WO2022179563A1 - Systèmes et compositions pour immunothérapies améliorées et leurs procédés - Google Patents

Systèmes et compositions pour immunothérapies améliorées et leurs procédés Download PDF

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WO2022179563A1
WO2022179563A1 PCT/CN2022/077683 CN2022077683W WO2022179563A1 WO 2022179563 A1 WO2022179563 A1 WO 2022179563A1 CN 2022077683 W CN2022077683 W CN 2022077683W WO 2022179563 A1 WO2022179563 A1 WO 2022179563A1
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cell
engineered
fold
heterologous
amino acid
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PCT/CN2022/077683
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English (en)
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Yanan YUE
Luhan Yang
Yangbin Gao
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Hangzhou Qihan Biotechnology Co., Ltd.
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Priority to CN202280016401.9A priority Critical patent/CN117157390A/zh
Priority to TW111108821A priority patent/TW202334394A/zh
Publication of WO2022179563A1 publication Critical patent/WO2022179563A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic 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
    • 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
    • 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
    • C12N2510/00—Genetically modified 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 treating cancer.
  • Some aspects of the present disclosure provide engineered immune cells (e.g., engineered natural killer (NK) cells) and methods of use thereof for treatment of cancer, such as, e.g., as hematologic malignancies or solid tumors.
  • engineered immune cells e.g., engineered natural killer (NK) cells
  • NK natural killer
  • the present disclosure provides an engineered cell, comprising: one or both of (i) a heterologous CD16 variant for enhanced CD16 signaling as compared to a control cell, and (ii) a heterologous variant of IL-15 or a receptor thereof; and a chimeric antigen receptor comprising an antigen binding moiety capable of binding to CD19.
  • the antigen binding moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 39, and a light chain variable region comprising an amino acid sequence having at least 80%identity with the polypeptide sequence of SEQ ID NO. 40.
  • the antigen binding moiety comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 41.
  • the chimeric antigen receptor comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 42.
  • the antigen binding moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 16, and a light chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 17.
  • the antigen binding moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 43, and a light chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 44.
  • the engineered cell is an engineered NK cell.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered cell is an engineered T cell.
  • the engineered cell is an engineered B cell.
  • the engineered cell is an engineered variant of an isolated stem cell or an induced stem cell.
  • the engineered cell comprises (ii) the heterologous variant of IL-15 or the receptor thereof.
  • the heterologous variant comprises a secretory IL-15 or a membrane-bound polypeptide.
  • the heterologous variant of IL-15 is the secretory IL-15 comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%sequence identity with the polypeptide sequence of SEQ ID NO. 24.
  • the heterologous variant of IL-15 is the membrane-bound polypeptide, wherein the membrane-bound polypeptide comprises at least a portion of IL-15 and at least a portion of IL-15 receptor.
  • the at least a portion of IL-15 and the at least a portion of IL-15 receptor is linked via a linker.
  • the linker is selected from the group consisting of (GGGGS) n, (EGKSSGSGSESKST) n, and (EGKSSGSGSESKST) n (GGGGS) n.
  • the n is any integer selected from 1-10.
  • the linker comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with any one selected from the group consisting of SEQ ID No. 18-20 and 55-60.
  • the membrane-bound polypeptide further comprises an intracellular signaling domain.
  • the intracellular signaling domain comprises an amino acid having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 21.
  • the membrane-bound polypeptide further comprises an extracellular signal peptide.
  • the extracellular signal peptide comprises an amino acid having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 22 or 31.
  • the membrane-bound polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with any one selected from the group consisting of SEQ ID NO. 13-15 and 52-54.
  • the engineered cell comprises (i) the heterologous CD16 variant.
  • the heterologous CD16 variant comprises F158V (F176V) .
  • the heterologous CD16 variant comprises S197P.
  • the heterologous CD16 variant comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 23.
  • the chimeric antigen receptor further comprises a hinge domain.
  • the hinge domain comprises an amino acid sequence derived from 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 chimeric antigen receptor further comprises a transmembrane domain.
  • the transmembrane domain comprises an amino acid sequence derived from 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 chimeric antigen receptor further comprises a costimulatory domain.
  • the costimulatory domain comprises an amino acid sequence derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.
  • the chimeric antigen receptor further comprises a signaling domain.
  • the signaling domain comprises an amino acid sequence derived from CD3 ⁇ , 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.
  • the present disclosure provides an engineered NK cell comprising a chimeric antigen receptor comprising an antigen binding moiety exhibiting specific binding to CD19, wherein the antigen binding moiety is characterized by a member selected from the group consisting of: (1) comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 39, and a light chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO.
  • the antigen binding moiety is characterized by (1) .
  • the antigen binding moiety comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 41.
  • the chimeric antigen receptor comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 42.
  • the antigen binding moiety is characterized by (2) .
  • the antigen binding moiety comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 35.
  • the chimeric antigen receptor comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 34.
  • the antigen binding moiety is characterized by (3) .
  • the antigen binding moiety comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 45 or 47.
  • the chimeric antigen receptor comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 46, 48, or 61.
  • the engineered cell further comprises a heterologous CD16 variant for enhanced CD16 signaling as compared to a control cell.
  • the heterologous CD16 variant comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 23.
  • the engineered cell further comprises a heterologous variant of IL-15 or a receptor thereof.
  • the heterologous variant of IL-15 or the receptor thereof is a secretory IL-15.
  • the secretory IL-15 comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%sequence identity with the polypeptide sequence of SEQ ID NO. 24.
  • the heterologous variant of IL-15 or the receptor thereof is a membrane-bound polypeptide.
  • the membrane-bound polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with any one selected from the group consisting of SEQ ID NO. 13-15 and 52-54.
  • the present disclosure provides one or more polynucleotides encoding a chimeric antigen receptor comprising an antigen binding moiety exhibiting specific binding to CD19, wherein the antigen binding moiety is characterized by a member selected from the group consisting of: (1) comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 39, and a light chain variable region comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO.
  • the antigen binding moiety is characterized by (1) .
  • the antigen binding moiety comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 41.
  • the chimeric antigen receptor comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 42.
  • the antigen binding moiety is characterized by (2) .
  • the antigen binding moiety comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 35.
  • the chimeric antigen receptor comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 34.
  • the antigen binding moiety is characterized by (3) .
  • the antigen binding moiety comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 45 or 47.
  • the chimeric antigen receptor comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 46, 48, or 61.
  • the one or more polynucleotides further encode a heterologous CD16 variant for enhanced CD16 signaling as compared to a control cell.
  • the heterologous CD16 variant comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with the polypeptide sequence of SEQ ID NO. 23.
  • the one or more polynucleotides further encode a heterologous variant of IL-15 or a receptor thereof.
  • the heterologous variant of IL-15 or the receptor thereof is a secretory IL-15.
  • the secretory IL-15 comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%sequence identity with the polypeptide sequence of SEQ ID NO. 24.
  • the heterologous variant of IL-15 or the receptor thereof is a membrane-bound polypeptide.
  • the membrane-bound polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or 100%identity with any one selected from the group consisting of SEQ ID NO. 13-15 and 52-54.
  • a single polynucleotide encodes: (i) the chimeric antigen receptor; and (ii) the heterologous CD16 variant and/or the heterologous variant of IL-15 or the receptor thereof.
  • a first polynucleotide molecule encodes (i) the chimeric antigen receptor but not (ii) the heterologous CD16 variant and/or the heterologous variant of IL-15 or the receptor thereof; and (B) a second polynucleotide molecule encodes (i) the heterologous CD16 variant and/or the heterologous variant of IL-15 or the receptor thereof, but not (ii) the chimeric antigen receptor.
  • the one or more polynucleotides are human codon optimized.
  • the one or more polynucleotides are part of at least one viral vector.
  • the at least one viral vector comprise an AAV vector.
  • the one or more polynucleotides are not part of a viral vector.
  • the present disclosure provides a composition comprising the one or more polynucleotides of any one of the preceding embodiments.
  • the present disclosure provides a composition comprising the engineered cell or the engineered NK cell of any one of the preceding embodiments.
  • the composition further comprises a separate therapeutic agent.
  • separate therapeutic agent is a chemotherapeutic agent.
  • the chemotherapeutic agent is an antibody.
  • the present disclosure provides a method for treating a condition of a subject, the method comprising administering to the subject the composition of any one of the preceding embodiments.
  • the condition of the subject is cancer or tumor.
  • the present disclosure provides a method of any one of the preceding embodiments, where (i) the separate therapeutic agent of the composition is administered to the subject prior to, simultaneously with, or subsequent to (ii) administration of the engineered cell or the engineered NK cell to the subject
  • an engineered cell comprising: (i) a CD16 variant for enhanced CD16 signaling as compared to a control cell, (ii) a heterologous IL-15, a heterologous IL-15 receptor or a fragment thereof, and (iii) a chimeric antigen receptor comprising an antigen binding moiety capable of binding to CD19.
  • the antigen binding moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 80%identity with SEQ ID NO. 16 and a light chain variable region comprising an amino acid sequence having at least 80%identity with SEQ ID NO. 17.
  • the engineered cell comprises a stem cell, a progenitor cell, and a specific cell.
  • the stem cell comprises an isolated stem cell and an induced stem cell.
  • the stem cell comprises an embryonic stem cell and an induced pluripotent stem cell.
  • the progenitor cell comprises an isolated progenitor cell and an induced progenitor cell.
  • he progenitor cell comprises a hematopoietic stem cell, a myeloid progenitor, a megakaryocyte progenitor, an erythrocyte progenitor and a lymphoid progenitor.
  • the specific cell comprises an isolated specific cell and an induced specific cell.
  • the specific cell comprises a T cell, a B cell and an NK cell.
  • the NK cell is derived from an isolated stem cell or an induced stem cell.
  • the NK cell is derived from an isolated progenitor cell or an induced progenitor cell.
  • a specific cell as disclosed herein may be a differentiated cell.
  • the heterologous IL-15 comprises a secretory IL-15 and a membrane-bound IL-15.
  • the heterologous IL-15 is a secretory IL-15 comprising an amino acid sequence having at least 80%identity with SEQ ID NO. 24.
  • the membrane bound IL-15 comprises at least a portion of IL-15 and at least a portion of IL-15 receptor.
  • the at least a portion of IL-15 and the at least a portion of IL-15 receptor is linked via a linker.
  • the linker is selected from the group consisting of (GGGGS) n, (EGKSSGSGSESKST) n, and (EGKSSGSGSESKST) n (GGGGS) n.
  • the n is any integer selected from 1-10.
  • the linker comprises an amino acid sequence having at least 80%identity with any one selected from the group consisting of SEQ ID No. 18-20.
  • the membrane bound IL-15 further comprises an intracellular signaling domain.
  • the intracellular signaling domain comprises an amino acid having at least 80%identity with SEQ ID NO. 21.
  • the membrane bound IL-15 further comprises an extracellular signal peptide.
  • the extracellular signal peptide comprises an amino acid having at least 80%identity with SEQ ID NO. 22.
  • the membrane bound IL-15 comprises an amino acid sequence having at least 80%identity with any one selected from the group consisting of SEQ ID NO. 13-15, and 52-54.
  • the CD16 variant is heterologous to the cell.
  • the CD16 variant comprises F158V (F176V) .
  • F158V may be due to counting from the N-terminal amino acid of the mature protein, excluding the signal peptide, and F158V and F176V may refer to the same amino acid substitution.
  • the CD16 variant further comprises S197P.
  • the CD16 variant comprises an amino acid sequence having at least 80%identity with SEQ ID NO. 23.
  • the chimeric antigen receptor further comprises a hinge domain.
  • the hinge domain comprises an amino acid sequence derived from 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 chimeric antigen receptor further comprises a transmembrane domain.
  • the transmembrane domain comprises an amino acid sequence derived from 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 chimeric antigen receptor further comprises a costimulatory domain.
  • the costimulatory domain comprises an amino acid sequence derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.
  • the chimeric antigen receptor further comprises a signaling domain.
  • the signaling domain comprises an amino acid sequence derived from CD3 ⁇ , 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.
  • a polynucleotide comprising: (i) a first sequence encoding a CD16 variant for enhanced CD16 signaling, (ii) a second sequence encoding a heterologous IL-15, and (iii) a third sequence encoding chimeric antigen receptor comprising an antigen binding moiety capable of binding to CD19.
  • the first sequence encoding the CD16 variant comprises a nucleic acid sequence having at least 80%identity with SEQ ID NO. 27.
  • the second sequence encoding the heterologous IL-15 comprises a nucleic acid sequence encoding a secretory IL-15 or a nucleic acid sequence encoding a membrane bound IL-15.
  • the nucleic acid sequence encoding the secretory IL-15 comprises a nucleic acid sequence having at least 80%identity with SEQ ID NO. 30.
  • the nucleic acid sequence encoding a membrane bound IL-15 comprises a nucleic acid sequence having at least 80%identity with SEQ ID NO. 28 or 29.
  • the third sequence comprises: (a) a nucleic acid sequence encoding a heavy chain variable region of the antigen binding moiety which has at least 80%identity with SEQ ID NO 25, and (b) a nucleic acid sequence encoding a light chain variable region of the antigen binding moiety which has at least 80%identity with SEQ ID NO 26.
  • an engineered cell comprising: (i) a CD16 variant for enhanced CD16 signaling as compared to a control cell, (ii) a heterologous IL-15, wherein the heterologous IL-15 is a membrane bound IL-15 comprising an amino acid sequence having at least 95%identity with any one selected from the group consisting of SEQ ID No. 13-15, and 52-54, and (iii) a chimeric antigen receptor comprising an antigen binding moiety capable of binding to CD19.
  • the engineered cell comprises a stem cell, a progenitor cell, and a specific cell.
  • the stem cell comprises an isolated stem cell and an induced stem cell.
  • the stem cell comprises an embryonic stem cell and an induced pluripotent stem cell.
  • the progenitor cell comprises an isolated progenitor cell and an induced progenitor cell.
  • the progenitor cell comprises a hematopoietic stem cell, a myeloid progenitor, a megakaryocyte progenitor, an erythrocyte progenitor and a lymphoid progenitor.
  • the specific cell comprises an isolated specific cell and an induced specific cell.
  • the specific cell comprises a T cell, a B cell and an NK cell.
  • the NK cell is derived from an isolated stem cell or an induced stem cell.
  • the NK cell is derived from an isolated progenitor cell or an induced progenitor cell.
  • the CD16 variant is heterologous to the cell.
  • the CD16 variant comprises F158V (F176V) .
  • the CD16 variant further comprises S197P.
  • the CD16 variant comprises an amino acid sequence having at least 80%identity with SEQ ID NO. 23.
  • the antigen binding moiety is an antibody selected from Fab, Fab’, F (ab’) 2, scFv, and sdAb.
  • the antigen binding moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 80%identity with SEQ ID NO. 16 and a light chain variable region comprising an amino acid sequence having at least 80%identity with SEQ ID NO. 17.
  • the chimeric antigen receptor further comprises a hinge domain.
  • the hinge domain comprises an amino acid sequence derived from 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 chimeric antigen receptor further comprises a transmembrane domain.
  • the transmembrane domain comprises an amino acid sequence derived from 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 chimeric antigen receptor further comprises a costimulatory domain.
  • the costimulatory domain comprises an amino acid sequence derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.
  • the chimeric antigen receptor further comprises a signaling domain.
  • the signaling domain comprises an amino acid sequence derived from CD3 ⁇ , 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.
  • a polynucleotide comprising: (i) a first sequence encoding a CD16 variant for enhanced CD16 signaling, (ii) a second sequence encoding a membrane bound IL-15, wherein the second sequence comprises a nucleic acid sequence having at least 95%identity with SEQ ID NO. 28 or 29, and (iii) a third sequence encoding a chimeric antigen receptor comprising an antigen binding moiety capable of binding to CD19.
  • an engineered cell comprising a heterologous IL-15, wherein the heterologous IL-15 is a membrane bound IL-15 comprising an amino acid sequence having at least 95%identity with any one selected from the group consisting of SEQ ID No. 13-15, and 52-54.
  • the present disclosure provides an engineered NK cell, comprising: (1) a secretory IL-15 that is heterologous to the engineered NK cell; and (2) one or more of: (i) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the cell, or (ii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, wherein the antigen is not CD19, wherein the engineered cell lacks a heterologous IL-15R.
  • the engineered NK cell further comprises a heterologous polynucleotide encoding the secretory IL-15.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell comprises the CD16 variant. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the chimeric polypeptide receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises both (i) the CD16 variant and (ii) the chimeric polypeptide receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell further comprises a heterologous polynucleotide encoding the secretory IL-15.
  • the antigen comprises one or more members selected from the group consisting of: BCMA, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100.
  • the antigen comprises a NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, AND ULBP6.
  • the present disclosure provides an engineered NK cell derived from an isolated stem cell or an induced stem cell, comprising a secretory IL-15 that is heterologous to the engineered NK cell, wherein the engineered cell lacks a heterologous IL-15R.
  • the engineered NK cell further comprises a heterologous polynucleotide encoding the secretory IL-15.
  • the present disclosure provides an engineered NK cell comprising a membrane-bound IL-15 that is heterologous to the engineered NK cell, wherein the engineered NK cell further comprise one or more of: (a) a heterologous cytokine that is not IL-15, (b) reduced expression or activity of endogenous immune regulator polypeptide, wherein the endogenous immune regulator polypeptide is not B2M, or (c) a safety switch (or inducible cell death moiety) .
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell comprises the heterologous cytokine that is not IL-15. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the reduced expression or activity of the endogenous immune regulator polypeptide, wherein the endogenous immune regulator polypeptide is not B2M. In some embodiments of any one of the engineered NK cells disclosed herein, wherein the engineered NK cell comprises safety switch. In some embodiments of any one of the engineered NK cells disclosed herein, wherein the engineered NK cell comprises two or more of (a) through (c) . In some embodiments of any one of the engineered NK cells disclosed herein, wherein the engineered NK cell comprises (a) through (c) .
  • the membrane bound IL-15 is a part of a chimeric membrane receptor comprising the IL-15 and at least a portion of IL-15R.
  • the chimeric membrane receptor comprises a sequence selected from the group consisting of (i) four or more adjacent repeats of SEQ ID NO. 9, (ii) SEQ ID NO. 10, (iii) SEQ ID NO. 11, (iv) a combination of SEQ ID NO. 9 and SEQ ID NO. 11, (v) SEQ ID NO. 12, (vi) SEQ ID NO. 13, and (vii) SEQ ID NO. 14) .
  • the present disclosure provides an engineered NK cell comprising a CD16 variant for enhanced CD16 signaling in the engineered NK cell, the CD16 variant comprising at least a portion of CD16 and at least a portion of CD64, wherein the engineered NK cell exhibits reduced expression or activity of an endogenous immune regulator polypeptide.
  • the engineered NK cell comprises a heterologous IL-15 or a fragment thereof.
  • the engineered NK cell comprises a receptor comprising a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the present disclosure provides an engineered NK cell comprising reduced activity of endogenous IL-17 signaling, wherein the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell comprises reduced expression of endogenous IL-17 or endogenous IL-17R. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression of endogenous IL-17 and endogenous IL-17R. In some embodiments of any one of the engineered NK cells disclosed herein, the endogenous IL-17 comprises IL-17A. In some embodiments of any one of the engineered NK cells disclosed herein, the endogenous IL-17 comprises IL-17F.
  • the endogenous IL-17R comprises IL-17RA, IL-17RB, IL-17RC, IL-17RD, or IL-17RE. In some embodiments of any one of the engineered NK cells disclosed herein, the endogenous IL-17R comprises IL-17RA.
  • the engineered NK cell exhibits enhanced expression profile of a NK cell marker as compared to a control cell without reduced expression or activity of endogenous IL-17.
  • the NK cell marker comprises human CD57 or killer immunoglobulin-like receptors (KIR) .
  • the engineered NK cell exhibits enhanced survival in the presence of tumor cells as compared to a control cell without reduced activity of endogenous IL-17 signaling.
  • the present disclosure provides an engineered NK cell comprising a heterologous STAT, wherein the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell further comprises a heterologous IL-15 or a fragment thereof.
  • the engineered NK cell further comprises a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell exhibits enhanced survival in the presence of tumor cells as compared to a control cell without the heterologous STAT.
  • the heterologous STAT comprises STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, or STAT6.
  • the heterologous STAT comprises STAT3.
  • the heterologous STAT comprises STAT5B.
  • the present disclosure provides an engineered NK cell comprising (1) reduced expression or activity of endogenous Killer-cell immunoglobulin-like receptor (KIR) and (2) one or more of (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, (b) a heterologous cytokine, (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell, (d) a heterologous immune regulator polypeptide, or (e) reduced expression or activity of endogenous immune regulator polypeptide.
  • KIR Killer-cell immunoglobulin-like receptor
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell comprises the chimeric polypeptide receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the heterologous cytokine. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the CD16 variant. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises heterologous immune regulator polypeptide. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the reduced expression or activity of endogenous immune regulator polypeptide.
  • the engineered NK cell comprises two or more of (a) through (e) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises three or more of (a) through (e) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises four or more of (a) through (e) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises all of (a) through (e) .
  • the KIR comprises KIR2D. In some embodiments of any one of the engineered NK cells disclosed herein, tthe KIR2D comprises KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, or KIR2DS5. In some embodiments of any one of the engineered NK cells disclosed herein, the KIR comprises KIR3D. In some embodiments of any one of the engineered NK cells disclosed herein, the KIR3D comprises KIR3DL1, KIR3DL2, KIR3DL3, or KIR3DS1.
  • the present disclosure provides an engineered NK cell comprising reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGF beta receptor, or (iv) endogenous SHIP2, wherein the engineered cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell comprises reduced expression or activity of the endogenous CD94. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of the endogenous CD96. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of the endogenous TGF beta receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of the endogenous SHIP2.
  • the engineered NK cell comprises reduced expression or activity of two or more of (i) through (iv) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of three or more of (i) through (iv) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of all of (i) through (iv) .
  • the present disclosure provides an engineered NK cell comprising reduced expression or activity of one or more of: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB, or (vi) endogenous NKG2DL, wherein the engineered cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell comprises reduced expression or activity of endogenous CD80. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of endogenous CD86. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of endogenous ICOSL. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of endogenous CD40L. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of endogenous MICA or MICB.
  • the engineered NK cell comprises reduced expression or activity of endogenous NKG2D. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of two or more of (i) through (vi) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of three or more of (i) through (vi) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of four or more of (i) through (vi) .
  • the engineered NK cell comprises reduced expression or activity of five or more of (i) through (vi) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises reduced expression or activity of (i) through (vi) .
  • the present disclosure provides an engineered NK cell comprising (1) reduced expression or activity of endogenous ICAM1 and (2) one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, (b) a heterologous cytokine, or (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell comprises the chimeric polypeptide receptor. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the heterologous cytokine. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the CD16 variant. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises two or more of (a) through (c) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises all of (a) through (c) .
  • the present disclosure provides an engineered NK cell comprising reduced expression or activity of endogenous ICAM1, wherein the engineered cell is derived from an induced stem cell.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the present disclosure provides an engineered NK cell comprising one or more of: (i) a heterologous PD-L2 or (ii) a heterologous TGF-beta.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell comprises the heterologous PD-L2. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the heterologous TGF-beta. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises (i) the heterologous PD-L2 and (ii) the heterologous TGF-beta.
  • the present disclosure provides an engineered NK cell comprising one or more of: (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, or (v) a heterologous CD59, wherein the engineered cell is derived from an isolated stem cell or an induced stem cell.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell comprises the heterologous CCL21. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the heterologous IL-10. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the heterologous CD46. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the heterologous CD55. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises the heterologous CD59.
  • the engineered NK cell comprises two or more of (i) through (v) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises three or more of (i) through (v) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises four or more of (i) through (v) . In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises all of (i) through (v) .
  • the present disclosure provides an engineered NK cell derived from an induced stem cell, comprising a heterologous cytokine that is not IL-15.
  • the heterologous cytokine that is not IL-15 comprises IL-21.
  • the present disclosure provides an engineered NK cell derived from an induced stem cell, comprising a heterologous IL-21.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the present disclosure provides an engineered NK cell comprising a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to CD38, wherein the engineered NK cell is derived from an isolated embryonic stem cell or an induced stem cell.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the present disclosure provides an engineered NK cell, comprising a chimeric polypeptide receptor comprising CD8 transmembrane domain and one or more of (i) 2B4 signaling domain and (ii) DAP10 signaling domain.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the chimeric polypeptide receptor comprises 2B4 signaling domain. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises DAP10 signaling domain. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises (i) 2B4 signaling domain and (ii) DAP10 signaling domain.
  • the present disclosure provides an engineered NK cell derived from an isolated stem cell or an induced stem cell, comprising a chimeric polypeptide receptor comprising one or more of (i) CD8 transmembrane domain, (ii) 2B4 signaling domain, or (iii) DAP10 signaling domain.
  • the engineered NK cell further comprises (i) a heterologous IL-15 or a fragment thereof and/or (ii) a heterologous IL-15R or a fragment thereof.
  • the chimeric polypeptide receptor comprises CD8 transmembrane domain. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises 2B4 signaling domain. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises DAP10 signaling domain. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises two or more of (i) through (iii) . In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises all of (i) through (iii) .
  • the engineered NK cell exhibits reduced expression or activity of endogenous CD38.
  • expression or activity of endogenous CD38 of the engineered NK cell is not modified.
  • the heterologous IL-15 or the fragment thereof is secreted by the engineered NK cell.
  • the heterologous IL-15 or the fragment thereof is membrane-bound.
  • the engineered NK cell comprises a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell comprises an additional chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen that is different. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen binding moiety of the chimeric polypeptide receptor is a multispecific binding moiety capable of specifically binding to two or more antigens that are different.
  • the antigen comprises one or more members selected from the group consisting of: BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100.
  • the antigen comprises a NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, AND ULBP6.
  • the engineered NK cell comprises a safety switch capable of effecting death of the engineered NK cell.
  • the safety switch comprises one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.
  • the engineered NK cell comprises a heterologous cytokine.
  • the heterologous cytokine comprises one or more members selected from the group consisting of IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21.
  • the heterologous cytokine is not IL15.
  • the engineered NK cell comprises a heterologous immune regulator polypeptide.
  • the heterologous immune regulator polypeptide comprises one or more members selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • the engineered NK cell exhibits reduced expression or activity of an endogenous immune regulator polypeptide.
  • the endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator.
  • the immune checkpoint inhibitor comprises one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4.
  • the immune checkpoint inhibitor comprises SHIP2.
  • the hypo-immunity regulator comprises one or more members selected from the group consisting of 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, and CD59.
  • the engineered NK cell comprises a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell.
  • the CD16 variant comprises a sequence selected from the group consisting of: SEQ ID NOs. 1-8.
  • the engineered NK cell exhibits enhanced cytotoxicity against a target cell as compared to a control cell.
  • the engineered NK cell induces reduced immune response in a host cell as compared to a control cell.
  • the host cell is an immune cell.
  • the isolated stem cell comprises an embryonic stem cell. In some embodiments of any one of the engineered NK cells disclosed herein, the induced stem cell comprises an induced pluripotent stem cell.
  • the present disclosure provides a method comprising (1) obtaining a cell from a subject; and (2) generating, from the cell, a subject engineered NK cell of any one of the engineered NK cells disclosed herein.
  • the present disclosure provides a method comprising administering to a subject in need thereof a population of NK cells comprising a subject engineered NK cell of any one of the engineered NK cells disclosed herein.
  • the method further comprises administering to the subject a separate therapeutic agent.
  • the separate therapeutic agent is a chemotherapeutic agent.
  • the present disclosure provides a composition comprising a subject engineered NK cell of any one of the engineered NK cells disclosed herein.
  • the present disclosure provides a composition
  • an engineered immune cell that comprises one or more members comprising (i) a heterologous IL-15 or a fragment thereof, (ii) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered immune cell, or (iii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen; and (2) a separate therapeutic agent capable of binding to CD20.
  • the engineered NK cell is derived from an isolated stem cell or an induced stem cell.
  • the isolated stem cell comprises an embryonic stem cell.
  • the induced stem cell comprises an induced pluripotent stem cell.
  • the engineered immune cell comprises the heterologous IL-15.
  • the heterologous IL-15 or the fragment thereof is secreted by the engineered NK cell.
  • the heterologous IL-15 or the fragment thereof is membrane-bound.
  • the engineered immune cell comprises the CD16 variant.
  • the CD16 variant comprises a sequence selected from the group consisting of: SEQ ID NOs. 1-8.
  • the engineered immune cell comprises the chimeric polypeptide receptor.
  • the engineered immune cell comprises two or more of (i) through (iii) . In some embodiments of any one of the compositions disclosed herein, the engineered immune cell comprises (i) through (iii) .
  • the engineered immune cell comprises an engineered NK cell. In some embodiments of any one of the compositions disclosed herein, the engineered immune cell comprises an engineered T cell.
  • the engineered NK cell exhibits reduced expression or activity of endogenous CD38. In some embodiments of any one of the compositions disclosed herein, expression or activity of endogenous CD38 of the engineered NK cell is not modified.
  • the antigen binding moiety of the chimeric polypeptide receptor is a multispecific binding moiety capable of specifically binding to two or more antigens that are different.
  • the antigen comprises one or more members selected from the group consisting of: BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100.
  • the antigen comprises a NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, AND ULBP6.
  • the engineered NK cell further comprises a safety switch capable of effecting death of the engineered NK cell.
  • the safety switch comprises one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, and B-cell CD20.
  • the engineered NK cell further comprises a heterologous cytokine.
  • the heterologous cytokine comprises one or more members selected from the group consisting of IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments of any one of the compositions disclosed herein, the heterologous cytokine is not IL15.
  • the engineered NK cell further comprises a heterologous immune regulator polypeptide.
  • the heterologous immune regulator polypeptide comprises one or more members selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • the engineered NK cell exhibits reduced expression or activity of an endogenous immune regulator polypeptide.
  • the endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator.
  • the immune checkpoint inhibitor comprises one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4.
  • the immune checkpoint inhibitor comprises SHIP2.
  • the hypo-immunity regulator comprises one or more members selected from the group consisting of 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, and CD59.
  • the engineered NK cell exhibits enhanced cytotoxicity against a target cell as compared to a control cell. In some embodiments of any one of the compositions disclosed herein, the engineered NK cell induces reduced immune response in a host cell as compared to a control cell.
  • the host cell is an immune cell.
  • the engineered NK cell further comprises a receptor comprising a heterologous IL-15R or a fragment thereof.
  • the present disclosure provides a method comprising: administering to a subject in need thereof a subject composition of any one of the compositions disclosed herein.
  • the separate therapeutic agent comprises a chemotherapeutic agent.
  • Figure 1A-1G illustrate engineered NK cells comprising a CD16 variant for enhanced CD16 signaling
  • Figure 2A-2G illustrate engineered NK cells comprising a chimeric antigen receptor against CD19
  • Figure 3A and 3B illustrate engineered T cells comprising heterologous human IL-15.
  • Figure 4A-4C illustrate design of aCD19-CAR, hnCD16 and IL-15RF.
  • Figure 5A-5B illustrate design of NK-019 construct and engineered NK cells expressing NK-019 construct
  • Figure 6A-6C illustrate enhanced NK cell activity conferred by aCD19-CAR
  • Figure 7A-7C illustrate enhanced ADCC conferred by hnCD16
  • Figure 8A-8B illustrate enhanced IL-15 signaling conferred by IL-15-RF
  • Figure 9 illustrates expression of NK-019 construct in NK92 cells
  • Figure 10 illustrates CD19-specific cytolytic ability of NK-019 NK92 cells against K562-CD19 cells
  • Figure 11A illustrates enhanced CD107a expression after human CD19+ stimulation in NK-019 NK92 cells
  • Figure 11B illustrates enhanced Interferon-gamma production by NK-019 NK92 cells after human CD19+ stimulation
  • Figure 12A-12C illustrate anti-tumor activity of NK-019 NK92 cells in Raji-NCG mouse model
  • Figure 13A-13B illustrates expression of NK-019 constructs in iPSCs.
  • Figure 14A-14B illustrate differentiation of NK-019 iPSCs into NK-019 iNK cells.
  • FIG. 15 illustrates differentiation and expansion of OI42 (QN-019a) transduced cells.
  • Figure 16A-16C illustrates anti-tumor activity of OI42 (QN-019a) NK cells in Raji and Nalm6 cells.
  • Figure 17 illustrates stable expression of hnCD16 on OI42 (QN-019a) cells.
  • Figure 18A-18B illustrates activation of STAT5 signaling pathway in OI42 (QN-019a) cells with IL-15RF.
  • FIG 19A-19B illustrates single and serial killing activity of OI42 (QN-019a) cells against Nalm6 cells.
  • FIGS. 20A-20B illustrates single and serial killing activity of OI42 (QN-019a) cells against Raji cells.
  • Figure 21A-21C illustrates anti-tumor activity of OI42 (QN-019a) cells in Nalm6 NOG tumor mouse model.
  • Figure 22 illustrates the karyotype of OI42 (QN-019a) cells.
  • Figure 23 illustrates stable expression of hnCD19 on KB15 cells.
  • Figure 24 illustrates anti-tumor activity of KB15 NK cells in Raji cells.
  • Figure 25A-25B illustrates activation of STAT5 signaling pathway in KB15 NK cells with IL-15RF.
  • Figure 26A-26C illustrates anti-tumor activity of KB15 cells in Raji tumor mouse model.
  • Figure 27 illustrates the karyotype of KB15 cells.
  • Figure 28 illustrates in vitro growth of KB15 NK cells with or without exogenous IL-2.
  • Figure 29A-29B illustrates the tumorgenicity of KB15 NK cells in nude mice.
  • a chimeric 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) .
  • reprogramming generally refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state.
  • a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state.
  • a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.
  • 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, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-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.
  • genomic DNA 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) .
  • 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
  • immune regulator polypeptide generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secretory polypeptide, cleavable polypeptide, non-cleavable polypeptide, etc. ) capable of regulating or controlling one or more attributes of an immune cell, such as a NK cell.
  • One or more attributes of an immune cell can comprise differentiation of the immune cell, immune cell morphology, expression of a polynucleotide or polypeptide construct within the immune cell, or activity of the immune cell (e.g., cytotoxic activity of an engineered NK cell against a diseased cell, such as a cancer cell) .
  • An immune regulator polypeptide can be endogenous to a host cell.
  • an immune regulator polypeptide can be heterologous to a hots cell.
  • controlling the one or more attributes of the immune cell can be mediated by downregulating expression of the immune regulator polypeptide (e.g., suppression, knock-down or knock-out) .
  • controlling the one or more attributes of the immune cell can be mediated by upregulating expression of the immune regulator polypeptide (e.g., upregulation of an endogenous gene or knock-in of a heterologous gene encoding the immune regulator polypeptide) .
  • controlling the one or more attributes of the immune cell can be mediated by maintaining expression of the immune regulator polypeptide for time period that is longer than a natural or normal expression profile of the immune regulator polypeptide in a host cell.
  • an immune regulator polypeptide can comprise a hypo-immunity regulator.
  • an immune regulator polypeptide can comprise an immune checkpoint inhibitor.
  • 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 checkpoint inhibitor generally refers to a group of molecules presented on a cell surface of an immune cell (e.g., T cells, myeloid cells, NK cells, B cells, etc. ) that can modulate immune response of the cell by down-regulating or inhibiting the immune response of the immune cell against a target cell, such as a cancer cell (i.e., anti-cancer or anti-tumor immune response) .
  • a target cell such as a cancer cell (i.e., anti-cancer or anti-tumor immune response) .
  • the target cell can express a receptor or a ligand of the immune checkpoint inhibitor presented on the surface of the immune cell, to engage with the immune checkpoint inhibitor and down-regulate or inhibit the immune response of the immune cells against the target cell.
  • down-regulating or inhibiting expression of the immune checkpoint inhibitor in the immune cell can, in some cases, enhance or prolong the immune response of the immune cell against a target cell.
  • 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.
  • T cells are part of the adaptive immune system and can be primed to recognize a specific threat by recognizing immune proteins (i.e., antigens) on a foreign cell surface.
  • immune proteins i.e., antigens
  • NK cells are part of the innate immune response and can respond to a broad range of objects that consider to be “non-self. ”
  • NK cells can attack their target cells without sensitization (i.e., antigen-specific priming) to eliminate foreign substances.
  • Unmodified NK cells derived from a subject can be cultured and expanded ex vivo, then administered to the subject as a treatment to attack their target cells, e.g., cancer cells.
  • target cells e.g., cancer cells.
  • NK cell-based therapies can be limited due to short half-life and/or poor proliferation of NK cells ex vivo or in vivo.
  • unmodified NK cells can be ineffective in targeting harder-to-treat cancers, such as myeloma or solid tumors.
  • ex vivo production of NK cells based on blood-derived stem cells e.g., HSCs
  • NK cells sourced and engineered to exhibit, for example, enhanced proliferation, half-life, and cytotoxic activity against target cells.
  • Immune cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered immune cell) .
  • Immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell.
  • Immune cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target.
  • the engineered Immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo.
  • the engineered Immune cells that are prepared ex vivo or in vitro 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. Alternatively, the engineered immune cells can be allogeneic to the subject.
  • engineered immune cells e.g., engineered NK cells
  • engineered immune cells disclosed herein can be derived from an isolated stem cell (e.g., isolated ESCs) .
  • engineered immune cells disclosed herein can be derived from induced stem cells (e.g., iPSCs) .
  • the stem cell disclosed herein can be an autologous cell or derived from the autologous cell.
  • the autologous cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the autologous cell can be obtained from the subject before the subject is found to have the condition.
  • the autologous cell can be an allogeneic cell, e.g., a universal stem cell with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs.
  • the autologous cell can be obtained from a healthy donor.
  • the engineered immune cell (e.g., engineered NK cell) can be an autologous cell.
  • the engineered immune cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the engineered immune cell can be obtained from the subject before the subject is found to have the condition.
  • the engineered immune cell can be an allogeneic cell, e.g., for a universal allogenic immunotherapy with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs.
  • the engineered immune cell can be obtained from a healthy donor.
  • T cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered T cell) .
  • T cells can be engineered to exhibit enhanced proliferation as compared to a control cell.
  • T cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target.
  • the engineered T cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo.
  • the engineered T cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) .
  • the engineered T cells can be autologous to the subject. Alternatively, the engineered T cells can be allogeneic to the subject.
  • NK cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered NK cell) .
  • NK cells can be engineered to exhibit enhanced proliferation as compared to a control cell.
  • NK cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target.
  • the engineered NK cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo.
  • the engineered NK cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) .
  • the engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the subject.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell.
  • the heterologous cytokine can comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) .
  • the engineered immune cell can further comprise one or both of: (i) a CD16 variant for enhanced CD16 signaling as compared to a control cell and (ii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen.
  • the antigen is not CD19.
  • the antigen binding moiety may not and need not exhibit any specific binding to CD19, but rather a specific binding to an antigen (e.g., one or more antigens) that is not CD19.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell.
  • the heterologous cytokine can comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) .
  • the engineered immune cell can further comprise one or both of: (i) a CD16 variant for enhanced CD16 signaling as compared to a control cell and (ii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to CD19.
  • the heterologous cytokine can comprise IL-15.
  • the heterologous IL-15 can comprise an amino acid sequence of SEQ ID NO. 53.
  • the engineered immune cell e.g., an engineered NK cell
  • a heterologous receptor that is a respective receptor of the heterologous cytokine as disclosed herein (e.g., heterologous IL-15 receptor (IL-15R, such as IL-15 ⁇ or IL-15 ⁇ ) for heterologous IL-15) .
  • the heterologous receptor can be an IL-15R ⁇ .
  • the heterologous IL-15R ⁇ can comprise an amino acid sequence of SEQ ID NO. 54.
  • the heterologous receptor can be a IL-15-IL-15R ⁇ fusion.
  • the heterologous IL-15-IL-15R ⁇ fusion can comprise an amino acid sequence of SEQ ID NO. 52.
  • the engineered immune cell may not and need not comprise any heterologous receptor that is a respective receptor of the heterologous cytokine.
  • the engineered immune cell comprising a heterologous IL e.g., IL-15
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell.
  • the heterologous cytokine can further comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) .
  • IL interleukin
  • the engineered immune cell may and need not comprise a heterologous receptor that is a respective receptor of the heterologous cytokine (e.g., a heterologous IL-15R) .
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be of the same species as that of the engineered immune cell (e.g., the engineered NK cell) .
  • both the heterologous cytokine and the engineered immune cell can be of human origin.
  • the heterologous cytokine can be of a different species than that of the engineered immune cell.
  • a heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be introduced to the engineered immune cell (e.g., engineered NK cell) by contacting a heterologous polynucleotide encoding the heterologous cytokine to the engineered immune cell.
  • the heterologous polynucleotide can be integrated into the engineered immune cell’s chromosome (e.g., nuclear chromosome) .
  • the heterologous polynucleotide may not and need not be integrated into the chromosome of the engineered immune cell.
  • a mRNA encoding a heterologous cytokine can be introduced (or inserted into) the engineered immune cell.
  • the heterologous polynucleotide is codon optimized to improve the expression of the heterologous cytokine.
  • the heterologous cytokine as disclosed herein can be a heterologous IL.
  • a heterologous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous ILs.
  • a heterologous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of heterologous ILs.
  • the heterologous IL can be a single type of heterologous IL.
  • Non-limiting examples of the heterologous 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 heterologous 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 engineered immune cell e.g., an engineered NK cell
  • the engineered immune cell can comprise at least a portion of heterologous human IL-15 (or a gene encoding thereof) .
  • the heterologous cytokine is a membrane bound IL-15 comprising the IL-15 and at least a portion of IL-15R.
  • the IL-15 and the at least a portion of IL-15R of the membrane bound IL-15 is linked via a linker.
  • Any suitable linker known in the art can be used in the membrane bound IL-15.
  • the linker is selected from the group consisting of (GGGGS) n, (EGKSSGSGSESKST) n, and (EGKSSGSGSESKST) n (GGGGS) n, and the n can be any integer selected from 1-10.
  • the linker is (GGGGS) n, and the n can be any integer selected from 1-10.
  • the linker is selected from GGGGS, (GGGGS) 2, (GGGGS) 3, (GGGGS) 4, (GGGGS) 5 or (GGGGS) 6.
  • the linker is (EGKSSGSGSESKST) n, and the n can be any integer selected from 1-10.
  • the linker is selected from the group consisting of EGKSSGSGSESKST, (EGKSSGSGSESKST) 2 or (EGKSSGSGSESKST) 3.
  • the linker is (EGKSSGSGSESKST) n (GGGGS) n, and the n can be any integer selected from 1-10.
  • the linker is EGKSSGSGSESKSTGGGGS or (EGKSSGSGSESKST) 2 GGGGS.
  • the linker comprises an amino acid sequence selected from the group consisting of SEQ ID No. 18-20, and 55-60.
  • the membrane bound IL-15 further comprises an intracellular signaling domain. Any suitable intracellular signaling domain known in the art can be used in the membrane bound IL-15 of the present application.
  • the membrane bound IL-15 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 21.
  • the membrane bound IL-15 comprises an amino acid sequence of SEQ ID NO. 21.
  • the membrane bound IL-15 further comprises an extracellular signal peptide. Any suitable extracellular signal peptide known in the art can be used in the membrane bound IL-15 of the present application.
  • the membrane bound IL-15 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 22.
  • the membrane bound IL-15 comprises an amino acid sequence of SEQ ID NO. 22.
  • the membrane bound IL-15 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any one selected from the group consisting of SEQ ID NO. 13-15, and 52-54.
  • the membrane bound IL-15 comprises an amino acid sequence of any one selected from the group consisting of SEQ ID NO. 13-15, and 52-54.
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine.
  • the heterologous cytokine may not and need not be secreted by the engineered immune cell.
  • the heterologous cytokine can be bound to a cell surface of the engineered immune 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 engineered immune 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.
  • the heterologous polynucleotide is codon optimized to improve the expression of the heterologous polypeptide.
  • a cleavable linker as disclosed herein 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 of the engineered immune cell (e.g., the engineered NK cell) .
  • the engineered immune 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 engineered immune 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 engineered immune 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.
  • An expression cassette as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein.
  • the expression cassette may not and need not be integrated into the genome of the engineered cell.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune 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
  • enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor can be characterized by an increase in phosphorylation of a downstream signaling protein 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
  • enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor can be characterized by an increased expression of a downstream gene 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
  • CD16 signaling e.g., constitutively activated signaling of CD16
  • engineered immune cell e.g., engineered NK cell
  • Enhanced CD16 signaling (e.g., constitutively activated signaling of CD16) of the engineered immune cell (e.g., engineered NK cell) as disclosed herein 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 engineered immune cell can be modified to enhance its signaling.
  • an enhanced signaling variant of CD16 can be artificially introduced to the engineered immune cell.
  • the engineered immune cell’s endogenous gene encoding CD16 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 engineered immune 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 heterologous CD16 variant comprises an amino acid sequence having at least 70%identity with SEQ ID NO. 23. In some cases, the heterologous CD16 variant comprises an amino acid sequence having at least 75%identity with SEQ ID NO. 23. In some cases, the heterologous CD16 variant comprises an amino acid sequence having at least 80% identity with SEQ ID NO. 23. In some cases, the heterologous CD16 variant comprises an amino acid sequence having at least 85%identity with SEQ ID NO. 23. In some cases, the heterologous CD16 variant comprises an amino acid sequence having at least 90%identity with SEQ ID NO. 23.
  • the heterologous CD16 variant comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 23. In some cases, the heterologous CD16 variant comprises an amino acid sequence of SEQ ID NO. 23.
  • a heterologous gene as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein.
  • the heterologous gene may not and need not be integrated into the genome of the engineered cell.
  • the enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell
  • the engineered immune 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 CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be greater than CD16 signaling 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 4-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,
  • enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the engineered immune cell can be characterized by an increase in phosphorylation of a downstream signaling protein 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 or
  • enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the engineered immune cell can be characterized by an increased expression of a downstream gene 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,
  • the CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the CD16 signaling of the engineered immune cell can be more prolonged (e.g., a longer duration of time of activated CD16 signaling) than CD16 signaling 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 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
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a heterologous cytokine as disclosed herein, wherein the heterologous cytokine is bound to a membrane (e.g., plasma membrane) of the engineered immune cell.
  • the heterologous cytokine can comprise a heterologous IL as disclosed herein (e.g., a heterologous IL-15) .
  • the engineered immune cell can further comprise one, two, or all of: (a) a different heterologous cytokine (e.g., a heterologous cytokine as disclosed herein, other than the one that is bound to the membrane of the subject cell) , (b) reduced expression or activity of an endogenous immune regulator polypeptide, and (c) a safety switch.
  • a different heterologous cytokine e.g., a heterologous cytokine as disclosed herein, other than the one that is bound to the membrane of the subject cell
  • the endogenous immune regulator polypeptide is not B2M.
  • the endogenous immune regulator can be, for example, a polypeptide other than B2M.
  • the engineered immune cell (e.g., an engineered NK cell) can comprise the different heterologous cytokine and one or both of (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and (c) the safety switch.
  • the engineered immune cell comprise the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and one or both of (a) the different heterologous cytokine and (c) the safety switch.
  • the engineered immune cell comprise the safety switch and one or both of (a) the different heterologous cytokine and (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) .
  • the engineered immune cell comprise all of (a) , (b) , and (c) .
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine as compared to a control cell, as disclosed herein.
  • the expression or activity of the endogenous immune regulator polypeptide can be reduced in the engineered immune cell (e.g., the engineered NK cell) , for example, via action of a gene editing moiety as disclosed herein.
  • the reduced expression or activity of the endogenous immune regulator polypeptide in the engineered immune cell can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation or dephosphorylation of a downstream signaling protein (e.g., SHP2, Ig ⁇ / ⁇ , Syk, etc. for PD1/PDL1 signaling) or (ii) expression of the endogenous immune regulator polypeptide (e.g., PD1) via Western blotting or PCR techniques.
  • a downstream signaling protein e.g., SHP2, Ig ⁇ / ⁇ , Syk, etc. for PD1/PDL1 signaling
  • a downstream signaling protein e.g., SHP2, Ig ⁇ / ⁇ , Syk, etc. for PD1/PDL1 signaling
  • expression of the endogenous immune regulator polypeptide e.g., PD1
  • reduced expression of the endogenous immune regulator polypeptide in the engineered immune cell can be characterized by a decrease in the expression of the endogenous immune regulator polypeptide (e.g., PD1) 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
  • reduced activity of the endogenous immune regulator polypeptide in the engineered immune cell can be characterized by a decrease in phosphorylation of a downstream signaling protein (e.g., SHP2 for PD1/PDL1 signaling) 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
  • a downstream signaling protein e.g., SHP2 for PD
  • reduced activity of the endogenous immune regulator polypeptide in the engineered immune cell can be characterized by an increase in phosphorylation of a downstream target signaling protein (e.g., Ig ⁇ / ⁇ or Syk for PD1/PDL1 signaling) 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-
  • a downstream target signaling protein e.g., Ig ⁇ / ⁇
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a CD16 variant as disclosed herein for enhanced CD16 signaling in the engineered NK cell.
  • the CD16 variant e.g., a heterologous CD16 variant
  • the engineered immune cell can further comprise reduced expression or activity of an endogenous immune regulator polypeptide as compared to a control cell, as disclosed herein.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced CD16 signaling as compared to a control cell, as disclosed herein.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) .
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • 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-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 engineered immune cell e.g., an engineered NK cell
  • the engineered immune 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.
  • reduced expression or activity of the endogenous cytokine e.g., the endogenous IL, such as IL-17
  • engineered immune cell e.g., engineered NK cell
  • reduced expression or activity of the endogenous cytokine can be characterized by a decrease in phosphorylation of a downstream signaling protein of the endogenous cytokine 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-
  • reduced expression or activity of the endogenous cytokine e.g., the endogenous IL, such as IL-17
  • engineered immune cell e.g., engineered NK cell
  • reduced expression or activity of the endogenous cytokine can be characterized by a decrease in the expression of a downstream gene of the endogenous cytokine 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 engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced expression profile of a specific cell marker for a committed immune cell (e.g., a NK cell marker) as compared to a control cell that does not exhibit the reduced activity of the endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) as disclosed herein.
  • a specific cell marker for committed NK cells can include CD57 or killer immunoglobulin-like receptors (KIR) .
  • KIR can comprise KIR2D and/or KIR3D.
  • KIR2D can comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and/or KIR2DS5.
  • KIR3D can comprise KIR3DL1, KIR3DL2, KIR3DL3, and/or KIR3DS1.
  • the enhanced expression profile of the specific cell marker for the committed immune cell (e.g., CD57 or KIR for NK cells) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.
  • the expression of the specific cell marker for a committed immune cell (e.g., CD57 or KIR or NK cells) in the engineered immune cell of the present disclosure can be greater than expression of the same by 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 4-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
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise one or both of: (i) a heterologous transcription factor (e.g., a heterologous STAT) , (ii) reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling as disclosed herein) , and (iii) reduced expression or activity of endogenous enzyme (e.g., a ligase, such as CBL-B) .
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the heterologous transcription factor can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous transcription factor.
  • the heterologous transcription factor can comprise at most 5, 4, 3, or 2 different types of transcription factor.
  • the heterologous transcription factor can have a single type of transcription factor.
  • the transcription factor can be involved in the engineered immune cell’s immune activity, proliferation, apoptosis, and/or differentiation.
  • the heterologous transcription factor for the engineered immune cell e.g., the engineered NK cell
  • STAT can include STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, STAT6, and modifications thereof.
  • STAT can comprise STAT3.
  • STAT can comprise STAT5B.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced survival in the presence of tumor cells as compared to a control cell without (i) the heterologous transcription factor (e.g., the heterologous STAT) or (ii) the reduced activity of endogenous cytokine signaling (e.g., endogenous IL-17 signaling) .
  • the heterologous transcription factor e.g., the heterologous STAT
  • endogenous cytokine signaling e.g., endogenous IL-17 signaling
  • the engineered immune cell can, in the presence of tumor cells, survive longer than 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, at least or up to about 40-fold, at least or up to about 50-fold
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell as disclosed herein (e.g., a NK cell marker, such as KIR) as compared to a control cell.
  • a specific endogenous cell marker is KIR.
  • the engineered immune cell can further comprise one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell, as disclosed herein, (d) an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell, and (e) reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein
  • a heterologous cytokine e.g., a heterologous IL, such as IL-15
  • the engineered immune cell can comprise the chimeric polypeptide receptor and one or more of (e.g., 1, 2, 3, or 4 of) : (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the engineered immune cell can comprise the heterologous cytokine and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the engineered immune cell can comprise the CD16 variant for enhanced CD16 signaling and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the CD16 variant for enhanced CD16 signaling and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the engineered immune cell can comprise the heterologous immune regulator and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the heterologous immune regulator e.g., 1, 2, 3, or 4 of
  • the engineered immune cell can comprise the reduced expression or activity of an endogenous immune regulator polypeptide and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (d) the heterologous immune regulator.
  • an endogenous immune regulator polypeptide e.g., 1, 2, 3, or 4 of
  • the reduced expression or activity of the specific endogenous cell marker for the committed immune cell e.g., KIR for NK cells
  • KIR for NK cells
  • the reduced expression or activity of the specific endogenous cell marker for the committed immune cell can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.
  • the expression of the specific endogenous cell marker for a committed immune cell (e.g., KIR or NK cells) in the engineered immune cell of the present disclosure can be less than expression of the same by 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 4-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
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of one or more endogenous immune checkpoint inhibitors (e.g., CD94, CD96, TGF beta receptor, SHIP2, etc. ) .
  • the engineered immune cell can exhibit reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous CD94 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous CD96 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous TGF beta receptor and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iv) endogenous SHIP (e.g., SHIP2) .
  • the engineered immune cell can exhibit reduced expression or activity of endogenous SHIP (e.g., SHIP2) and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iii) endogenous TGF beta receptor.
  • endogenous SHIP e.g., SHIP2
  • endogenous CD94 e.g., 1, 2, or all of
  • the reduced expression or activity of the immune checkpoint inhibitor (e.g., CD94, CD96, TGF beta receptor, SHIP2, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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
  • the reduced expression or activity of the endogenous CD94 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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,
  • the reduced expression or activity of the endogenous CD96 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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,
  • the reduced expression or activity of the endogenous TGF beta receptor in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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-
  • the reduced expression or activity of the endogenous SHIP (e.g., SHIP2) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise one or more hypo-immunity regulators.
  • the engineered immune cell exhibits reduced expression or activity of one or more hypo-immunity regulators from: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB, or (vi) endogenous NKG2DL.
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the reduced expression or activity of the endogenous hypo-immunity regulator (e.g., CD80, CD86, ICOSL, CD40L, MICA, MICB, NKG2DL, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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-
  • the reduced expression or activity of the endogenous CD80 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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,
  • the reduced expression or activity of the endogenous CD86 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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,
  • the reduced expression or activity of the endogenous ICOSL in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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
  • the reduced expression or activity of the endogenous hypo-immunity regulator CD40L in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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
  • the reduced expression or activity of the endogenous MICA or MICB in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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
  • the reduced expression or activity of the endogenous NKG2DL in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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-
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) .
  • the engineered immune cell further comprises one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, and (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell.
  • a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein
  • a heterologous cytokine e.g., a heterologous IL, such as IL-15
  • CD16 variant for enhanced CD16 signaling as compared to a control cell.
  • the engineered immune cell (e.g., the engineered NK cell) comprises a chimeric polypeptide receptor as disclosed herein and one or both of: (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling.
  • the heterologous cytokine e.g., a heterologous IL, such as IL-15
  • the engineered immune cell e.g., the engineered NK cell
  • the heterologous cytokine e.g., a heterologous IL, such as IL-15
  • the engineered immune cell comprise the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling.
  • the engineered immune cell (e.g., the engineered NK cell) comprises the CD16 variant for enhanced CD16 signaling and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein.
  • the heterologous cytokine e.g., a heterologous IL, such as IL-15
  • the reduced expression or activity of the endogenous ICAM1 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by 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 4-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
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the reduced expression or activity of the endogenous ICAM1 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell, as disclosed herein.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell.
  • the immune regulator polypeptide comprises a hypo-immunity regulator.
  • the hypo-immunity regulator can be PDL2.
  • the hypo-immunity regulator can be TGF-beta.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell.
  • the immune regulator polypeptide comprises a hypo-immunity regulator.
  • the hypo-immunity regulator can comprise one or more members from: (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CCL21 and one or more of (e.g., 1, 2, 3, or all of) : (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous IL-10 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CD46 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CD55 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (v) a heterologous CD59.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the heterologous CD59 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (iv) a heterologous CD55.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a heterologous cytokine (e.g., a heterologous IL) , as disclosed herein that is not IL-15.
  • the heterologous cytokine comprises IL-21 or variants thereof.
  • the engineered immune cell can be derived from an induced stem cell (e.g., iPSC) .
  • a control cell can be a cell can be an immune cell, such as a NK cell, used for comparison purposes.
  • a control cell can be a cell that does not comprise a heterologous cytokine (e.g., IL-15) .
  • a control cell can be a cell that does not comprise a CD16 variant for enhanced CD16 signaling.
  • a control cell can be a cell that a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen.
  • a control cell can be a cell that comprises a heterologous IL-15R.
  • a control cell can be a cell that does not comprise a membrane bound heterologous cytokine (e.g., IL-15) .
  • a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous immune regulator polypeptide.
  • a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous cytokine (e.g., IL-17) or a receptor thereof (e.g., IL-17R) .
  • a control cell can be a cell that does not comprise a heterologous transcription factor (e.g., STAT) .
  • a heterologous transcription factor e.g., STAT
  • a control cell can be a cell that does not exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell (e.g., a NK cell marker, such as KIR) .
  • a control cell can be a cell that does not comprise a heterologous immune regulator polypeptide.
  • a control cell can be a cell that does not exhibit reduced expression or activity of one or more of: endogenous CD94, endogenous CD96, endogenous TGF beta receptor, or endogenous SHIP2.
  • a control cell can be a cell that does not exhibit reduced expression or activity of one or more of: endogenous CD80, endogenous CD86, endogenous ICOSL, endogenous CD40L, endogenous MICA or MICB, or endogenous NKG2DL.
  • a control cell can be a cell that does not exhibit reduced expression or activity of ICAM1.
  • a control cell can be a cell that does not comprise a heterologous PDL2 or heterologous TGF beta.
  • a control cell can be a cell that does not comprise one or more of: heterologous CCL21, heterologous IL-10, heterologous CD46, heterologous CD55, or heterologous CD59.
  • a control cell can be a cell that does not comprise heterologous IL-21. In some cases, a control cell can be a cell that is not derived from a cell line. In some cases, a control cell can be a cell that is not derived from an isolated ESC. In some cases, a control cell can be a cell that is not derived from an iPSC.
  • the engineered immune cell can comprise a heterologous cytokine (e.g., a heterologous IL, such as IL-15) as disclosed herein.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell comprises a heterologous receptor that is a respective receptor of the heterologous cytokine (e.g., a heterologous IL-15R) , as disclosed herein.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced signaling of the endogenous signaling pathway induced by the heterologous cytokine and/or the heterologous receptor (e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R) as disclosed herein.
  • the heterologous cytokine and/or the heterologous receptor e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R
  • the engineered immune cell can exhibit reduced expression or activity of endogenous CD38 as compared to a control cell.
  • Such engineered immune 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
  • any one of the engineered immune cell e.g., the engineered NK cell
  • expression or activity of endogenous CD38 of the engineered immune cell may not and need not be modified.
  • Such engineered immune cell may be used to treat a subject who has or is suspected of having a disease (e.g., cancer, tumor) that is not multiple myeloma.
  • the engineered immune cell can comprise a heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or the fragment thereof can be secreted by the engineered immune cell.
  • the secretory heterologous IL-15 comprises an amino acid sequence having at least 70%identity with SEQ ID NO. 24. In some embodiments, the secretory heterologous IL-15 comprises an amino acid sequence having at least 75%identity with SEQ ID NO. 24. In some embodiments, the secretory heterologous IL-15 comprises an amino acid sequence having at least 80%identity with SEQ ID NO. 24. In some embodiments, the secretory heterologous IL-15 comprises an amino acid sequence having at least 85%identity with SEQ ID NO. 24. In some embodiments, the secretory heterologous IL-15 comprises an amino acid sequence having at least 90%identity with SEQ ID NO. 24.
  • the secretory heterologous IL-15 comprises an amino acid sequence having at least 95%identity with SEQ ID NO. 24. In some embodiments, the secretory heterologous IL-15 comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 24. In some embodiments, the secretory heterologous IL-15 comprises an amino acid sequence of SEQ ID NO. 24.
  • the engineered immune cell can comprise a heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or the fragment thereof can be bound to a cell surface membrane of the engineered immune cell.
  • the engineered immune cell can comprise at least one chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as provided in the present disclosure.
  • the engineered immune cell can comprise a plurality of different chimeric polypeptide receptors to specifically bind a plurality of different antigens.
  • the engineered immune cell can comprise at least one chimeric polypeptide receptor that comprises a plurality of antigen binding moieties to specifically bind a plurality of different antigens.
  • the engineered immune cell can comprise a safety switch capable of effecting death of the engineered immune cell.
  • the engineered immune 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 engineered immune cell (e.g., administered to a subject comprising the engineered immune 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 engineered immune 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 engineered immune cells e.g., the engineered NK cells
  • the engineered immune 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.
  • the engineered immune cell can comprise heterologous immune receptor polypeptide.
  • the immune regulator polypeptide can comprise one or more members selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • the engineered immune cell can exhibits reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprises an immune checkpoint inhibitor or a hypo-immunity regulator (or both) .
  • the immune checkpoint inhibitor as disclosed herein can comprise one or more members selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGF beta receptor, and 2B4.
  • the immune checkpoint inhibitor can comprise SHIP2.
  • the hypo-immunity regulator as disclosed herein can comprise one or more members selected from the group consisting of 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, and CD59.
  • the engineered immune cell can comprise a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered immune cell.
  • the engineered immune cell can exhibit enhanced cytotoxicity against a target cell as compared to a control cell.
  • the engineered immune 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
  • the engineered immune cell can induce reduced immune response from separate immune cells (e.g., separate T cells and/or B-cells in vitro, or a host’s immune cells upon administration of the engineered immune 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 engineered immune cell to the host
  • the engineered immune cell as disclosed herein can reduce the immune response from the separate immune cells 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 50-fold,
  • the engineered immune 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 engineered immune 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 engineered immune cell to the host
  • the half-life of the engineered immune 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
  • the engineered immune cell can effect enhanced function or pathological condition of a bodily tissue of a subject as compared to a control cell.
  • treatment with the engineered immune 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,
  • the engineered immune 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 engineered immune 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
  • 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 engineered immune 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.
  • a heterologous gene can be operatively coupled to (e.g., for knock-in) a constitutive, inducible, temporal, tissue-specific, and/or cell type-specific promoter.
  • a promoter of interest can include CMV, EF1a, PGK, CAG, and UBC.
  • any one of the engineered immune cell e.g., the engineered NK cell
  • TME tumor microenvironment gene
  • having reduced expression or activity of a TME can enhance the engineered immune cell’s immune activity against a target cell.
  • a TME gene may be an immune checkpoint inhibitor.
  • Non- limiting examples of the TME can include: NKG2A, NKG2D, PD1, CTLA4, LAG3, TIM3, TIGIT, KIR2D, CD94, CD96, TGF beta receptor, 2B4, and SHIP2.
  • any one of the engineered immune cell e.g., the engineered NK cell
  • can exhibit one or more heterologous genes e.g., knocked-in
  • enhanced function CD137, CD80, CD86, DAP10 (e.g., with or without point mutation) .
  • any one of the engineered immune cell e.g., the engineered NK cell
  • endogenous genes for, e.g., hypo-immunity: B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) .
  • any one of the engineered immune cell e.g., the engineered NK cell
  • can exhibit one or more heterologous genes e.g., knocked-in for, e.g., hypo-immunity: HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • heterologous genes e.g., knocked-in for, e.g., hypo-immunity: HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59.
  • any one of the engineered immune cell e.g., the engineered NK cell
  • can exhibit one or more heterologous genes e.g., knocked-in: CD3, CD4, CD80, 41BBL, and CD131.
  • the engineered immune cell (e.g., the engineered NK cell) of the present disclosure 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 engineered immune cell can be engineered to express a chimeric polypeptide receptor transiently or permanently.
  • a recombinant chimeric polypeptide receptor can be delivered to the engineered immune cell via, e.g., a liposome, and be incorporated into the engineered immune cell via membrane fusion.
  • a heterologous polynucleotide construct encoding the chimeric polypeptide receptor can be delivered to the engineered immune cell.
  • the heterologous polynucleotide construct i.e., a gene
  • encoding the heterologous polynucleotide construct can be incorporated into the chromosome of the engineered immune cell (i.e., chromosomal gene) or, alternatively, may not or need not be integrated into the chromosome of the engineered immune 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 engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be inactivated.
  • a function of the endogenous TCR of the engineered immune 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 in the engineered immune cell (e.g., engineered NK cell) 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 can be a CD8 ⁇ hinge.
  • the CD8 ⁇ hinge can comprise an amino acid sequence of SEQ ID NO. 36.
  • the hinge domain can be a CD28 hinge.
  • the CD28 hinge can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with the polypeptide sequence of SEQ ID NO. 49.
  • a transmembrane domain of a CAR in the engineered immune cell 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 transmembrane domain can be a CD8 ⁇ transmembrane domain. In some cases, the CD8 ⁇ transmembrane domain can comprise an amino acid sequence of SEQ ID NO. 37. In some embodiments, the transmembrane domain can be a CD28 transmembrane domain. In some cases, the CD28 transmembrane domain can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with the polypeptide sequence of SEQ ID NO. 50.
  • 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 signal peptide of a CAR in the engineered immune cell can comprise a full length or at least a portion of the native or modified polypeptide 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 signal peptide can be cleaved (e.g., self-cleaved) from a mature CAR protein.
  • the signal peptide can be a CD8 ⁇ signal peptide.
  • the CD8 ⁇ signal peptide can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with the polypeptide sequence of SEQ ID NO. 31.
  • 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 in the engineered immune cell (e.g., engineered NK cell) 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 can be a CD3 ⁇ signal domain.
  • the CD3 ⁇ signaling domain can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with the polypeptide sequence of SEQ ID NO. 38.
  • the signaling domain CAR in the engineered immune cell e.g., engineered NK cell
  • the signaling domain CAR in the engineered immune cell 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 co-stimulatory domain can be a 2B4 co-stimulatory domain.
  • the 2B4 co-stimulatory domain can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with the polypeptide sequence of SEQ ID NO. 32.
  • the co-stimulatory domain can be a CD28 co-stimulatory domain.
  • the CD28 co-stimulatory domain can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with the polypeptide sequence of SEQ ID NO. 51.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell comprises the chimeric polypeptide receptor (e.g., CAR) that comprises at least CD8 transmembrane domain and one or more of: (i) 2B4 signaling domain and (ii) DAP10 signaling domain.
  • 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.
  • he 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, alemt
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain
  • the antigen binding domain can be capable of binding specifically and preferentially to an antigen comprising one or more members selected from the group comprising BCMA, CD20, CD22, CD30, CD33, CD38, CD70, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100.
  • the NKG2D ligand comprises one or more members selected from the group comprising of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain
  • the antigen binding domain can be capable of binding specifically and preferentially to CD19.
  • Any suitable antigen binding domain capable of binding specifically and preferentially to CD19 can be used in the chimeric polypeptide receptor of the present application.
  • antigen binding domain is selected from Fab, Fab’, F (ab’) 2, scFv, and sdAb.
  • the antigen binding domain capable of binding specifically and preferentially to CD19 comprises a heavy chain variable region, light chain variable region or a combination thereof.
  • the heavy chain variable region of the antigen binding domain capable of binding specifically and preferentially to CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 16. In some embodiments, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO. 16.
  • the heavy chain variable region of the antigen binding domain capable of binding specifically and preferentially to CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 39.
  • the heavy chain variable region comprises an amino acid sequence of SEQ ID NO. 39.
  • the heavy chain variable region of SEQ ID NO. 39 can be a “Type 2” anti-CD19 heavy chain variable region.
  • the heavy chain variable region of the antigen binding domain capable of binding specifically and preferentially to CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 43.
  • the heavy chain variable region comprises an amino acid sequence of SEQ ID NO. 43.
  • the heavy chain variable region of SEQ ID NO. 43 can be a “Type 3” anti-CD19 heavy chain variable region.
  • the light chain variable region of the antigen binding domain capable of binding specifically and preferentially to CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 17. In some embodiments, the light chain variable region comprises an amino acid sequence of SEQ ID NO. 17.
  • the light chain variable region of the antigen binding domain capable of binding specifically and preferentially to CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 40.
  • the light chain variable region comprises an amino acid sequence of SEQ ID NO. 40.
  • the light chain variable region of SEQ ID NO. 40 can be a “Type 2” anti-CD19 light chain variable region.
  • the light chain variable region of the antigen binding domain capable of binding specifically and preferentially to CD19 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 44.
  • the light chain variable region comprises an amino acid sequence of SEQ ID NO. 44.
  • the light chain variable region of SEQ ID NO. 44 can be a “Type 3” anti-CD19 light chain variable region.
  • the heavy chain variable region and the light chain variable region can be fused together by a linker sequence.
  • the linker sequence can be any one of SEQ ID NO. 18-20, and 55-60.
  • An anti-CD19 scFv comprising (i) the heavy chain variable region comprising the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with of SEQ ID NO. 16 and (ii) the light chain variable region comprising the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 17, a CAR construct comprising such scFv, or a cell comprising such CAR may be generally be referred to as “Type 1” or “KB15” throughout the present disclosure.
  • An anti-CD19 scFv comprising (i) the heavy chain variable region comprising the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 39 and (ii) the light chain variable region comprising the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 40, a CAR construct comprising such scFv, or a cell comprising such CAR may be generally be referred to as “Type 2” or “OI42” throughout the present disclosure.
  • An anti-CD19 scFv comprising (i) the heavy chain variable region comprising the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 43 and (ii) the light chain variable region comprising the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO. 44, a CAR construct comprising such scFv, or a cell comprising such CAR may be generally be referred to as “Type 3” throughout the present disclosure.
  • an antigen binding domain can be a scFv.
  • the scFv can be a Type 1 anti-CD19 scFv comprising (i) the anti-CD19 heavy chain variable region having the sequence of SEQ ID NO. 16, (ii) the anti-CD19 light chain variable region of SEQ ID NO. 17, and (iii) the linker sequences of GGGGSGGGGSGGGGS.
  • the Type 1 anti-CD19 scFv can have an amino acid sequence of SEQ ID NO. 35.
  • the scFv can be a Type 2 anti-CD19 scFv comprising (i) the heavy chain variable region having the sequence of SEQ ID NO. 39, (ii) the light chain variable region of SEQ ID NO. 40, and (iii) the linker sequence of GGGGSGGGGSGGGGS.
  • the Type 2 anti-CD19 scFv can have an amino acid sequence of SEQ ID NO. 41.
  • the scFv can be a Type 3 anti-scFv.
  • a first example of Type 3 anti-CD19 scFv can comprise (i) the heavy chain variable region having the sequence of SEQ ID NO. 43, (ii) the light chain variable region of SEQ ID NO. 44, and (iii) the linker sequence of GSTSGSGKPGSGEGSTKG.
  • the first example of Type 3 anti-CD19 scFv can comprise an amino acid sequence of SEQ ID NO. 45.
  • a second example of Type 3 anti-CD19 scFv can comprise (i) the heavy chain variable region having the sequence of SEQ ID NO. 43, (ii) the light chain variable region of SEQ ID NO. 44, and (iii) the linker sequence of GGGGSGGGGSGGGGS.
  • the second example of Type 3 anti-CD19 scFv can comprise an amino acid sequence of SEQ ID NO. 47.
  • the engineered immune cell (e.g., the engineered NK cell) as disclosed can comprise a chimeric polypeptide receptor (e.g., TFP or CAR) that comprises an antigen binding domain capable of specifically binding an antigen of a target cell.
  • the engineered CAR immune cell e.g., the engineered CAR NK cell
  • the engineered anti-CD19 CAR immune cell e.g., the engineered anti-CD19 CAR NK cell
  • the Type 1 anti-CD19 CAR with a CD8 ⁇ signal peptide can comprise an amino acid sequence of SEQ ID NO. 33.
  • the engineered anti-CD19 CAR immune cell e.g., the engineered CAR NK cell
  • the Type 1 anti-CD19 CAR without a CD8 ⁇ signal peptide can comprise an amino acid sequence of SEQ ID NO. 34.
  • the engineered anti-CAR immune cell (e.g., the engineered CAR NK cell) can have a Type 2 anti-CD19 CAR comprising (i) the anti-CD19 heavy chain variable region having the sequence of SEQ ID NO. 39, (ii) the anti-CD19 light chain variable region of SEQ ID NO. 40, and (iii) the linker sequence of GGGGSGGGGSGGGGS.
  • the Type 2 anti-CD19 CAR can have an amino acid sequence of SEQ ID NO. 42.
  • the engineered anti-CAR immune cell (e.g., the engineered CAR NK cell) can have a Type 3 anti-CD19 CAR.
  • a first example of Type 3 anti-CD19 CAR can comprise (i) the heavy chain variable region having the sequence of SEQ ID NO. 43, (ii) the light chain variable region of SEQ ID NO. 44, and (iii) the linker sequence of GSTSGSGKPGSGEGSTKG.
  • the first example of Type 3 anti-CD19 CAR can comprise an amino acid sequence of SEQ ID NO. 46.
  • a second example of Type 3 anti-CD19 CAR can comprise (i) the heavy chain variable region having the sequence of SEQ ID NO. 43, (ii) the light chain variable region of SEQ ID NO.
  • Type 3 anti-CD19 can comprise an amino acid sequence of SEQ ID NO. 48.
  • a third example of Type 3 anti-CD19 CAR can comprise (i) the heavy chain variable region having the sequence of SEQ ID NO. 43, (ii) the light chain variable region of SEQ ID NO. 44, (iii) the linker sequence of GGGGSGGGGSGGGGS, and (iv) the CD8 ⁇ signal peptide of SEQ ID NO. 31.
  • the third example of Type 3 anti-CAR can comprise an amino acid sequence of SEQ ID NO. 61.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain capable of specifically binding an antigen of a target cell, and the engineered immune cell can exhibit reduced expression or activity of an endogenous gene encoding the same antigen of the chimeric polypeptide receptor.
  • a population of the engineered immune cells can avoid targeting and killing each other, e.g., upon administration to a subject in need thereof.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38.
  • the engineered immune cell ’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.
  • the subject engineered immune cells comprising the chimeric polypeptide receptor against CD38 can be capable of targeting and effecting death (or degradation) of plasma cells.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38.
  • the engineered immune cell is an engineered NK cell that is derived from an isolated ESC or an induced stem cell (e.g., iPSC) .
  • the engineered immune cell’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.
  • any of the components described herein can be fused together using a linker sequence (e.g., fusing light and heavy chains of scFv, or fusing IL-15 and IL-15R) .
  • the linker sequence can be any one of SEQ ID NO. 18-20, and 55-60.
  • any one of the engineered immune cell e.g., the engineered NK cell disclosed herein can be derived from an isolated stem cell (e.g., an ESC) or an induced stem cell (iPSC) .
  • the isolated stem cell or the induced stem cell can be modified (e.g., genetically modified) to generate the engineered immune cell.
  • pluripotency of stem cells can be determined, in part, by assessing pluripotency characteristics of the cells.
  • Pluripotency characteristics can include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers including, but not limited to SSEA1 (mouse only) , SSEA3/4, SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or CD50; (iv) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm) ; (v) teratoma formation consisting of the three somatic lineages; and (
  • stem cells e.g., ESCs or iPSCs
  • the stem cells can be genetically modified to express any one of the heterologous polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation.
  • the stem cells can be genetically modified to reduce expression or activity of any one of the endogenous genes or polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation.
  • such genetically modified CD34+ hematopoietic stem cell is or is a source of any one of the engineered immune cell of the present disclosure.
  • stem cells as disclosed herein can be cultured in APEL media with ROCKi (Y-27632) (e.g., at about 10 micromolar ( ⁇ M) ) , SCF (e.g., at about 40 nanograms per milliner (ng/mL) of media) , VEGF (e.g., at about 20 ng/mL of media) , and BMP-4 (e.g., at about 20 ng/mL of media) to differentiate into CD34+ hematopoietic stem cells.
  • ROCKi Y-27632
  • SCF e.g., at about 40 nanograms per milliner (ng/mL) of media
  • VEGF e.g., at about 20 ng/mL of media
  • BMP-4 e.g., at about 20 ng/mL of media
  • the CD34+ hematopoietic stem cells (e.g., genetically modified with one or more features of any one of the engineered immune cell of the present disclosure) can be induced to differentiate in to a committed immune cell, such as T cells or NK cells.
  • a committed immune cell such as T cells or NK cells.
  • the induced differentiation process generates any one of the engineered NK cell of the present disclosure.
  • genetically modified CD34+ hematopoietic stem cells are cultured in the presence of IL-3 (e.g., about 5 ng/mL) , IL-7 (e.g., about 20 ng/mL) , IL-15 (e.g., about 10 ng/mL) , SCF (e.g., about 20 ng/mL) , and Flt3L (e.g., about 10 ng/mL) to differentiate into CD45+NK cells.
  • IL-3 e.g., about 5 ng/mL
  • IL-7 e.g., about 20 ng/mL
  • IL-15 e.g., about 10 ng/mL
  • SCF e.g., about 20 ng/mL
  • Flt3L e.g., about 10 ng/mL
  • the CD45+ NK cells can be expanded in culture, e.g., in a media comprising IL-2, mbIL-21 aAPC using Gas Permeable Rapid Expansion (G-Rex) platform.
  • G-Rex Gas Permeable Rapid Expansion
  • iPSC-derived NK cells as disclosed herein can be cultured with one or more heterologous cytokines comprising Il-2, IL-15, or IL-21. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with (e.g., for cell expansion) one or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21.
  • iPSC-derived NK cells as disclosed herein can be cultured with two or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21 (e.g., IL-2 and IL-15, IL-2 and IL-21, or IL-15 and IL-21) , either simultaneously or sequentially in any order.
  • iPSC-derived NK cells as disclosed herein can be cultured with all of Il-2, IL-15, and IL-21, either simultaneous or sequentially in any order.
  • the gene editing moiety as disclosed herein can comprise a CRISPR-associated polypeptide (Cas) , zinc finger nuclease (ZFN) , zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN) , transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP) , an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.
  • Cas CRISPR-associated polypeptide
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • RBP RNA-binding proteins
  • Argonaute protein any derivative thereof, any variant thereof, or any fragment thereof.
  • the actuator moiety comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein.
  • the actuator moiety comprises an RBP complexed with a gRNA which is able to form a complex with a Cas protein.
  • the gRNA comprises a targeting segment which exhibits at least 80%sequence identity to a target polynucleotide.
  • the Cas protein substantially lacks DNA cleavage activity.
  • a suitable gene editing moiety comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN) ; transcription activator-like effector nucleases (TALEN) ; meganucleases; RNA-binding proteins (RBP) ; CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo) , archaeal Argonaute (aAgo) , and
  • Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD) , Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12) , Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1 (CasA) , Cse2 (CasB) , Cse3 (CasE) , Cse4 (CasC) , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, C
  • the gene editing moiety as disclosed herein can be fused with an additional functional moiety (e.g., to form a fusion moiety) , and non-limiting examples of a function of the additional functional moiety can include methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristo
  • gene editing e.g., knock in
  • delivery of heterologous genetic material can be achieved other viral and non-viral based gene transfer methods can be used to introduce nucleic acids in host cells (e.g., stem cells, hematopoietic stem cells, etc. as disclosed herein) .
  • host cells e.g., stem cells, hematopoietic stem cells, etc. as disclosed herein
  • Such methods can be used to administer nucleic acids encoding polypeptide molecules of the present disclosure to cells in culture (or in a host organism) .
  • Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell.
  • Non-viral vector delivery systems can include DNA plasmids, RNA (e.g. a transcript of a vector described herein) , naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • RNA or DNA viral based systems can be used to target specific cells and traffick the viral payload to the nucleus of the cell.
  • Viral vectors can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo) . Alternatively, viral vectors can be administered directly (in vivo) to the subject.
  • Viral based systems can include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome can occur with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, which can result in long term expression of the inserted transgene.
  • Methods of non-viral delivery of nucleic acids can include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used.
  • antisense oligonucleotides can be utilized to suppress or silence a target gene expression.
  • Non-limiting examples of antisense oligonucleotides can include short hairpin RNA (shRNA) , microRNA (miRNA) , and small interfering RNA (siRNA) .
  • the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be combined with a co-therapeutic agent to treat a subject in need thereof.
  • the engineered immune 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
  • a composition comprising (a) any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein and (b) a co-therapeutic agent (i.e., a separate therapeutic agent) (e.g., an antibody, such as anti-CD20 antibody or anti-PD1 antibody) .
  • a co-therapeutic agent i.e., a separate therapeutic agent
  • an antibody such as anti-CD20 antibody or anti-PD1 antibody
  • the engineered immune cell can comprise one or more of: (i) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) as disclosed herein, (ii) a CD16 variant for enhanced CD16 signaling as disclosed herein, and (iii) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclose herein.
  • the co-therapeutic agent comprises an anti-CD20 antibody.
  • the engineered immune cell can comprise the heterologous cytokine (e.g., IL-15) as disclosed herein and one or both of: (ii) the CD16 variant for enhanced CD16 signaling and (iii) the chimeric polypeptide receptor comprising the antigen binding moiety.
  • heterologous cytokine e.g., IL-15
  • the engineered immune cell can comprise the CD16 variant for enhanced CD16 signaling and one or both of: (i) the heterologous cytokine (e.g., IL-15) and (iii) the chimeric polypeptide receptor comprising the antigen binding moiety.
  • the heterologous cytokine e.g., IL-15
  • the chimeric polypeptide receptor comprising the antigen binding moiety.
  • the engineered immune cell can comprise the chimeric polypeptide receptor comprising the antigen binding moiety and one or both of: (i) the heterologous cytokine (e.g., IL-15) and (ii) the CD16 variant for enhanced CD16 signaling.
  • the heterologous cytokine e.g., IL-15
  • the CD16 variant for enhanced CD16 signaling e.g., CD16 signaling.
  • 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 ( 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 the above; as well as combinations 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 engineered immune cell (e.g., the 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 engineered immune cell can be administered to the subject for adaptive immunotherapy.
  • the subject can be treated (e.g., administered with) a population of engineered immune 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.
  • engineered immune cells e.g., engineered NK cells
  • the present disclosure provides a method comprising (a) obtaining a cell from a subject; and (b) generating, from the cell, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein.
  • the cell obtained from the subject is ESC.
  • the cell e.g., a fibroblast, such as an adult skin fibroblast
  • the 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 NK cells comprising any one of the engineered immune cell (e.g., the engineered NK cell) 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 engineered immune cell (e.g., the 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 engineered immune cell (e.g., the 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 engineered immune cell (e.g., 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
  • a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to an antigen (e.g., CD33) as disclosed herein
  • a heterologous cytokine e.g., IL-15
  • CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat AML.
  • 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) .
  • BCL B-cell leukemia
  • any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein that comprises one or more of: (i) a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to CD19 as disclosed herein, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) a CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat 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.
  • Example 1 Engineered NK cells
  • Table 1 illustrates examples of engineered NK cells with or without genetic modifications, along with possible functions, and therapeutic indications.
  • therapeutic indications can include acute myeloid leukemia (AML) , multiple myeloma (MM) , Myelodysplastic syndrome (MDS) , B cell leukemia, T cell leukemia, solid tumor, and blood cancer.
  • NK cells can be engineered to exhibit enhanced CD16 signaling.
  • NK92 cells were engineered to exhibit enhanced CD16 signaling.
  • the engineered NK92 cells were modified to express CD64/CD16A fusion protein (SEQ ID NO. 1) and CD16 variant (SEQ ID NO. 24) (i.e., hnCD16) .
  • the resulting hnCD16 NK92 cells were validated by identifying enhanced expression of both CD16 (e.g., via anti-CD16-PE antibody) and CD64 (e.g., via anti-CD64-APC/AF700 antibody) using fluorescence-activated cell sorting (FACS) , as shown in Figure 1A. Wild-type (WT) NK92 cells were used as control.
  • the hnCD16 construct sequence can comprise “FHVS” (SEQ ID NO. 2) .
  • the hnCD16 construct sequence can comprise “WFHVS” (SEQ ID NO. 3) .
  • the hnCD16 construct sequence can comprise “FHVSF” (SEQ ID NO. 4) .
  • the hnCD16 construct sequence can comprise “WFHVSF” (SEQ ID NO. 5) .
  • the hnCD16 construct sequence can comprise “VWFHVSFC” (SEQ ID NO. 6) .
  • the hnCD16 construct sequence can comprise “PVWFHVSFCL” (SEQ ID NO. 7) .
  • the hnCD16 construct sequence can comprise “TPVWFHVSFCLV” (SEQ ID NO. 8) .
  • the hnCD16 NK92 cells were cultured alone (unstimulated, control) or in the presence of K562 cells capable of activating NK cells (K562) or phorbol 12-myristate 13-acetate (PMA) to activate CD16 and induce cleavage thereof.
  • K562 cells capable of activating NK cells
  • PMA phorbol 12-myristate 13-acetate
  • Figure 1B data revealed that the hnCD16 NK92 cells were highly resistant to the activation-induced cleavage of CD16a, as compared to peripheral blood (PB) NK cells as a control ( Figure 1B) .
  • PB peripheral blood
  • Figure 1B for example, treatment with PMA marginally reduced the percentage of CD16+ cells from 92%to 85%for the hnCD16 NK92 cells, whereas the same treatment reduced the percentage of CD16+ cells from 96%to 25% ( Figure 1B) .
  • hnCD16 NK92 cells Persistency of hnCD16 in the hnCD16 NK92 cells was also confirmed by using anait-CD64 antibody (Figure 1C) . Also, it was observed that hnCD16 NK92 cells did not downregulate endogenous CD16 expression upon stimulation (e.g., K652 or PMA) ( Figure 1D and 1E) .
  • the target cells (Raji cells) were treated with (i) the hnCD16 NK92 cells and (ii) either anti-CD20 antibody or hIgG as a control.
  • NK cells can be engineered to comprise at least (i) a heterologous transcription factor (e.g., STAT) and (ii) reduced expression or activity of an endogenous cytokine receptor (e.g., endogenous IL receptor, such as IL-17R) .
  • a heterologous transcription factor e.g., STAT
  • an endogenous cytokine receptor e.g., endogenous IL receptor, such as IL-17R
  • NK cells are generated from isolated ESCs or iPSCs.
  • the NK cells are engineered to express a heterologous STAT (e.g., STAT3 and/or STAT5B) .
  • a gene encoding the heterologous STAT is incorporated into the NK cell’s genome via either viral transduction or via action of a gene editing moiety as disclosed herein.
  • the NK cells are also engineered to exhibit reduced expression or activity of endogenous IL-17R (i.e., STAT3 + IL-17R - NK cells) .
  • NK cells with either one of (i) the heterologous STAT and (ii) reduced expression or activity of IL-17R, or non-engineered NK cells are used as a control.
  • the engineered STAT3 + IL-17R - NK cells can be cultured in vitro to assess viability and growth (or proliferative capacity) of the engineered STAT3 + IL-17R - NK cells in absence of an exogenous cytokine.
  • the NK cells are cultured in culture medium without the addition of exogenous cytokines for 3-6 weeks.
  • the engineered STAT3 + IL-17R - NK cells exhibit a significantly higher number of NK cells as compared to the control cells, indicating the enhanced survival and persistency of the engineered STAT3 + IL-17R - NK cells in vitro.
  • the engineered STAT3 + IL-17R - NK cells can be administered in NCG mice having a Raji xenograft model.
  • NCG mice are triple immunodeficient and lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function to host the xenograft model.
  • the engineered STAT3 + IL-17R - NK cells and the control cells are each administered into the respective Raji xenograft model mice via intravenous (IV) tail vein injection, at a dose of about 1 ⁇ 10 6 cells per animal.
  • mice injected with the engineered STAT3 + IL-17R - NK cells exhibit higher NK cell concentrations in the peripheral blood from about 7 days to about 28 days post-infusion, demonstrating the enhanced survival and persistency of the engineered STAT3 + IL-17R - NK cells in vivo.
  • NK92 cells were engineered to express anti-CD19 CAR, then cultured in the presence of CD19+ Raji cells to assess targeting of the Raji cells by the engineered anti-CD19 NK cells.
  • Wild type (WT) NK92 cells were used as control.
  • the anti-CD19 CAR NK cells exhibited enhanced cytotoxicity against the Raji cells (as ascertained by a reduced number of alive Raji cells) as compared to the control ( Figure 2A and 2B) .
  • the anti-CD19 CAR NK cells exhibited enhanced expression of endogenous CD107a (indicative of cytotoxic granule release) as compared to the control ( Figure 2C and 2D) .
  • the anti-CD19 CAR NK cells exhibited enhanced cytokine production (e.g., IFN-gamma and/or TNF-alpha production) as compared to the control ( Figure 2E-2G) .
  • NK92 cells were engineered with (i) hIL-15 knock in or (ii) hIL-15-hIL15R fusion polypeptide knock in.
  • Two variants of the hIL-15-hIL15R fusion polypeptide were tested.
  • the first variant i.e., hIL15-IL15Ra fused-1 or “fus1”
  • the first variant was designed with a linker between hIL-15 and hIL15R, which linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 9) , e.g., “GGGGSGGGGSGGGGSGGGGSGGGGGGSGGGGS” (SEQ ID NO. 10) .
  • the second variant (i.e., hIL15-IL15Ra fused-2 or “fus2” ) was designed with a linker between hIL-15 and hIL15R, which linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 9) and one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “EGKSSGSGSESKST” (SEQ ID NO. 11) , e.g., “EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS” (SEQ ID NO. 12) .
  • NK92 cells with either of the hIL-15-hIL15R fusion polypeptide variant knocked-in were positive for hIL-15 ( Figure 3A) .
  • the engineered NK92 cells expressing either variant of the hIL-15-hIL15R fusion polypeptide for enhanced IL-15 signaling exhibited longer persistency as compared to control NK92 cells engineered express secretory form of IL-15.
  • Western blotting analysis revealed increased phosphorylation of IL-15-stimulated STAT5 in the NK92 cells expressing either hIL15-IL15Ra fused-1 (fus1) or hIL15-IL15Ra fused-2 (fus2) , as compared to the secretory IL-15 (IL15) (Figure 3B) .
  • hIL15-IL15Ra fused-1 sequence (SEQ ID NO. 13) :
  • hIL15-IL15Ra fused-2 sequence (SEQ ID NO. 14) :
  • Example 7 Functional analysis of aCD19-CAR, hnCD16 and IL-15 in vitro.
  • the aCD19-CAR comprising a humanized anti-CD19 scFv, a human CD8a hinge, a CD8a transmembrane domain, a 2B4 costimulatory and a CD3 ⁇ signaling domain was designed as shown in Figure 4A, in which VH and VL sequences of the anti-CD19 scFv used in aCD19-CAR are as shown in SEQ ID NO. 16 and SEQ ID NO. 17.
  • SEQ ID NO. 16 may be referred to as a Type 1 anti-CD19 heavy chain variable region herein.
  • SEQ ID NO. 17 may be referred to as a Type 1 anti-CD19 light chain variable region herein.
  • NK92 cells were engineered to express the aCD19-CAR, then cultured in the presence of K562 (CD19negative) and K562-CD19 (CD19positive) cells to assess targeting of K562-CD19 cells by the engineered aCD19-CAR NK cells.
  • Wild type (WT) NK92 cells were used as control.
  • FIG. 6A FACS analysis in Figure 6A illustrates expression of aCD19-CAR at cell surface of the NK-CAR NK92 cells.
  • Figure 6B illustrates aCD19-CAR NK92 cells displayed CD19 antigen-specific killing against K562-CD19 cells at effector: target (E: T) ratios of 0.2: 1 and 1: 1.
  • Figure 6C illustrates aCD19-CAR NK92 cells displayed higher IFN- ⁇ production upon the antigen-specific stimulation of K562-CD19 cells compared to WT-NK92 cells at E: T ratios of 0.2: 1 and 1: 1.
  • E target
  • T target
  • FIG. 6C illustrates aCD19-CAR NK92 cells displayed higher IFN- ⁇ production upon the antigen-specific stimulation of K562-CD19 cells compared to WT-NK92 cells at E: T ratios of 0.2: 1 and 1: 1.
  • hnCD16 of human CD16 variant with F158V and S197P mutations was designed as Figure 4B. Then NK92 cells were engineered to express the hnCD16 to enhanced CD16 signaling. The resulting NK92 cells were highly resistant to the activation-induced cleavage of CD16a and have superior in vitro ADCC against CD19-expressing Raji cells.
  • FIG. 7A FACS analysis illustrates expression of hnCD16 (human CD16 variant with F158V and S197P mutations) at cell surface of NK92 cells.
  • Figure 7B illustrates hnCD16-expressing NK92 cells were highly resistant to the activation-induced cleavage of CD16a, and PMA/Ionomycin was used as a positive control for stimulation.
  • Figure 7C illustrates hnCD16-expressing NK92 cells displayed superior in vitro ADCC against CD19-expressing Raji cells at different E: T ratios.
  • IL-15-RF membrane bound heterologous IL-15
  • IL-15-RF membrane bound heterologous IL-15
  • Figure 4C NK92 cells were engineered to express the IL-15-RF (hIL15-IL15Ra fused-1 as described in Example 6, SEQ ID No. 13) .
  • IL-15-RF was expressed and anchored on NK92 cell surface.
  • Western analysis result in Figure 8B also illustrates increased phosphorylation of IL-15-stimulated STAT5 and STAT3, indicating the activation of IL-15 downstream pathway in NK92 cells expressing IL-15-RF and secretory IL-15 (SEQ ID NO. 24) .
  • WT NK92 cells with addition of exogenous IL-15 served as positive control.
  • Example 8 Design of NK-019 construct comprising aCD19-CAR, hnCD16 and IL-15RF, and expression of NK-019 construct in NK92 cells.
  • NK-019 construct comprising aCD19-CAR, hnCD16, and IL-15 is as shown is Figure 5A. Then NK92 cells were transfected with three different NK-019 constructs:
  • NK-019w, NK-019 construct comprising aCD19-CAR, hnCD16 and secreted IL-15 (SEQ ID No. 25) ;
  • NK-019wf1 NK-019 construct comprising aCD19-CAR, hnCD16 and hIL15-IL15Ra fused-1 (SEQ ID No. 13) ;
  • NK-019wf2 NK-019 construct comprising aCD19-CAR, hnCD16 and hIL15-IL15Ra fused-2 (SEQ ID No. 14) .
  • aCD19-CAR, hnCD16 and IL-15 components in NK92 cells was detected by FACs, respectively.
  • aCD19-CAR, hnCD16 and IL-15 components of NK019 construct were successfully expressed on NK92 cells simultaneously.
  • Example 9 Efficacy of NK cells expressing NK-019 against CD19-expressing target cells.
  • K562 (CD19negative) and K562-CD19 (CD19positive) cells were used as target cells in the efficacy assays.
  • NK92 cells were transfected with NK-019w, NK-019wf1 and NK-019wf2, then cultured in the presence of K562 (CD19negative) and K562-CD19 (CD19positive) cells to assess targeting of K562-CD19 cells by the engineered NK cells.
  • Wild type (WT) NK92 cells were used as control.
  • NK92 cells transfected with different NK-019 constructs all displayed hCD19-antigen-specific cytolytic ability to K562-CD19 cells, in which NK92 cells transfected with NK-019w showed highest cytolytic activity.
  • NK92 cells transfected with NK-019w, NK-019wf1 and NK-019wf2 exhibited enhanced expression of endogenous CD107a as compared to the control, indicating cytotoxic granule release of NK cells.
  • the NK92 cells transfected with NK-019w, NK-019wf1 and NK-019wf2 exhibited enhanced IFN-gamma production as compared to the control, as shown in Figure 11B.
  • Example 10 Anti-tumor activity of NK-019 NK92 cells in Raji NCG mouse model.
  • NCG mice were subject to 100 cGy irradiation on day -2 and injected with Raji-Luc cells expressing luciferase on day -1 through IV (5x10 5 cells/mouse) .
  • the mice were divided into five groups, in which four of the groups were injected with NK92 cells, and NK92 cells expressing NK-019w (w-NK019-NK92) , NK-019wf1 (wf1-NK019-NK92) and NK-019wf2 (wf2-NK019-NK92) through IV (5x10 6 cells/mouse) , as shown in Figure 12A.
  • NK92 cells expressing NK-019w, NK-019wf1 and NK-019wf2 significantly suppressed expansion of CD19 positive tumors in NCG mice, indicating in vivo anti-tumor activity of NK-019 NK92 cells.
  • Example 11 Expression of NK-019 in iPSCs and differentiation of iPSCs into iNK cells.
  • Exemplary NK-019 construct comprising aCD19-CAR, hnCD16, and IL-15 flanked by a PiggyBac transposon ITR was used as donor vector for transgene integration.
  • SSEA-4 is a pluripotency marker of iPSC.
  • Figure 13A illustrates expression of SSEA-4 on the gene-edited iPSCs, indicating pluripotency of edited iPSC cells.
  • iPSCs expressing NK-019w were differentiated into CD34+iHSCs.
  • CD34+ iHSCs were further differentiated into mature iNK cells (CD45+and CD56+) with consistently expression of aCD19-CAR, hnCD16 and IL-15RF on cell surface, as shown in Figure 14B.
  • Example 12 Functional analysis of engineered OI42 NK cells.
  • Engineered NK cells were generated from iPSCs, similarly as described in Example 4 and Example 11. As illustrated in FIG. 15, iPSCs were engineered (e.g., transduced via AAV, CRSIPR-Cas9 mediated insertion) to comprise (e.g., express) an anti-CD19 CAR, a heterologous CD16 variant for enhanced CD16 signaling (e.g., hnCD16) , and a heterologous IL construct for enhanced IL-15 signaling (e.g., IL-15RF) . Subsequently, the iPSCs differentiated to HSCs, and further differentiated to NKs. Following differentiation to NK cells, the culture of NK cells was then expanded.
  • the anti-CD19 CAR in this example, comprised scFv having the amino acid sequence of the Type 2 scFv as disclosed herein (e.g., SEQ ID NO. 42) .
  • Exemplary OI42 (QN-019a) construct comprising aCD19-CAR, hnCD16, and IL-15RF was used as donor vector for transgene integration.
  • the OI42 construct was inserted via CRISPR-Cas9 mediated site-specific insertion and one copy of the transgene was inserted into the iPSCs genome.
  • SSEA-4 was included as a pluripotency marker for iPSCs.
  • gene edited iPSCs expressed SSEA-4, indicating the edited iPSC cells were pluripotent.
  • edited iPSCs expressed the TRA-1-81 epitope, showing that the iPSCs remained undifferentiated.
  • iPSCs expressing OI42 were differentiated into CD34+ iHSCs.
  • CD34+ iHSCs were further differentiated into iNK cells (CD56+) with expression of aCD-19-CAR on cell surface.
  • the resulting expanded NK cells consistently expressed aCD19-CAR, hnCD16, and IL-15RF on cell surface, as shown in Figure 15.
  • OI42 NK cells The level of aCD19-CAR expression on the cell surface of OI42 (QN-019a) NK cells was measured using a labeled CD19 antibody.
  • OI42 NK cells and unmodified umbilical cord blood NK cells (CB-NK) were incubated either with or without the labeled CD19 antibody.
  • OI42 NK cells incubated with the labeled CD19 antibody had the highest level of CD19 expression of all the conditions tested, as shown in Figure 16A, showing that OI42 NK cells expressed CD19.
  • OI42 NK cells were cultured in the presence of CD19-expressing GFP+ Nalm6 cells (Figure 16B) or CD19-expressing GFP+ Raji cells ( Figure 16C) at an effector: target (E: T) ratio of 1: 1.
  • E target
  • Nalm6 and Raji cells were incubated with CB-NK, unmodified iNK cells (QN-001) , or without any NK cells.
  • pictures of the co-cultured cells were taken every 3 hours, the fluorescence area value of the GFP channel was recorded, and the target cell numbers via the fluorescence area value was calculated.
  • OI42 NK cells showed the highest cytolytic activity in both Nalm6 and Raji cells, respectively.
  • OI42 NK cells were stimulated for 4 hours with different stimulation conditions and the level of CD16 expression on cell surface was measured. Stimulation conditions included were: unstimulated cells, co-culture with Raji cells + IgG, co-culture with Raji cells + anti-CD20 antibody, co-culture with K562 cells, and stimulation with Propidium monoazide (PMA) /ionomycin. The percent of CD16+ NK cells was then calculated by dividing the percent of CD16 positive cells after stimulation by the percent of unstimulated CD16+NK cells. In all stimulation conditions tested, OI42 NK cells showed consistent expression of CD16 on cell surface, as shown in Figure 17.
  • the level of phosphorylated STAT5 was measured in OI42 NK (QN-019a) and QN-001 cells.
  • OI42 NK cells and QN-001 cells were incubated with or without IL-2 for 24 hours before measuring the level of p-STAT5.
  • the cells were alternatively incubated with an isotype instead of an antibody against p-STAT5.
  • OI42 NK cells were able to consistently express p-STAT5 levels with or without the addition of IL-2.
  • OI42 (QN-019a) NK cells were incubated with GFP+ Nalm6 cells at a E: T ratio of 1: 1 and pictures of the co-cultured cells were taken every 3 hours and the fluorescence area value of the GFP channel was recorded. Negative control conditions included: Nalm6 cells cultured alone, CB-NK cells cultured with Nalm6 cells, and QN-001 cells cultured with Nalm6 cells. As shown in Figure 19A, OI42 NK cells showed the highest cytolytic activity in Nalm6 cells.
  • OI42 NK cells were incubated with GFP+ Nalm6 cells at a E: T ratio of 1: 1. Every 24 hours, new tumor cells were added to the lysis system. During the co-culture, pictures were taken every 3 hours, the fluorescence area value of the GFP channel was recorded, and the target cell numbers via the fluorescence area value were calculated. As shown in Figure 19B, OI42 NK cells showed the highest cytolytic ability in Nalm6 cells at every addition of new tumor cells.
  • OI42 NK cells were incubated with GFP+ Raji cells at a E: T ratio of 3: 1 and pictures of the co-cultured cells were taken every 3 hours and the fluorescence area value of the GFP channel was recorded.
  • Negative control conditions included: Raji cells cultured alone, CB-NK cells cultured with Raji cells, and QN-001 cells cultured with Raji cells.
  • OI42 NK cells showed the highest cytolytic activity in Raji cells.
  • OI42 NK cells were incubated with GFP+ Raji cells at a E: T ratio of 3: 1. Every 24 hours, new tumor cells were added to the lysis system. During the co-culture, pictures were taken every 3 hours, the fluorescence area value of the GFP channel was recorded, and the target cell numbers via the fluorescence area value were calculated. As shown in Figure 20B, OI42 NK cells showed the highest cytolytic ability in Raji cells at every addition of new tumor cells.
  • NOG mice were intravenously injected with 1x10 5 luciferase expressing Nalm6 cells at day -1 as shown in Figure 21A.
  • mice were given either intravenous injections of 1x10 7 CB-NK cells or OI42 (QN-019a) NK cells.
  • An additional negative control group was included that did not introduce any heterologous NK cells to the mice.
  • mice were given 1 ⁇ g per mouse (/mouse) of IL-15 once a day.
  • From days 0-21 mice were given 100,000 IU/mouse of IL-2 every 2-3 days. Mice were imaged using In Vivo Imaging System (IVIS) weekly.
  • IVIS In Vivo Imaging System
  • Karyotype characterization was performed using G-banded metaphase cells, as shown in Figure 22. A total of 2 images were scanned and 50 cells were analyzed. The data showed that OI42 (QN-019a) NK cells chromosome number was equal to 46. The sex chromosomes were made up by two X chromosomes, which matched the karyotype seen in women. No obvious chromosomal abnormalities of the OI42 NK cells were found.
  • Example 13 Tumorigenicity of OI42 NK cells.
  • mice may be injected subcutaneously with either tumor cells or OI42 (QN-019a) NK cells.
  • the mice may be monitored for multiple months following injection.
  • the body weight of the mice can be monitored and the change in body weight may be calculated.
  • the tumor volume in each injected mouse can be monitored and measured.
  • Pathological samples may be taken from the mice to stain and image portions of tissues.
  • Example #14 Functional analysis of engineered KB15 NK cells.
  • Engineered NK cells were generated from iPSCs, similarly as described in Example 4 and Example 11.
  • iPSCs were engineered (e.g., transduced via AAV, CRSIPR-Cas9 mediated insertion) to comprise (e.g., express) an anti-CD19 CAR, a heterologous CD16 variant for enhanced CD16 signaling (e.g., hnCD16) , and a heterologous IL construct for enhanced IL-15 signaling (e.g., IL-15RF) .
  • the iPSCs differentiated to HSCs, and further differentiated to NKs. Following differentiation to NK cells, the culture of NK cells was then expanded.
  • the anti-CD19 CAR in this example, comprised scFv having the amino acid sequence of the Type 3 scFv as disclosed herein (e.g., SEQ ID NO. 45, 47) .
  • Exemplary KB15 (QN-019) construct comprising aCD19-CAR, hnCD16, and IL-15RF was used as donor vector for transgene integration.
  • the KB15 construct was inserted via PiggyBac mediated transgene random insertion and four copies of the transgene was inserted into the iPSCs genome.
  • the KB15+ iPSCs differentiated to CD34+ iHSCs, and then further differentiated to CD56+ iNK cells.
  • the iNKs cells were expanded for 7 days.
  • KB15 NK cells, QN-001 cells, or CB-NK cells were stimulated with different tumor cells or reagents for 4 hours.
  • the different stimulation conditions included were: unstimulated, cultured with Raji cells, cultured with Raji cells + anti-CD20 antibody, cultured with K562 cells, and stimulated with PMA/Ionomycin. Stimulation with PMA/Ionomycin served as a positive control for CD16 cleavage from the cell membrane.
  • hnCD16 transgene was stably expressed in KB15 NK cells across all the stimulation conditions tested.
  • KB15 NK cells To test the ability of KB15 NK cells to lyse tumor cells via antibody-dependent cellular cytotoxicity (ADCC) , GFP+ Raji cells were first incubated with Rituximab (anti-CD20 antibody) for 1 hour at 4°C. Rituximab was then washed off and effector cells (KB15, QN-001, or CB-NK) were added to the corresponding wells at an E: T ratio of 0.3: 1.
  • Negative controls included Raji cells not incubated with Rituximab with no effector cells added and Raji cells incubated with Rituximab with no effector cells added. Images were taken every three hours for a total of 48 hours.
  • the killing capacity of the effector cells was evaluated by calculating the area of green fluorescence intensity during the imaging time course. As shown in Figure 24, KB15 NK cells, with or without prior Rituximab incubation, showed the highest cytolytic activity in Raji cells.
  • the level of phosphorylated STAT5 was measured in KB15 NK cells and QN-001 cells.
  • KB15 NK cells and QN-001 cells were incubated with or without IL-2 for 24 hours before measuring the level of p-STAT5.
  • the cells were alternatively incubated with an isotype instead of an antibody against p-STAT5.
  • Figure 25A and 25B KB15 NK cells were able to consistently express p-STAT5 levels with or without the addition of IL-2.
  • NOG mice were intravenously injected with 1x10 5 luciferase expressing Raji cells at day 0 as shown in Figure 26A.
  • mice were given intravenous injections of 1x10 7 KB15 NK cells.
  • An additional negative control group was included that did not introduce any heterologous NK cells to the mice.
  • mice were given 1 ⁇ g/mouse of IL-15 once a day.
  • mice were given 100,000 IU/mouse of IL-2 every 2-3 days. Mice were imaged using In Vivo Imaging System (IVIS) weekly.
  • IVIS In Vivo Imaging System
  • Karyotype characterization was performed using G-banded metaphase cells, as shown in Figure 27. A total of 252 images were scanned and 50 cells were analyzed. The data showed that KB15 NK cells chromosome number was equal to 46. The sex chromosomes were made up by two X chromosomes, which matched the karyotype seen in women. No obvious chromosomal abnormalities of the KB15 NK cells were found.
  • KB15 NK cells and QN-001 cells were cultured with or without exogenous IL-2 (200U/ml) . Cell number was counted every 3-4 days and used to evaluate cell growth and cell death. As shown in Figure 28, no abnormal in vitro growth was seen in KB15 NK cells when cultured with or without IL-2.

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Abstract

La présente invention concerne des cellules modifiées comprenant un ou les deux éléments suivants : (i) un variant hétérologue de CD16 pour une signalisation CD16 améliorée par rapport à une cellule témoin, et (ii) un variant hétérologue de IL-15 ou un récepteur de celui-ci; et un récepteur chimérique à l'antigène comprenant un fragment de liaison à l'antigène capable de se lier à CD19, ainsi que les produits et utilisations correspondants.
PCT/CN2022/077683 2021-02-24 2022-02-24 Systèmes et compositions pour immunothérapies améliorées et leurs procédés WO2022179563A1 (fr)

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