclosed herein, such as human IL-15 (or a gene encoding thereof) .
  • the cell e.g., an engineered NK cell
  • the cell can comprise at least a portion of heterologous variant of a receptor of any one of the IL as disclosed herein, such as human IL-15 receptor (IL-15R) (or a gene encoding thereof) .
  • IL-15R human IL-15 receptor
  • heterologous cytokine e.g., the heterologous IL
  • the heterologous cytokine can be a secretory cytokine.
  • the heterologous cytokine may not and need not be secreted by the cell.
  • the heterologous cytokine can be bound to a cell surface of the cell.
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine.
  • An expression cassette encoding the heterologous cytokine can be introduced to the cell.
  • the expression cassette can further encode an additional heterologous polypeptide, e.g., a heterologous receptor.
  • a first polynucleotide sequence encoding the heterologous cytokine and a second polynucleotide sequence encoding the additional heterologous polypeptide (e.g., the heterologous receptor) can be coupled to each other via a polynucleotide linker encoding a cleavage linker.
  • the heterologous receptor can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15) .
  • the expression cassette may not and need not encode any additional heterologous polypeptide other than the heterologous cytokine.
  • a cleavable linker or cleavage linker can comprise a self-cleaving peptide, such as a self-cleaving 2A peptide.
  • Self-cleaving peptides can be found in members of the Picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV) , Thosea asigna virus (TaV) and porcine tescho virus-1 (PTV-I) , and cardioviruses such as Theilovirus (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis viruses.
  • Non-limiting examples of the self-cleaving 2A peptide can include “F2A” , “E2A” , “P2A” , “T2A” , and functional variants thereof.
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be bound to a cell surface the cell (e.g., the engineered NK cell) .
  • the cell can be genetically modified such that a heterologous polynucleotide sequence encoding the heterologous cytokine is coupled to a gene encoding an endogenous transmembrane protein of the cell.
  • the endogenous transmembrane protein can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15) .
  • an expression cassette encoding a heterologous fusion polypeptide comprising (i) the heterologous cytokine that is coupled to (ii) a heterologous receptor can be introduced to the cell.
  • the heterologous cytokine may not and need not be cleavable from the heterologous receptor.
  • Non-limiting examples of the heterologous receptor can include a respective receptor of the heterologous cytokine (e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15) , or a different receptor such as a common gamma chain ( ⁇ C) receptor or a modification thereof.
  • the cell e.g., the engineered NK cell
  • the cell can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine (e.g., the heterologous IL, such as the heterologous IL-15) and/or the heterologous receptor (e.g., the heterologous IL receptor, such as the heterologous IL-15R) as disclosed herein.
  • the enhanced signaling of the endogenous signaling pathway as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., JAK3, STAT3, STAT5, etc.
  • a downstream gene e.g., Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc, Ifng, etc. for IL-15/IL-15R
  • PCR polymerase chain reaction
  • the one or more desired characteristics can comprise reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) .
  • endogenous cytokine signaling e.g., endogenous IL signaling, such as endogenous IL-17 signaling
  • the engineered NK cell can be treated with inhibitors (e.g., small molecule inhibitors) of the endogenous cytokine signaling.
  • the engineered NK cell can comprise reduced expression of endogenous IL (e.g., endogenous IL-17) or endogenous receptor thereof (e.g., via indel or transgene mutation, via transient or permanent suppression, etc. ) .
  • the engineered NK cell can comprise reduced expression of endogenous IL-17.
  • the engineered NK cell can comprise reduced expression of endogenous IL-17R.
  • the engineered NK cell can comprise reduced expression of endogenous IL-17 and endogenous IL-17R.
  • the endogenous cytokine as disclosed herein can be an endogenous IL.
  • An endogenous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of endogenous ILs.
  • An endogenous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of endogenous ILs.
  • the endogenous IL can be a single type of endogenous IL.
  • Non-limiting examples of the endogenous IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36.
  • the endogenous IL can be IL-17.
  • Non-limiting examples of endogenous Il-17 can include IL-17A, IL-17F, and natural mutations thereof.
  • the cell e.g., an engineered NK cell
  • the cell as disclosed herein can exhibit reduced expression or activity of IL-17A or IL-17F.
  • an endogenous gene encoding the endogenous cytokine e.g., an endogenous IL, such as IL-17
  • an endogenous cytokine e.g., an endogenous IL, such as IL-17
  • a gene editing moiety as disclosed herein.
  • the endogenous receptor can be a respective receptor of any cytokine as disclosed herein (e.g., a respective receptor of any IL as disclosed herein) .
  • the endogenous receptor can be a respective receptor of IL (e.g., IL-17R for IL-7 signaling) .
  • IL-17R can include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof.
  • the endogenous IL-17R comprises IL-17RA.
  • the reduced expression or activity of the endogenous cytokine e.g., an endogenous IL, such as IL-17
  • endogenous receptor thereof as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc. for IL-17) or (ii) expression of a downstream gene via Western blotting or PCT techniques.
  • a downstream signaling protein e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc.
  • a downstream gene of IL cytokine can include a chemokine (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc. ) , a cytokine (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc. ) , an acute phase response molecule (e.g., SAA, CRP, lipocalin 2/24p3, etc. ) , and/or an enzyme (e.g., a metalloproteinase, such as MMP1, MMP3, MMP9, MMP13) .
  • a chemokine e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc.
  • a cytokine e.g., IL-6, TNFa, G-CSF, GM-CSF, etc.
  • the cell can induce immune response towards a target cell.
  • the target can be, for example, a diseased cell, a cancer cell, a tumor cell, etc.
  • regulation of a target gene can be regulated by a gene editing moiety as disclosed herein.
  • the regulation can be enhancing (or activating) or reducing (or inhibiting) the expression level and/or the activity level of the target gene.
  • the regulation can comprise cleaving at least a portion of the target gene via action of the gene editing moiety (e.g., CRISPR-Cas system with a guide nucleic acid molecule, such as a guide RNA molecule) , to edit the at least the portion of the target gene.
  • the regulation may not require cleaving any portion of the target gene via action of the gene editing moiety.
  • the gene editing moiety e.g., dCas-activator or dCas-inhibitor fusion protein, along with a guide nucleic acid molecule
  • the gene editing moiety can form a complex with the target gene, to either (A) activate expression of the target gene or (B) hinder or inhibit expression of the target gene.
  • the cell can exhibit enhanced cytotoxicity against a target cell as compared to a control cell.
  • the cell as disclosed herein can exhibit cytotoxicity (e.g., in vitro, ex vivo, or in vivo) against a target cell or a target population of cells that is greater than that of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to
  • the cell can exhibit enhanced half-life upon exposure to separate immune cells (e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the cell to the host) as compared to a control cell.
  • separate immune cells e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the cell to the host
  • the half-life of the cells can be greater than that of the control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold
  • the population of mesodermal cells having HE potential or the population of HE cells can be characterized by exhibiting expression of CD31 and/or CD34.
  • the population of mesodermal cells having HE potential or the population of HE cells can be characterized by expressing a greater level of CD31 and/or CD34 as compared to a control population of cells (e.g., cells not having been subjected to the any of the methods provided herein, such as non-treated cells or cells differentiated via a conventional method as provided herein) , by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 100%.
  • Expression of CD31 and/or CD34 can be ascertained by, for example, monitoring the number of the cells positive for CD31
  • any of the methods of the present disclosure can be utilized to generate a population of cells comprising (i) the population of mesodermal cells having HE potential or (ii) the population of HE cells, and a proportion of (i) or (ii) in the generated population of cells can be at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or substantially about 100%.
  • the population of NK cells can be characterized by exhibiting expression of CD56.
  • the population of NK cells can be characterized by expressing a greater level of CD56 as compared to a control population of cells (e.g., cells not having been subjected to the any of the methods provided herein, such as non-treated cells or cells differentiated via a conventional method as provided herein) , by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 100%.
  • Expression of CD56 can be ascertained by, for example, monitoring the number of the cells positive for CD56 over time (e.g., via FACS) or measuring secretion of CD56 from cells.
  • the population of NK cells can be characterized by exhibiting expression of an activating NK receptor (e.g., an endogenous activating NK receptor) .
  • the population of NK cells can be characterized by expressing a greater level of activating NK receptor as compared to a control population of cells (e.g., cells not having been subjected to the any of the methods provided herein, such as non-treated cells or cells differentiated via a conventional method as provided herein) , by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 100%.
  • Expression of activating NK receptor can be ascertained by, for example, monitoring the number of the cells positive for activating NK receptor over
  • the population of NK cells can be characterized by exhibiting expression of an NKp30.
  • the population of NK cells can be characterized by expressing a greater level of NKp30 as compared to a control population of cells (e.g., cells not having been subjected to the any of the methods provided herein, such as non-treated cells or cells differentiated via a conventional method as provided herein) , by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 100%.
  • Expression of NKp30 can be ascertained by, for example, monitoring the number of the cells positive for NKp30 over time (e.g., via FACS) or measuring secretion of NKp30
  • the population of NK cells can be characterized by exhibiting expression of NKG2A.
  • the population of NK cells can be characterized by expressing a greater level of NKG2A as compared to a control population of cells (e.g., cells not having been subjected to the any of the methods provided herein, such as non-treated cells or cells differentiated via a conventional method as provided herein) , by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 100%.
  • Expression of NKG2A can be ascertained by, for example, monitoring the number of the cells positive for NKG2A over time (e.g., via FACS) or measuring secretion of NKG2A from
  • the population of NK cells can be characterized by exhibiting expression of NKp44.
  • the population of NK cells can be characterized by expressing a greater level of NKp44 as compared to a control population of cells (e.g., cells not having been subjected to the any of the methods provided herein, such as non-treated cells or cells differentiated via a conventional method as provided herein) , by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 100%.
  • Expression of NKp44 can be ascertained by, for example, monitoring the number of the cells positive for NKp44 over time (e.g., via FACS) or measuring secretion of NKp44 from cells.
  • any of the methods of the present disclosure can be utilized to generate a population of cells comprising (i) the population of NK cells expressing CD56, or (ii) the population of NK cells expressing the activating NK receptor (e.g., NKp30, NKG2A, NKp44, etc.
  • the activating NK receptor e.g., NKp30, NKG2A, NKp44, etc.
  • a proportion of (i) or (ii) in the generated population of cells can be at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or substantially about 100%.
  • the cell can effect enhanced function or pathological condition of a bodily tissue of a subject as compared to a control cell.
  • treatment with the cell can effect enhanced function or pathological condition of a bodily tissue of a subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least
  • the cell can effect delayed degeneration of function or pathological condition of a bodily tissue of a subject as compared to a control cell.
  • treatment with the cell can effect delayed degeneration of function or pathological condition of a bodily tissue of a subject by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about
  • the bodily tissue can comprise one or more members selected from the group consisting of blood, plasma, serum, urine, perilymph fluid, feces, saliva, semen, amniotic fluid, cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomit, bone, heart, thymus, artery, blood vessel, lung, muscle, stomach, intestine, liver, pancreas, spleen, kidney, gall bladder, thyroid gland, adrenal gland, mammary gland, ovary, prostate gland, testicle, skin, adipose, eye, brain, infected tissue, diseased tissue, malignant tissue, calcified tissue, and healthy tissue.
  • the bodily tissue can comprise one or more members selected from the group consisting of blood, plasma, serum, urine, perilymph fluid, feces, saliva, semen, amniotic fluid, cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomit, bone, heart,
  • the cell can induce immune response towards a target cell.
  • the target can be, for example, a diseased cell, a cancer cell, a tumor cell, etc.
  • regulation of a target gene can be regulated by a gene editing moiety as disclosed herein.
  • the regulation can be enhancing (or activating) or reducing (or inhibiting) the expression level and/or the activity level of the target gene.
  • the regulation can comprise cleaving at least a portion of the target gene via action of the gene editing moiety (e.g., CRISPR-Cas system with a guide nucleic acid molecule, such as a guide RNA molecule) , to edit the at least the portion of the target gene.
  • the regulation may not require cleaving any portion of the target gene via action of the gene editing moiety.
  • the gene editing moiety e.g., dCas-activator or dCas-inhibitor fusion protein, along with a guide nucleic acid molecule
  • the gene editing moiety can form a complex with the target gene, to either (A) activate expression of the target gene or (B) hinder or inhibit expression of the target gene.
  • a heterologous polynucleotide e.g., a heterologous gene
  • a constitutive, inducible, temporal, tissue-specific, and/or cell type-specific promoter e.g., for knock-in
  • a promoter of interest can include CMV, EF1a, PGK, CAG, and UBC.
  • Non-limiting examples of an insertion site can include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, B2M, GAPDH, TCR, RUNX1, TAP1, 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, and TIGIT.
  • the cell e.g., the engineered immune cell such as the engineered NK cell
  • the cell can comprise a chimeric polypeptide receptor as disclosed herein (e.g., at least 1, 2, 3, 4, 5, or more different types of chimeric polypeptide receptors) .
  • the cell can be engineered to express a chimeric polypeptide receptor transiently or permanently.
  • a recombinant chimeric polypeptide receptor can be delivered to the cell via, e.g., a liposome, and be incorporated into the cell via membrane fusion.
  • a heterologous polynucleotide construct e.g., DNA or RNA
  • encoding the chimeric polypeptide receptor can be delivered to the cell.
  • heterologous polynucleotide construct i.e., a gene
  • encoding the heterologous polynucleotide construct can be incorporated into the chromosome of the cell (i.e., chromosomal gene) or, alternatively, may not or need not be integrated into the chromosome of the cell as disclosed herein.
  • a chimeric polypeptide receptor can comprises a T cell receptor fusion protein (TFP) .
  • T cell receptor fusion protein or “TFP” generally refers to a recombinant polypeptide construct comprising (i) one or more antigen binding moieties (e.g., monospecific or multispecific) , (ii) at least a portion of TCR extracellular domain, (iii) at least a portion of TCR transmembrane domain, and (iv) at least a portion of TCR intracellular domain.
  • an endogenous T cell receptor (TCR) of the cell e.g., the engineered immune cell such as the engineered NK cell
  • TCR T cell receptor
  • a function of the endogenous TCR of the cell can be inhibited by an inhibitor.
  • a gene encoding a subunit of the endogenous TCR can be inactivated (e.g., edited via action of the gene editing moiety as disclosed herein) such that the endogenous TCR is inactivated.
  • the gene encoding the subunit of endogenous TCR can be one or more of: TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • a chimeric polypeptide receptor can comprises a chimeric antigen receptor (CAR) .
  • CAR chimeric antigen receptor
  • the term “chimeric antigen receptor” or “CAR” generally refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as “an intracellular or intrinsic signaling domain” ) comprising a functional signaling domain derived from a stimulatory molecule.
  • the stimulatory molecule may be the zeta chain associated with the T cell receptor complex.
  • the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule.
  • the costimulatory molecule may comprise 4-1BB (i.e., CD137) , CD27, and/or CD28.
  • the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • a CAR may be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof.
  • First-generation CARs include an antigen binding domain with specificity for a particular antigen (e.g., an antibody or antigen-binding fragment thereof such as an scFv, a Fab fragment, a VHH domain, or a VH domain of a heavy-chain only antibody) , a transmembrane domain derived from an adaptive immune receptor (e.g., the transmembrane domain from the CD28 receptor) , and a signaling domain derived from an adaptive immune receptor (e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3 ⁇ receptor or Fc ⁇ RI ⁇ ) .
  • an adaptive immune receptor e.g., one or more (e.g., three) ITAM domains derived from the intracellular region of the CD3 ⁇ receptor or Fc ⁇ RI ⁇
  • Second-generation CARs modify the first-generation CAR by addition of a co-stimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from co-stimulatory receptors that act alongside T-cell receptors such as CD28, CD137/4-1BB, and CD134/OX40) , which abrogates the need for administration of a co-factor (e.g., IL-2) alongside a first-generation CAR.
  • Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3 ⁇ -CD28-OX40, or CD3 ⁇ -CD28-41BB) .
  • Fourth-generation CARs modify second-or third-generation CARs by the addition of an activating cytokine (e.g., IL-12, IL-23, or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more of the costimulatory domains and the CD3 ⁇ ITAM domain) or under the control of a CAR-induced promoter (e.g., the NFAT/IL-2 minimal promoter) .
  • a CAR may be a new generation CAR system that is different than the first-, second-, third-, or fourth-generation CAR system as disclosed herein.
  • a hinge domain (e.g., the linker between the extracellular antigen binding domain and the transmembrane domain) of a CAR as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
  • a transmembrane domain of a CAR as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
  • the hinge domain and the transmembrane domain of a CAR as disclosed herein can be derived from the same protein (e.g., CD8) .
  • the hinge domain and the transmembrane domain of the CAR as disclosed herein can be derived from different proteins.
  • a signaling domain of a CAR can comprise at least or up to about 1 signaling domain, at least or up to about 2 signaling domains, at least or up to about 3 signaling domains, at least or up to about 4 signaling domains, at least or up to about 5 signaling domains, at least or up to about 6 signaling domains, at least or up to about 7 signaling domains, at least or up to about 8 signaling domains, at least or up to about 9 signaling domains, or at least or up to about 10 signaling domains.
  • a signaling domain (e.g., a signaling peptide of the intracellular signaling domain) of a CAR as disclosed herein can comprise a full length or at least a portion of a polypeptide of CD3 ⁇ , 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.
  • the signaling domain CAR as disclosed herein can comprise a full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.
  • the cell e.g., the engineered immune cell such as the engineered NK cell
  • the chimeric polypeptide receptor e.g., CAR
  • the engineered cell e.g., the engineered NK cell
  • the chimeric polypeptide receptor e.g., TFP or CAR
  • the 2B4 signaling domain can be flanked by the CD8 transmembrane domain and the DAP10 signaling domain.
  • the DAP10 signaling domain can be flanked by the CD8 transmembrane domain and the 2B4 signaling domain.
  • the chimeric polypeptide receptor as disclosed herein can further comprise yet an additional signaling domain derived from CD3 ⁇ .
  • An antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor can be a cell surface marker, a secreted marker, or an intracellular marker.
  • Non-limiting examples of an antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor (e.g., TFP or CAR) as disclosed herein can include ADGRE2, carbonic anhydrase IX (CA1X) , CCRI, CCR4, carcinoembryonic antigen (CEA) , CD3 ⁇ , CD5, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD133, CD138, CD269 (BCMA) , CD S, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen) , epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM)
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include 1-40- ⁇ -amyloid, 4-1BB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1) , B7-H3, Bacillus anthracis anthrax, BAFF, beta-amyloid, B-lymphoma cell, C242 antigen, C5, CA-125, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX) , cardiac myosin, CCL11 (eotaxin-1) , CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin) , CD15, CD152, CD154 (CD40L)
  • coli shiga toxin type-1 E. coli shiga toxin type-2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain-B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptor, ganglioside GD2, GD2, GD3 ganglioside, glypican 3, GMCSF receptor ⁇ -chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factor receptor kinase, human TNF, human beta-amyloid, ICAM-1 (CD54) , IFN- ⁇
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include 707-AP, a biotinylated molecule, a-Actinin-4, abl-bcr alb-b3 (b2a2) , abl-bcr alb-b4 (b3a2) , adipophilin, AFP, AIM-2, Annexin II, ART-4, BAGE, b-Catenin, bcr-abl, bcr-abl p190 (e1a2) , bcr-abl p210 (b2a2) , bcr-abl p210 (b3a2) , BING-4, CAG-3, CAIX, CAMEL, Caspase-8, CD171, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v7/8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an antibody, a fragment thereof, or a variant thereof.
  • antibody can be a natural antibody (e.g., naturally secreted by a subject’s immune cell, such as B cells) , a synthetic antibody, or a modified antibody.
  • the antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an Fc domain of an antibody from the group comprising 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtu
  • the cell e.g., the engineered immune cell such as the engineered NK cell
  • the cell can be combined with a co-therapeutic agent to treat a subject in need thereof.
  • the cell can be administered to the subject prior to, concurrent with, or subsequent to administration of the co-therapeutic agent to the subject.
  • the present disclosure provides a composition comprising (a) any one of the cell (e.g., the engineered immune cell such as the engineered NK cell) disclosed herein and (b) a co-therapeutic agent (i.e., a separate therapeutic agent) .
  • a co-therapeutic agent i.e., a separate therapeutic agent
  • Non-limiting examples of a co-therapeutic agent can include cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, for example, anti-CD20 antibodies, anti-PD1 antibodies (e.g., Pembrolizumab) platelet derived growth factor inhibitors (e.g., GLEEVEC TM (imatinib mesylate) ) , a COX-2 inhibitor (e.g., celecoxib) , interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR- ⁇ , BlyS, APRIL, BCMA receptor (s) , TRAIL/Apo2, other bioactive and organic chemical agents, and the like.
  • anti-CD20 antibodies e.g., Pembrolizumab
  • platelet derived growth factor inhibitors e.g
  • cytotoxic agent generally refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • a cytotoxic agent can include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu) , chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide) , doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin.
  • radioactive isotopes e.g., At211, I131, I125,
  • Non-limiting examples of a chemotherapeutic agent can include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone) ; delta-9-tetrahydrocannabinol (dronabinol, );beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan CPT-11 (irinotecan, ) , acetyl
  • ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill. ) , and docetaxel ( -Poulenc Rorer, Antony, France) ; chloranbucil; gemcitabine 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine oxaliplatin; leucovovin; vinorelbine novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoids such as retinoic acid; capecitabine pharmaceutically acceptable salts, acids or derivatives of any of
  • chemotherapeutic agent can also include “anti-hormonal agents” or “endocrine therapeutics” that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves.
  • Examples include anti-estrogens and selective estrogen receptor modulators (SERMs) , including, for example, tamoxifen (including tamoxifen) , raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs) ; agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as and ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example,
  • chemotherapeutic agents includes bisphosphonates such as clodronate (for example, or ) , etidronate, NE-58095, zoledronic acid/zoledronate, alendronate, pamidronate, tiludronate, or risedronate; as well as troxacitabine (a1, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR) ; vaccines such as vaccine and gene therapy vaccines, for example, vaccine, vaccine, and vaccine; topoisomerase 1 inhibitor; rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016)
  • Examples of a chemotherapeutic agent can also include antibodies such as alemtuzumab (Campath) , bevacizumab ( Genentech) ; cetuximab ( Imclone) ; panitumumab ( Amgen) , rituximab ( Genentech/Biogen Idec) , pertuzumab ( 2C4, Genentech) , trastuzumab ( Genentech) , tositumomab (Bexxar, Corixia) , and the antibody drug conjugate, gemtuzumab ozogamicin ( Wyeth) .
  • antibodies such as alemtuzumab (Campath) , bevacizumab ( Genentech) ; cetuximab ( Imclone) ; panitumumab ( Amgen) , rituximab ( Genentech/Biogen Idec) , pertuzumab ( 2C4, Genentech) ,
  • Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, feMzumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolov
  • Examples of a chemotherapeutic agent can also include “tyrosine kinase inhibitors” such as an EGFR-targeting agent (e.g., small molecule, antibody, etc. ) ; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724, 714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI) ; dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline) , an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis) ; pan-HER inhibitors such as canertinib (CI-1033; Pharmacia) ; Raf-1 inhibitors such as antis
  • Examples of a chemotherapeutic agent can also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, opr
  • Examples of a chemotherapeutic agent can also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene
  • growth inhibitory agent generally refers to a compound or composition which inhibits growth and/or proliferation of a cell (e.g., a cell whose growth is dependent on PD-L1 expression) either in vitro or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase) , such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine) , taxanes, and topoisomerase II inhibitors such as the anthracycline antibiotic doxorubicin ( (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy- ⁇ -L-lyxo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5, 12-naphthacenedione) , epirubicin, daunorubicin, etoposide, and bleomycin.
  • doxorubicin (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy- ⁇ -L-lyxo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5
  • paclitaxel and docetaxel are anticancer drugs both derived from the yew tree.
  • Docetaxel Rhone-Poulenc Rorer
  • paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • the cell e.g., engineered immune cell, such as engineered NK cell
  • the cell can be generated from an isolated stem cell (e.g., isolated ESCs, iPSCs, etc. ) .
  • the cell (e.g., engineered immune cell, such as engineered NK cell) of the present disclosure can be used (e.g., administered) to treat a subject in need thereof.
  • the subject can have or can be suspected of having a condition, such as a disease (e.g., cancer, tumor, tissue degeneration, fibrosis, etc. ) .
  • a cell e.g., a stem cell or a committed adult cell
  • the generated cell can be administered to the subject for adaptive immunotherapy.
  • the subject can be treated (e.g., administered with) a population of the cells (e.g., engineered NK cells) of the present disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses.
  • a population of the cells e.g., engineered NK cells
  • the present disclosure provides a method comprising (a) obtaining an initial cell from a subject; and (b) generating, from the initial cell, any one of the cells (e.g., engineered immune cell, such as engineered NK cell) disclosed herein.
  • the initial cell obtained from the subject is ESC.
  • the initial cell e.g., a fibroblast, such as an adult skin fibroblast
  • the initial cell is modified and transformed into an iPSC.
  • the present disclosure provides a method comprising administering to a subject in need thereof a population of the cell (e.g., engineered immune cell, such as engineered NK cell) as disclosed herein.
  • the method can further comprise administering to the subject a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .
  • a co-therapeutic agent e.g., a chemotherapeutic agent, anti-CD20 antibody, etc.
  • the present disclosure provides a method comprising administering to a subject in need thereof any one of the composition disclosed herein.
  • the composition can comprise (i) any one of the cell (e.g., engineered immune cell, such as engineered NK cell) disclosed herein and (ii) a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .
  • Any one of the methods disclosed herein can be utilized to treat a target cell, a target tissue, a target condition, or a target disease of a subject.
  • a target disease can be a viral, bacterial, and/or parasitic infection; inflammatory and/or autoimmune disease; or neoplasm such as a cancer and/or tumor.
  • a target cell can be a diseased cell.
  • a diseased cell can have altered metabolic, gene expression, and/or morphologic features.
  • a diseased cell can be a cancer cell, a diabetic cell, and an apoptotic cell.
  • a diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
  • a variety of target cells can be killed using any one of the cells (e.g., engineered immune cell, such as engineered NK cell) disclosed herein.
  • a target cell can include a wide variety of cell types.
  • a target cell can be in vitro.
  • a target cell can be in vivo.
  • a target cell can be ex vivo.
  • a target cell can be an isolated cell.
  • a target cell can be a cell inside of an organism.
  • a target cell can be an organism.
  • a target cell can be a cell in a cell culture.
  • a target cell can be one of a collection of cells.
  • a target cell can be a mammalian cell or derived from a mammalian cell.
  • a target cell can be a rodent cell or derived from a rodent cell.
  • a target cell can be a human cell or derived from a human cell.
  • a target cell can be a prokaryotic cell or derived from a prokaryotic cell.
  • a target cell can be a bacterial cell or can be derived from a bacterial cell.
  • a target cell can be an archaeal cell or derived from an archaeal cell.
  • a target cell can be a eukaryotic cell or derived from a eukaryotic cell.
  • a target cell can be a pluripotent stem cell.
  • a target cell can be a plant cell or derived from a plant cell.
  • a target cell can be an animal cell or derived from an animal cell.
  • a target cell can be an invertebrate cell or derived from an invertebrate cell.
  • a target cell can be a vertebrate cell or derived from a vertebrate cell.
  • a target cell can be a microbe cell or derived from a microbe cell.
  • a target cell can be a fungi cell or derived from a fungi cell.
  • a target cell can be from a specific organ or tissue.
  • a target cell can be a stem cell or progenitor cell.
  • Target cells can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc. ) .
  • Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc.
  • Clonal cells can comprise the progeny of a cell.
  • a target cell can comprise a target nucleic acid.
  • a target cell can be in a living organism.
  • a target cell can be a genetically modified cell.
  • a target cell can be a host cell.
  • a target cell can be a totipotent stem cell, however, in some embodiments of this disclosure, the term “cell” may be used but may not refer to a totipotent stem cell.
  • a target cell can be a plant cell, but in some embodiments of this disclosure, the term “cell” may be used but may not refer to a plant cell.
  • a target cell can be a pluripotent cell.
  • a target cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage but may not be able to differentiate into any other non-hematopoietic cell.
  • a target cell may be able to develop into a whole organism.
  • a target cell may or may not be able to develop into a whole organism.
  • a target cell may be a whole organism.
  • a target cell can be a primary cell.
  • cultures of primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more.
  • Cells can be unicellular organisms. Cells can be grown in culture.
  • a target cell can be a diseased cell.
  • a diseased cell can have altered metabolic, gene expression, and/or morphologic features.
  • a diseased cell can be a cancer cell, a diabetic cell, and a apoptotic cell.
  • a diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
  • the target cells may be harvested from an individual by any method.
  • leukocytes may be harvested by apheresis, leukocytapheresis, density gradient separation, etc.
  • Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy.
  • An appropriate solution may be used for dispersion or suspension of the harvested cells.
  • Such solution can generally be a balanced salt solution, (e.g. normal saline, phosphate-buffered saline (PBS) , Hank's balanced salt solution, etc.
  • PBS phosphate-buffered saline
  • Buffers can include HEPES, phosphate buffers, lactate buffers, etc.
  • Cells may be used immediately, or they may be stored (e.g., by freezing) . Frozen cells can be thawed and can be capable of being reused. Cells can be frozen in a DMSO, serum, medium buffer (e.g., 10%DMSO, 50%serum, 40%buffered medium) , and/or some other such common solution used to preserve cells at freezing temperatures.
  • Non-limiting examples of cells which can be target cells include, but are not limited to, lymphoid cells, such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell) , Natural killer cell, cytokine induced killer (CIK) cells (see e.g.
  • myeloid cells such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil) , Monocyte/Macrophage, Red blood cell (Reticulocyte) , Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell) , parathyroid (Parathyroid chief cell, Oxyphil cell) , adrenal (Chromaffin cell) , pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia) , Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph) ; cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, granulocyte,
  • Apocrine sweat gland cell odoriferous secretion, sex-hormone sensitive
  • Gland of Moll cell in eyelid specialized sweat gland
  • Sebaceous gland cell lipid-rich sebum secretion
  • Bowman's gland cell in nose washes olfactory epithelium
  • Brunner's gland cell in duodenum enzymes and alkaline mucus
  • Seminal vesicle cell secretes seminal fluid components, including fructose for swimming sperm
  • Prostate gland cell secretes seminal fluid components
  • Bulbourethral gland cell massbourethral gland cell
  • Bartholin's gland cell vaginal lubricant secretion
  • Gland of Littre cell Gland of Littre cell
  • Uterus endometrium cell (carbohydrate secretion)
  • Isolated goblet cell of respiratory and digestive tracts micus secretion
  • Duct cell (of seminal vesicle, prostate gland, etc. ) , Epithelial cells lining closed internal body cavities, Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell) , Megakaryocyte (platelet precursor) , Monocyte, Connective tissue macrophage (various types) , Epidermal Langerhans cell, Osteoclast (in bone) , Dendritic cell (in lymphoid tissues) , Microglial cell (in central nervous system) , Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types) ,
  • the target cell is a cancer cell.
  • cancer cells include cells of cancers including Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma
  • the targeted cancer cell represents a subpopulation within a cancer cell population, such as a cancer stem cell.
  • the cancer is of a hematopoietic lineage, such as a lymphoma.
  • the antigen can be a tumor associated antigen.
  • the target cell e.g., B cells
  • the target cell as disclosed herein is associated or is suspected of being associated with an autoimmune disease.
  • the subject being treated with any one of the cell (e.g., engineered immune cell, such as engineered NK cell) of the present disclosure can have or can be suspected of having an autoimmune disease.
  • Non-limiting examples of an autoimmune disease can include acute disseminated encephalomyelitis (ADEM) , acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplantation rejection, anti-GBM/Anti-TBM nephritis, antiphospholipid syndrome (APS) , autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED) , autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP) , autoimmune thyroid disease, autoimmune urticaria, axonal &neuronal neuropathies, Balo
  • the autoimmune disease comprises one or more members selected from the group comprising rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE) , myasthenia gravis, multiple sclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) , Waldenstrom's macroglobulinemia (WM) , chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) , Hashimoto's Encephalopathy (HE) , Hashimoto's Thyroiditis, Graves' Disease, Wegener's Granulomatosis, and antibody-mediated transplantation rejection (e.g., for tissue transplant
  • the target disease is acute myeloid leukemia (AML) .
  • AML acute myeloid leukemia
  • the target disease is non-Hodgkin’s lymphoma (NHL) .
  • the target disease is chronic lymphocytic leukemia (CLL) .
  • CLL chronic lymphocytic leukemia
  • the target disease is B-cell leukemia (BCL) .
  • the target disease is non-small-cell lung carcinoma (NSCLC) .
  • NSCLC non-small-cell lung carcinoma
  • the target cells form a tumor (i.e., a solid tumor) .
  • a tumor treated with the methods herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20%in size, and/or do not metastasize) .
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks.
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.
  • the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more.
  • the tumor is completely eliminated, or reduced below a level of detection.
  • a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment.
  • a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment.
  • a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.
  • compositions e.g., comprising one or more proteins, such as growth factors, and/or one or more small molecules
  • methods as provided herein can be used to generate a population of immune cells (e.g., Natural killer (NK) cells, T cells, Natural killer T (NKT) cells, etc. ) from a population of progenitor cells, such as stem cells (e.g., induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, etc. ) .
  • NK Natural killer
  • T cells T cells
  • Natural killer T (NKT) cells etc.
  • progenitor cells such as stem cells (e.g., induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, etc. ) .
  • stem cells e.g., induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells
  • the generation of the population of immune cells as disclosed herein can be a single step (e.g., use of a single composition) , or can comprise a plurality of steps (e.g., using a single composition or a plurality of different compositions) .
  • a plurality of different compositions can be used to direct differentiation of a stem cell towards an immune cell in a plurality of steps, such as one or more of: (1) stem cell to mesodermal cells, (2) mesodermal cells to mesodermal cells having enhanced hemogenic endothelium (HE) potential, (3) mesodermal cells with such enhanced HE potential to hemogenic endothelial cells, and/or (4) hemogenic endothelial cells to a differentiated immune cells (e.g., NK cells, T cells, NKT cells, etc. ) .
  • a differentiated immune cells e.g., NK cells, T cells, NKT cells, etc.
  • iPSCs Induced pluripotent stem cells
  • APEL2 Albumin Polyvinylalcohol Essential Lipids 2
  • CHIR GSK-3 ⁇ / ⁇ inhibitor
  • the iPSCs are cultured in this first composition (e.g., for about 48 hours (2 days) ) . While in this first composition, the iPSCs differentiate into mesodermal cells.
  • the first composition is removed, and the mesodermal cells are washed.
  • the mesodermal cells are cultured in APEL2 media supplemented with 10 ng/ml bone morphogenetic protein 4 (BMP4) , 50 ng/ml vascular endothelial growth factor (VEGF) , 50 ng/ml basic fibroblast growth factor, and 10 ⁇ M transforming growth factor ⁇ (TGF- ⁇ ) /activin signaling inhibitor SB431542.
  • BMP4 bone morphogenetic protein 4
  • VEGF vascular endothelial growth factor
  • TGF- ⁇ basic fibroblast growth factor
  • the mesodermal cells are cultured in this second composition for 96 hours (4 days) . While in this composition, the mesodermal cells differentiate into mesodermal cells having hemogenic endothelium (HE) potential.
  • HE hemogenic endothelium
  • the mesodermal cells with HE potential are cultured in Serum-Free Expansion Medium II (SFEMII) supplemented with 20 ng/ml BMP4, 5 ng/ml VEGF, 20 ng/ml interleukin-7 (IL-7) , 10 ng/ml interleukin-15 (IL-15) , 10 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3L) , 5 ng/ml interleukin-3 (IL-3) , and 40 ng/ml stem cell factor (SCF) .
  • the mesodermal cells are cultured in this third composition for 96 hours (4 days) . While in this third composition, the mesodermal cells with HE potential differentiate into hemogenic endothelial (HE) cells.
  • the third composition is removed, and the HE cells are washed.
  • the HE cells are cultured in SFEMII media supplemented with 20 ng/ml IL-7, 10 ng/ml FLTL3, 10 ng/ml IL-15, and 20 ng/ml SCF.
  • the HE cells are cultured in this four composition for up to 40-45 days. While in this fourth composition, the HE cells differentiate into NK cells.
  • compositions components and protocol time periods are shown in Table 1, and the components and protocol time periods for conventional method of differentiating NK cells are shown in Table 2.
  • Example 2 Generating and Validating Differentiation of iPSCs to Natural Killer (NK) Cells
  • the generation of the population of immune cells as disclosed herein can be a single step (e.g., use of a single composition) , or can comprise a plurality of steps (e.g., using a single composition or a plurality of different compositions) .
  • a plurality of different compositions can be used to direct differentiation of a stem cell towards an immune cell in a plurality of steps, such as one or more of: (1) stem cell to mesodermal cells, (2) mesodermal cells to hemogenic endothelial cells, and/or (3) hemogenic endothelial cells to a differentiated immune cells (e.g., NK cells, T cells, NKT cells, etc. ) .
  • the composition s components and protocol time periods are shown in Table 3.
  • Each step of the differentiation process can be validated to ensure proper generation of an immune cell (e.g., NK cell) .
  • the iPSC pluripotency was confirmed by the expression of cell surface markers SSEA4 and TRA-1-81. As shown in FIG. 1B and FIG. 1C, nearly 100%of the iPSCs were positive for SSEA4 alone and TRA-1-8 alone, and 99.63%of iPSCs were positive for both SSEA4 and TRA-1-8, as assessed via flow cytometry. This indicated that the iPSC pluripotency was well maintained.
  • HE cells Hemogenic endothelial cells
  • iPSCs were dissociated with TrypLE and passaged at a density of 5,000 cells/ml in iPSC culture medium, including ROCK inhibitor Y27632.
  • Mesoderm formation was induced by incubating iPSCs with CHIR for two days.
  • concentrations of CHIR greater than 3 ⁇ M e.g., 6 ⁇ M and 10 ⁇ M of CHIR
  • both concentrations of 6 ⁇ M and 10 ⁇ M of CHIR produced comparable efficiency of hemogenic endothelial cell induction (80.34%and 77.75%, respectively) , as assessed by expression of CD34.
  • NK cells Natural Killer (NK) cells
  • iPSCs were differentiated to NK cells with compositions and time periods described in Table 3.
  • Flow cytometry with CD45 and CD56 markers revealed that the addition of CHIR alone during the first 48 hours of mesoderm induction was sufficient for NK differentiation, compared to when BMP4 and/or Activin are added (Table 5) .
  • NK cell archetypal phenotypic markers e.g., CD56, NK cell receptor NKp30, and inhibitory NK cell receptor NKG2A
  • NK cell archetypal phenotypic markers e.g., CD56, NK cell receptor NKp30, and inhibitory NK cell receptor NKG2A
  • FIG. 3A 85.06%of the cells were CD56-positive at day 21 of differentiation.
  • FIG. 3B 30.41%of the cells were NKp30-positive at day 21 of differentiation.
  • FIG. 3C 34.11%of the cells were NKG2A-positive at day 21 of differentiation.
  • Flow cytometry data was quantified, as shown in FIG. 3D.
  • NK cell archetypal phenotypic markers e.g., CD56, NK cell receptor NKG2A, and inhibitory NK cell receptor NKp44
  • NK cell archetypal phenotypic markers e.g., CD56, NK cell receptor NKG2A, and inhibitory NK cell receptor NKp44
  • FIG. 4A 94.13%of the cells were CD56-positive at day 30 of differentiation.
  • FIG. 4B 52.52%of the cells were NKG2A-positive at day 30 of differentiation.
  • FIG. 4C 29.91%of the cells were NKp44-positive at day 30 of differentiation.
  • Flow cytometry data was quantified, as shown in FIG. 4D.
  • NK cell archetypal phenotypic markers e.g., CD56, NK cell receptor NKG2A, and inhibitory NK cell receptor NKp44
  • NK cell archetypal phenotypic markers e.g., CD56, NK cell receptor NKG2A, and inhibitory NK cell receptor NKp44
  • FIG. 5A 99.32%of the cells were CD56-positive at day 37 of differentiation.
  • FIG. 5B 45.49%of the cells were NKG2A-positive at day 37 of differentiation.
  • FIG. 5C 39.47%of the cells were NKp44-positive at day 37 of differentiation.
  • Flow cytometry data was quantified, as shown in FIG. 5D.
  • NK cell archetypal phenotypic markers at different days of differentiation showed that purity of differentiated NK cells was improved with more days of differentiation. Furthermore, yield was calculated as the number of NK cells (iNK) differentiated from one iPSC. As shown in FIG. 6, higher yield was achieved on day 37 than day 30 (696 versus 3, 956 folds) .
  • the method disclosed herein e.g., Table 3 generated more iNK from a single iPSC than by using the conventional method (Table 2) .
  • compositions of matter disclosed herein in the composition section of the present disclosure may be utilized in the method section including methods of use and production disclosed herein, or vice versa.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Virology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des méthodes de génération d'une cellule immunitaire, telle qu'une cellule immunitaire modifiée. Selon un aspect, l'invention concerne une méthode in vitro de génération de cellules mésodermiques dérivées de cellules souches. Ladite méthode peut comprendre la mise en contact d'une population de cellules souches avec une composition comprenant un activateur de la voie de signalisation Wnt/β-caténine (activateur). L'activateur peut être suffisant pour transformer la population de cellules souches en une population de cellules mésodermiques.
PCT/CN2023/073482 2022-01-28 2023-01-27 Méthodes et compositions pour immunothérapies cellulaires WO2023143475A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CNPCT/CN2022/074827 2022-01-28
CN2022074827 2022-01-28
CN2022142848 2022-12-28
CNPCT/CN2022/142848 2022-12-28

Publications (1)

Publication Number Publication Date
WO2023143475A1 true WO2023143475A1 (fr) 2023-08-03

Family

ID=87470617

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/073482 WO2023143475A1 (fr) 2022-01-28 2023-01-27 Méthodes et compositions pour immunothérapies cellulaires

Country Status (2)

Country Link
TW (1) TW202340453A (fr)
WO (1) WO2023143475A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160143949A1 (en) * 2014-11-25 2016-05-26 President And Fellows Of Harvard College Methods for generation of podocytes from pluripotent stem cells and cells produced by the same
CN106459918A (zh) * 2014-04-24 2017-02-22 得克萨斯州大学系统董事会 施加诱导多能干细胞产生过继细胞疗法产品
WO2021032851A1 (fr) * 2019-08-20 2021-02-25 Adaptimmune Limited Procedes de production de cellules progenitrices hemogeniques a partir de cellules souches pluripotentes
WO2021113744A1 (fr) * 2019-12-06 2021-06-10 Fate Therapeutics, Inc. Amélioration d'une cellule immunitaire effectrice dérivée d'ipsc à l'aide de petits composés
US20220025330A1 (en) * 2019-01-22 2022-01-27 Washington University Compositions and methods for generating hematopoietic stem cells (hscs)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459918A (zh) * 2014-04-24 2017-02-22 得克萨斯州大学系统董事会 施加诱导多能干细胞产生过继细胞疗法产品
US20160143949A1 (en) * 2014-11-25 2016-05-26 President And Fellows Of Harvard College Methods for generation of podocytes from pluripotent stem cells and cells produced by the same
US20220025330A1 (en) * 2019-01-22 2022-01-27 Washington University Compositions and methods for generating hematopoietic stem cells (hscs)
WO2021032851A1 (fr) * 2019-08-20 2021-02-25 Adaptimmune Limited Procedes de production de cellules progenitrices hemogeniques a partir de cellules souches pluripotentes
WO2021113744A1 (fr) * 2019-12-06 2021-06-10 Fate Therapeutics, Inc. Amélioration d'une cellule immunitaire effectrice dérivée d'ipsc à l'aide de petits composés

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WEN-JUN YANG, PING HU: "Study on mesodermal cell lineages", CHINESE BULLETIN OF LIFE SCIENCES, vol. 28, no. 8, 24 August 2016 (2016-08-24), pages 849 - 855, XP093080941, DOI: 10.13376/j.cbls/2016112 *

Also Published As

Publication number Publication date
TW202340453A (zh) 2023-10-16

Similar Documents

Publication Publication Date Title
US20230013642A1 (en) Toxicity Management for Anti-Tumor Activity of CARs
WO2022095902A1 (fr) Systèmes et procédés pour des immunothérapies améliorées
TWI787599B (zh) 嵌合抗原和ｔ細胞受體及使用方法
TWI785009B (zh) Ｃｄ７０結合分子及使用彼之方法
TW201902493A (zh) 使用併入最佳化多功能t細胞之嵌合受體t細胞之治療
EP4240835A1 (fr) Systèmes et procédés de régulation de l'expression ou de l'activité des gènes
WO2023143475A1 (fr) Méthodes et compositions pour immunothérapies cellulaires
WO2022179562A1 (fr) Récepteurs chimériques à l'antigène dans des cellules immunitaires
WO2023093763A1 (fr) Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules
WO2023078288A1 (fr) Systèmes et procédés pour des immunothérapies améliorées
WO2022179563A1 (fr) Systèmes et compositions pour immunothérapies améliorées et leurs procédés
WO2023147777A1 (fr) Systèmes et procédés pour des immunothérapies améliorées
WO2023078287A1 (fr) Systèmes et procédés pour des immunothérapies améliorées
WO2023147776A1 (fr) Systèmes et procédés pour des immunothérapies améliorées
WO2024183750A1 (fr) Nouvelle cellule immunitaire et son utilisation pour le traitement de maladies
US20210338725A1 (en) Treatment methods
US20220135642A1 (en) Cells, compositions and methods for enhancing immune function
CA3152930A1 (fr) Ciblage d'epha3 et ses utilisations
OA19499A (en) Chimeric antigen and T cell receptors and methods of use.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23746353

Country of ref document: EP

Kind code of ref document: A1