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WO2025106552A1 - Méthodes et compositions pour le traitement de la leucémie - Google Patents

Méthodes et compositions pour le traitement de la leucémie Download PDF

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WO2025106552A1
WO2025106552A1 PCT/US2024/055742 US2024055742W WO2025106552A1 WO 2025106552 A1 WO2025106552 A1 WO 2025106552A1 US 2024055742 W US2024055742 W US 2024055742W WO 2025106552 A1 WO2025106552 A1 WO 2025106552A1
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cells
genetically engineered
dose
subject
administration
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David Richard SHOOK
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Nkarta Inc
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Nkarta Inc
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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
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • 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 A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/15—Non-antibody based
    • 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 A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 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
    • C12N2510/00—Genetically modified cells

Definitions

  • NKT.112WO PATENT METHODS AND COMPOSITIONS FOR TREATMENT OF LEUKEMIA CROSS-REFERENCE TO RELATED APPLICATIONS [0001]
  • This application claims priority to United States Provisional Application No. 63/598,865, filed November 14, 2023, the entire contents of which is incorporated by reference herein.
  • FIELD [0002]
  • Several embodiments disclosed herein relate to methods and compositions comprising genetically engineered cells for treating a leukemia.
  • the methods and compositions are for treating acute myeloid leukemia (AML).
  • the cells are generally engineered to express chimeric receptors such as chimeric antigen receptors (CARs) that bind to a ligand of the NKG2D receptor.
  • CARs chimeric antigen receptors
  • features of the methods include an improvement in the safety and/or clinical outcomes of subjects treated in accord with the provided methods.
  • BACKGROUND [0003]
  • Various cell therapy methods are available for treating diseases and conditions.
  • adoptive cell therapies including those involving administration of cells expressing chimeric receptors specific for a disease or condition of interest, such as chimeric antigen receptors (CARs), can be effective in the treatment of cancer and other diseases and conditions.
  • CARs chimeric antigen receptors
  • Improved methods are needed, however, to increase the safety, efficacy, or both, of such methods. Provided are methods and uses that meet such needs.
  • the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells and prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy comprising fludarabine (Flu) and cytarabine (Flu/Ara- C).
  • Flu fludarabine
  • cytarabine Flu/Ara- C
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, prior to administration of the first dose of the genetically engineered NK
  • the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, prior to administration of the first dose of the genetically engineered NK
  • the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered
  • the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered
  • At least about 15% of all subjects treated according to the method exhibit complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi) at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, at least about 15% of all subjects treated according to the method exhibit complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi) at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered
  • the response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered
  • At least about 25% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi), and the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete hematologic recovery
  • At least about 25% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi), and the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 25%, and the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 25%, and the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the second dose of the genetically engineered NK cells is administered to the subject about 7 days after administration of the first dose of the genetically engineered cells.
  • the third dose of genetically engineered NK cells is administered to the subject about 7 days after administration of the second dose of the genetically engineered NK cells.
  • each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells.
  • the lymphodepleting therapy comprises administration of five doses of Flu. In some embodiments, each dose of Flu comprises between about 10 mg/m 2 and about 40 mg/m 2 . In some embodiments, the lymphodepleting therapy comprises administration of five doses of Ara-C. In some embodiments, each dose of Ara-C comprises between about 1 g/m 2 and about 4 g/m 2 .
  • the first doses of Flu and Ara-C are each given 7 days prior to initiation of the dosing cycle; the second doses of Flu and Ara-C are each given 6 days prior to initiation of the dosing cycle; the third doses of Flu and Ara-C are each given 5 days prior to initiation of the dosing cycle; the fourth doses of Flu and Ara-C are each given 4 days prior to initiation of the dosing cycle; and the fifth doses of Flu and Ara-C are each given 3 days prior to initiation of the dosing cycle.
  • each dose of Flu comprises about 30 mg/m 2 .
  • each dose of Ara-C comprises about 2 g/m 2 .
  • the AML is a relapsed/refractory AML (r/r AML).
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetic
  • the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lympho
  • At least about 25% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi), and the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete hematologic recovery
  • At least about 25% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi), and the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete hematologic recovery
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lympho
  • the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lympho
  • At least about 15% of all subjects treated according to the method exhibit complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi) at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, at least about 15% of all subjects treated according to the method exhibit complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi) at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lympho
  • the response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lympho
  • At least about 25% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi), and the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete hematologic recovery
  • At least about 25% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi), and the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 25%, and the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 25%, and the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • (i) the overall survival rate (OS) of all subjects treated according to the method is at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%; and (ii) the survival of each subject is determined at a time point that is about or at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%; and (ii) the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%; and (ii) the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • At least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi); and (ii) the best response of each subject is determined at a time point that is about or at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete hematologic recovery
  • At least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi); and (ii) the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete hematologic recovery
  • At least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi); and (ii) the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete hematologic recovery
  • At least about 15% of all subjects treated according to the method exhibit complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi) at a time point that is about or at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, at least about 15% of all subjects treated according to the method exhibit complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi) at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • CR complete remission
  • CRh complete remission with partial recovery of peripheral blood counts
  • CRi complete remission with incomplete hematologic recovery
  • no more than about 20%, no more than about 15%, no more than about 10%, or no more than about 5% of subjects treated according to the methods exhibited cytokine release syndrome (CRS), immune cell associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GVHD).
  • CRS cytokine release syndrome
  • ICANS immune cell associated neurotoxicity syndrome
  • GVHD graft-versus-host disease
  • the dosing cycle is about 28 days.
  • the method comprises administering an additional dosing cycle as a consolidation treatment.
  • the clinical response is a partial response (PR).
  • the clinical response is a complete response with incomplete hematologic recovery (CRi).
  • the clinical response is a complete response (CR).
  • the method comprises administering an additional dosing cycle as retreatment.
  • the method comprises administration of between one dosing cycle and five dosing cycles.
  • the subject is administered the lymphodepleting therapy prior to each dosing cycle.
  • the subject has less than or equal to 5% peripheral blasts. In some embodiments, subject does not have evidence of extramedullary disease.
  • the method further comprises administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, a therapeutic agent that increases expression of a NKG2D ligand in the subject, and any combination thereof.
  • the therapeutic agent is a chemotherapeutic agent.
  • the therapeutic agent is a monoclonal antibody.
  • the therapeutic agent is a NK cell engager. In some embodiments, the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells.
  • subject has been treated with at least one prior line of therapy. In some embodiments, the subject has been treated with one, two, three, or four prior lines of therapy. In some embodiments, the subject has been treated with one prior line of therapy. In some embodiments, the subject has been treated with two prior lines of therapy. In some embodiments, the subject has been treated with three prior lines of therapy. In some embodiments, the subject has been treated with four prior lines of therapy. [0039] In some embodiments, the subject has an ECOG of 0-2. In some embodiments, the subject has an ECOG of 0.
  • the subject has an ECOG of 1. In some embodiments, the subject has an ECOG of 2. In some embodiments, the subject is 18 years of age or older.
  • the chimeric receptor comprises an extracellular antigen-binding domain that binds a ligand of NKG2D, a transmembrane domain, and an intracellular signaling domain.
  • the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 26.
  • the transmembrane domain comprises a CD8 transmembrane region.
  • the intracellular signaling domain comprises a co-stimulatory domain and a CD3zeta.
  • the co-stimulatory domain comprises an OX40 domain.
  • the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 39.
  • the genetically engineered NK cells express a membrane-bound interleukin 15 (mbIL15).
  • the mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40.
  • the population of engineered NK cells are allogeneic to the subject.
  • a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis.
  • each dose of the engineered NK cells is administered to the subject on an outpatient basis.
  • the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the time point is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engine
  • NK cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed or refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m
  • FIG.1 shows a non-limiting example of a dosing regimen for treating an acute myeloid leukemia (AML) with NKG2D receptor-expressing natural killer (NKG2D NK) cells following lymphodepletion with fludarabine and cytarabine (Flu/Ara-C).
  • FIG. 3 shows clinical responses in patients treated according to the non-limiting dosing regimen.
  • the mainstay of treatment is an attempt to induce complete remission (CR) with intensive chemotherapy, often followed by allogeneic hematopoietic cell transplantation (HCT) in younger patients who are fit and able to tolerate toxicities (National Comprehensive Cancer Network (NCCN) 2019.
  • Intensive therapy results in a median overall survival (OS) of approximately 24 months (uptodate.com/contents/induction-therapy-for-acute-myeloid-leukemia-in-younger-adults; retrieved 2019 Mar 03).
  • Older patients who are unfit either due to age and/or comorbidities are not candidates for intensive therapy or HCT and can receive various low intensity regimens or best supportive care (NCCN 2019).
  • HMAs Hypomethylating agents
  • Ara-C low dose cytarabine
  • Reported CR rates with venetoclax combinations are approximately 21% to 54% (uptodate.com/contents/acute-myeloid-leukemia-treatment-and-outcomes-in-older-adults; retrieved 2019 Mar 03).
  • an immune cell such as an NK cell
  • a chimeric receptor such as a NKG2D- based chimeric receptor, including as encoded by a nucleic acid as described herein.
  • Targeted therapy is a cancer treatment that employs certain drugs that target specific genes or proteins found in cancer cells or cells supporting cancer growth, (like blood vessel cells) to reduce or arrest cancer cell growth.
  • genetic engineering has enabled approaches to be developed that harness certain aspects of the immune system to fight cancers.
  • a patient’s own immune cells are modified to specifically eradicate that patient’s type of cancer.
  • Various types of immune cells can be used, such as T cells, Natural Killer (NK cells), or combinations thereof, as described in more detail below.
  • polynucleotides, polypeptides, and vectors that encode chimeric receptors that comprise a target binding moiety (e.g., an extracellular binder of a ligand expressed by a cancer cell) and a cytotoxic signaling complex are also provided for herein.
  • some embodiments include a polynucleotide, polypeptide, or vector that encodes, for example an activating chimeric receptor comprising an NKG2D-based extracellular domain that binds a NKG2D ligand, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6, among others, to facilitate targeting of an immune cell to a cancer and exerting cytotoxic effects on the NKG2D ligand-expressing cell (e.g., cancer cell).
  • engineered immune cells e.g., NK cells
  • chimeric receptors for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6, among others.
  • engineered immune cells e.g., NK cells
  • polynucleotides and vectors that encode such NKG2D chimeric receptors are also provided herein, in several embodiments. Also provided are methods of treating a cancer (e.g., AML) and uses of NKG2D chimeric receptor-expressing immune cells.
  • Engineered Cells for Immunotherapy [0069]
  • cells of the immune system are engineered to express a chimeric receptor, such that they have enhanced cytotoxic effects against target cells, such as NKG2D ligand- expressing cancer cells.
  • a cell of the immune system may be engineered to include NKG2D- based chimeric receptor as described herein.
  • white blood cells or leukocytes are used, since their native function is to defend the body against growth of abnormal cells and infectious disease.
  • white bloods cells include granulocytes and agranulocytes (presence or absence of granules in the cytoplasm, respectively).
  • Granulocytes include basophils, eosinophils, neutrophils, and mast cells.
  • Agranulocytes include lymphocytes and monocytes.
  • Cells such as those that follow or are otherwise described herein may be engineered to include a chimeric receptor, such as a NKG2D ligand-directed chimeric receptor, or a nucleic acid encoding the chimeric receptor.
  • the cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • mbIL15 membrane-bound interleukin 15
  • the polynucleotide encoding the chimeric receptor also encodes the mbIL15 domain, wherein the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding the mbIL15 domain are separated by a nucleic acid sequence encoding a ribosomal skip element.
  • the immune cells engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15.
  • Monocytes for Immunotherapy Monocytes are a subtype of leukocyte. Monocytes can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes are associated with the adaptive immune system and serve the main functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of cellular material, or entire cells, followed by digestion and destruction of the engulfed cellular material. In several embodiments, monocytes are used in connection with one or more additional engineered cells as disclosed herein.
  • monocytes engineered to express a NKG2D-based chimeric receptor that targets a NKG2D ligand, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6 (among others).
  • the monocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the polynucleotide encoding the chimeric receptor also encodes the mbIL15 domain, wherein the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding the mbIL15 domain are separated by a nucleic acid sequence encoding a ribosomal skip element.
  • the monocytes engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.
  • the monocytes are derived from a donor. In some embodiments, the donor does not have a cancer. In several embodiments, the monocytes are autologous to the subject.
  • the monocytes are derived from the subject. In some embodiments, the monocytes are allogeneic to the subject.
  • Lymphocytes for Immunotherapy the other primary sub-type of leukocyte include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity). While B cells are engineered according to several embodiments, disclosed herein, several embodiments also relate to engineered T cells and/or engineered NK cells (mixtures of T cells and NK cells are used in some embodiments, either from the same donor, or from different donors).
  • lymphocytes engineered to express a NKG2D-based chimeric receptor that targets a NKG2D ligand, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6 (among others).
  • the lymphocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the polynucleotide encoding the chimeric receptor also encodes the mbIL15 domain, wherein the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding the mbIL15 domain are separated by a nucleic acid sequence encoding a ribosomal skip element.
  • the lymphocytes engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.
  • the lymphocytes are derived from a donor. In some embodiments, the donor does not have a cancer. In several embodiments, the lymphocytes are autologous to the subject.
  • the lymphocytes are derived from the subject. In some embodiments, the lymphocytes are allogeneic to the subject.
  • T Cells for Immunotherapy T cells are distinguishable from other lymphocytes sub-types (e.g., B cells or NK cells) based on the presence of a T-cell receptor on the cell surface. T cells can be divided into various different subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cell, mucosal associated invariant T cells and gamma delta T cells. In some embodiments, a specific subtype of T cell is engineered. In some embodiments, a mixed pool of T cell subtypes is engineered.
  • CD4+ T cells are engineered.
  • CD8+ T cells are engineered.
  • regulatory T cells are engineered.
  • gamma delta T cells are engineered.
  • a mixed pool of T cell subtypes is engineered.
  • CD4+ and CD8+ T cells are engineered.
  • specific techniques such as use of cytokine stimulation are used to enhance expansion/collection of T cells with a specific marker profile. For example, in several embodiments, activation of certain human T cells, e.g.
  • CD4+ T cells CD8+ T cells is achieved through use of CD3 and/or CD28 as stimulatory molecules.
  • a method of treating or preventing cancer comprising administering T cells expressing the NKG2D-based chimeric receptor.
  • cancer e.g., AML
  • T cells engineered to express a NKG2D-based chimeric receptor that targets a NKG2D ligand for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6 (among others).
  • the T cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the polynucleotide encoding the chimeric receptor also encodes the mbIL15 domain, wherein the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding the mbIL15 domain are separated by a nucleic acid sequence encoding a ribosomal skip element.
  • the T cells engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.
  • the T cells are derived from a donor.
  • the donor does not have a cancer.
  • the T cells are autologous to the subject.
  • the T cells are derived from the subject.
  • the T cells are allogeneic to the subject.
  • NK Cells for Immunotherapy [0077] In several embodiments, there is provided a method of treating a cancer (e.g., AML), comprising administering natural killer (NK) cells expressing a NKG2D-based chimeric receptor.
  • a cancer e.g., AML
  • NK natural killer
  • NK cells engineered to express a NKG2D-based chimeric receptor that targets a NKG2D ligand, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6 (among others).
  • the NK cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the polynucleotide encoding the chimeric receptor also encodes the mbIL15 domain, wherein the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding the mbIL15 domain are separated by a nucleic acid sequence encoding a ribosomal skip element.
  • the NK cells engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.
  • the NK cells are derived from a donor. In some embodiments, the donor does not have a cancer. In several embodiments, the NK cells are autologous cells to the subject.
  • the NK cells are derived from the subject. In some embodiments, the NK cells are allogeneic to the subject. [0080] In several embodiments, NK cells are preferred because the natural cytotoxic potential of NK cells is relatively high. In several embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, leading to an even more effective activity against target cells (e.g., tumor or other diseased cells). [0081] In several embodiments, immortalized NK cells are used and are subject to engineering, as disclosed herein. In some embodiments, the NK cells are derived from cell line NK-92.
  • NK-92 cells are derived from NK cells, but lack major inhibitory receptors displayed by normal NK cells, while retaining the majority of activating receptors.
  • NK-92 cells are used in combination with NK cells as disclosed herein.
  • NK- 92 cells are used in combination with T cells as disclosed herein.
  • Hematopoietic Stem Cells for Cancer Immunotherapy the immune cells comprise hematopoietic stem cells (HSCs).
  • HSCs hematopoietic stem cells
  • MICA MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6 (among others).
  • the HSCs are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the polynucleotide encoding the chimeric receptor also encodes the mbIL15 domain, wherein the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding the mbIL15 domain are separated by a nucleic acid sequence encoding a ribosomal skip element.
  • the HSCs engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.
  • the HSCs are derived from a donor.
  • the donor does not have a cancer.
  • the HSCs are autologous to the subject.
  • the HSCs are derived from the subject.
  • the HSCs are allogeneic to the subject.
  • Induced Pluripotent Stem Cells [0084]
  • the immune cells comprise NK, T, or other immune cells derived from pluripotent stem cells (iPSCs).
  • iPSCs are used, in several embodiments, to leverage their ability to differentiate and derive into non-pluripotent cells, including, but not limited to, CD34 cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells comprising one or several genetic modifications at selected sites through differentiating iPSCs or less differentiated cells comprising the same genetic modifications at the same selected sites.
  • the iPSCs are used to generate iPSC-derived NK or T cells.
  • iPSCs engineered to express a NKG2D-based chimeric receptor that targets a NKG2D ligand, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6 (among others).
  • the iPSCs are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the polynucleotide encoding the chimeric receptor also encodes the mbIL15 domain, wherein the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding the mbIL15 domain are separated by a nucleic acid sequence encoding a ribosomal skip element.
  • the iPSCs engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.
  • the engineered iPSCs are differentiated into NK cells.
  • the iPSCs are derived from a donor. In some embodiments, the donor does not have a cancer.
  • the iPSCs are autologous to the subject. In several embodiments, the iPSCs are derived from the subject. In some embodiments, the iPSCs are allogeneic to the subject. II. Chimeric Receptor Constructs [0087] Provided herein are chimeric receptor constructs comprising an extracellular antigen- binding domain comprising a fragment of NKG2D, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the transmembrane domain comprises a hinge and a transmembrane region. In some embodiments, the intracellular signaling domain comprises a primary signaling domain (e.g., CD3 zeta) and a co-stimulatory domain.
  • a primary signaling domain e.g., CD3 zeta
  • the chimeric receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.
  • NK cells expressing the same and uses thereof to treat a leukemia (e.g., r/r AML).
  • Extracellular Antigen-Binding Domains [0088] Some embodiments of the compositions and methods described herein relate to a chimeric receptor that includes an extracellular antigen-binding domain as described herein.
  • the antigen-binding domain is derived from or comprises wild-type or non-wild-type sequence of an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab')2, a single domain antibody (sdAb), a vH or vL domain, a camelid VHH domain, or a non-immunoglobulin scaffold such as a DARPIN, an affibody, an affilin, an adnectin, an affitin, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a kunitz domain, an Armadillo repeat protein, an autoantigen, a receptor or a ligand.
  • a non-immunoglobulin scaffold such as a DARPIN, an affibody, an affilin, an adnectin, an
  • the tumor-binding domain contains more than one antigen-binding domain.
  • antigen-binding domain shall be given its ordinary meaning, and shall also refer to a protein comprising an antigen-binding fragment that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding fragment to adopt a conformation that promotes binding of the antigen-binding protein to the antigen.
  • the antigen is a cancer antigen or a fragment thereof.
  • the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen.
  • the antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds to the antigen or from the light chain of an antibody that binds to the antigen. In still some embodiments, the antigen-binding fragment comprises all six CDRs from an antibody that binds to the antigen (three from the heavy chain and three from the light chain). In several embodiments, the antigen-binding fragment comprises one, two, three, four, five, or six CDRs from an antibody that binds to the antigen, and in several embodiments, the CDRs can be any combination of heavy and/or light chain CDRs.
  • the antigen-binding fragment in some embodiments is an antibody fragment.
  • an antigen-binding domain comprises an antibody, antibody fragment (e.g., an antigen-binding fragment of an antibody), antibody derivative, and/or antibody analog.
  • antigen-binding domains include, but are not limited to, a single-chain variable fragment (scFv), a nanobody (e.g. VH domain of camelid heavy chain antibodies; VHH fragment,), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a Fd fragment, and a complementarity determining region (CDR) fragment.
  • scFv single-chain variable fragment
  • a nanobody e.g. VH domain of camelid heavy chain antibodies; VHH fragment,
  • Fab fragment e.g. VH domain of camelid heavy chain antibodies
  • Fab' fragment e.g. VH domain of camelid heavy chain antibodies
  • F(ab')2 fragment e.g. Fab' fragment
  • Fv fragment e.g., a F(
  • Antibody fragments may compete for binding of a target antigen with an intact (e.g., native) antibody and the fragments may be produced by the modification of intact antibodies (e.g. enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technologies or peptide synthesis.
  • the antigen-binding domain can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer.
  • the antigen-binding domain comprises one or more antibody fragments incorporated into a single polypeptide chain or into multiple polypeptide chains.
  • antigen-binding domains can include, but are not limited to, a diabody; an intrabody; a domain antibody (single VL or VH domain or two or more VH domains joined by a peptide linker;); a maxibody (2 scFvs fused to Fc region); a triabody; a tetrabody; a minibody (scFv fused to CH3 domain); a peptibody (one or more peptides attached to an Fc region); a linear antibody (a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions); a small modular immunopharmaceutical; and immunoglobulin fusion proteins (e.g.
  • the antigen-binding domain has the structure of an immunoglobulin.
  • immunoglobulin shall be given its ordinary meaning, and shall also refer to a tetrameric molecule, with each tetramer comprising two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • Human light chains are classified as kappa and lambda light chains.
  • Kappa (K) and lambda ( ) light chains refer to the two major antibody light chain isotypes.
  • a light chain may include a polypeptide comprising, from amino terminus to carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL).
  • Heavy chains are classified as mu ( ), delta ( ), gamma ( ), alpha ( ), and epsilon ( ), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • An antibody “heavy chain” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • the IgM has subclasses including, but not limited to, IgM1 and IgM2.
  • the heavy chains in IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), whereas the heavy chains in IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4).
  • the immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes.
  • the antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain (e.g., between the light and heavy chain) and between the hinge regions of the antibody heavy chains.
  • the antigen-binding domain is or comprises an antibody.
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen.
  • Antibodies can be monoclonal or polyclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.
  • Antibodies can be tetramers of immunoglobulin molecules.
  • the antibody may be “humanized”, “chimeric” or non-human.
  • An antibody may include an intact immunoglobulin of any isotype, and includes, for instance, chimeric, humanized, human, and bispecific antibodies.
  • An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains.
  • Antibody sequences can be derived solely from a single species, or can be “chimeric,” that is, different portions of the antibody can be derived from two different species as described further below.
  • the term “antibody” also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains provided the antibodies retain the same or similar binding and/or function as the antibody comprised of two full length light and heavy chains.
  • antibodies having 1, 2, 3, 4, or 5 amino acid residue substitutions, insertions or deletions at the N-terminus and/or C-terminus of the heavy and/ or light chains are included in the definition provided that the antibodies retain the same or similar binding and/or function as the antibodies comprising two full length heavy chains and two full length light chains.
  • antibodies examples include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies. There is provided, in some embodiments, monoclonal and polyclonal antibodies.
  • polyclonal antibody shall be given its ordinary meaning, and shall also refer to a population of antibodies that are typically widely varied in composition and binding specificity.
  • mAb monoclonal antibody
  • Monoclonal antibodies bind to the antigen at a particular epitope on the antigen.
  • the antigen-binding domain is or comprises a fragment or antigen- binding fragment of an antibody.
  • antibody fragment refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either vL or vH), camelid vHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Patent No.6,703,199, which describes fibronectin polypeptide mini bodies).
  • An antibody fragment may include a Fab, Fab’, F(ab’)2, and/or Fv fragment that contains at least one CDR of an immunoglobulin that is sufficient to confer specific antigen binding to a cancer antigen (e.g., CD19).
  • Antibody fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. [00101] In some embodiments, Fab fragments are provided.
  • a Fab fragment is a monovalent fragment having the VL, VH, CL and CH1 domains;
  • a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region;
  • a Fd fragment has the VH and CH1 domains;
  • an Fv fragment has the VL and VH domains of a single arm of an antibody; and
  • a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain.
  • these antibody fragments can be incorporated into single domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.
  • the antibodies comprise at least one CDR as described herein.
  • single-chain variable fragments There is also provided for herein, in several embodiments, single-chain variable fragments.
  • single-chain variable fragment shall be given its ordinary meaning, and shall also refer to a fusion protein in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site).
  • a “single-chain variable fragment” is not an antibody or an antibody fragment as defined herein.
  • Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is configured to reduce or not allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain.
  • a linker that is configured to reduce or not allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain.
  • Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites.
  • tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
  • the antigen-binding domain comprises one or more CDRs.
  • CDR shall be given its ordinary meaning, and shall also refer to the complementarity determining region (also termed “minimal recognition units” or “hypervariable region”) within antibody variable sequences.
  • the CDRs permit the antigen-binding protein to specifically bind to a particular antigen of interest.
  • the CDRs in each of the two chains typically are aligned by the framework regions to form a structure that binds specifically to a specific epitope or domain on the target protein.
  • naturally-occurring light and heavy chain variable regions both typically conform to the following order of these elements: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.
  • the order is typically: FW-H1, CDR-H1, FW-H2, CDR- H2, FW-H3, CDR-H3, and FW-H4 from N-terminus to C-terminus.
  • FW-L1, CDR-L1, FW-L2, CDR-L2, FW-L3, CDR-L3, FW-L4 from N-terminus to C-terminus.
  • a numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883.
  • CDRs Complementarity determining regions
  • FR framework regions
  • Other numbering systems for the amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol.29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol.309(3):657- 670; 2001).
  • the binding domains disclosed herein may utilize CDRs defined according to any of these systems.
  • the CDRs may be defined in accordance with any of Kabat, Chothia, extended, IMGT, Paratome, AbM, and/or conformational definitions, or a combination of any of the foregoing. Any of the CDRs, either separately or within the context of variable domains, can be interpreted by one of skill in the art under any of these numbering systems as appropriate.
  • One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen-binding protein.
  • the antigen-binding domains provided herein comprise one or more CDR(s) as part of a larger polypeptide chain.
  • the antigen-binding domains covalently link the one or more CDR(s) to another polypeptide chain. In some embodiments, the antigen-binding domains incorporate the one or more CDR(s) noncovalently. In some embodiments, the antigen-binding domains may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure comprises a polypeptide or portion thereof that is sufficient to form a conformationally stable structural support, or framework, or scaffold, which is able to display one or more sequences of amino acids that bind to an antigen (e.g., CDRs, a variable region, etc.) in a localized surface region.
  • an antigen e.g., CDRs, a variable region, etc.
  • Such structures can be a naturally occurring polypeptide or polypeptide “fold” (a structural motif), or can have one or more modifications, such as additions, deletions and/or substitutions of amino acids, relative to a naturally occurring polypeptide or fold.
  • the scaffolds can be derived from a polypeptide of a variety of different species (or of more than one species), such as a human, a non-human primate or other mammal, other vertebrate, invertebrate, plant, bacteria or virus.
  • the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains.
  • those framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain and/or tendamistat domains.
  • antigen-binding domains with more than one binding site are also provided, in some embodiments, antigen-binding domains with more than one binding site.
  • the binding sites are identical to one another while in some embodiments the binding sites are different from one another.
  • an antibody typically has two identical binding sites, while a “bispecific” or “bifunctional” antibody has two different binding sites.
  • bispecific antigen-binding domain or antibody will bind to two different epitopes, which can reside on the same or different protein targets.
  • a bispecific chimeric receptor e.g., CAR
  • CAR can impart to an engineered cell the ability to target multiple tumor markers.
  • compositions and methods described herein relate to a NKG2D-based chimeric receptor that includes an extracellular antigen-binding domain that binds a ligand of NKG2D.
  • the antigen-binding domain binds to, for example MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6.
  • Natural killer Group 2 member D is an NK cell activating receptor that recognizes a variety of ligands expressed on cells. The surface expression of various NKG2D ligands is generally low in healthy cells but is upregulated upon, for example, malignant transformation.
  • Non-limiting examples of ligands recognized by NKG2D include, but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, as well as other molecules expressed on target cells that control the cytolytic or cytotoxic function of NK cells.
  • engineered immune cells such as NK cells are leveraged for their ability to recognize and destroy tumor cells.
  • NK cells express both inhibitory and activating receptors on the cell surface. Inhibitory receptors bind self-molecules expressed on the surface of healthy cells (thus preventing immune responses against “self” cells), while the activating receptors bind ligands expressed on abnormal cells, such as tumor cells.
  • NK cells are engineered to express a chimeric receptor comprising full length NKG2D (e.g., human NKG2D) as an extracellular component to recognize a NKG2D ligand on the surface of a cancer cell.
  • full length NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 27.
  • the antigen-binding domain is encoded by SEQ ID NO: 27.
  • full length NKG2D comprises the amino acid sequence of SEQ ID NO: 42.
  • the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 42.
  • NK cells are engineered to express a chimeric receptor comprising a fragment of NKG2D (e.g., a fragment of human NKG2D) as an extracellular component to recognize a NKG2D ligand on the surface of a cancer cell.
  • the fragment of NKG2D comprises all or a fragment of the extracellular domain of NKG2D.
  • the fragment of NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 25.
  • the antigen- binding domain is encoded by SEQ ID NO: 25.
  • the fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26.
  • the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 26.
  • the full length NKG2D, or functional fragment thereof is human NKG2D.
  • full length NKG2D is human full length NKG2D.
  • the fragment of NKG2D is a fragment of human NKG2D. Additional information about chimeric receptors for use in the presently disclosed methods and compositions can be found in PCT Patent Publication No. WO/2018/183385, which is incorporated in its entirety by reference herein.
  • cells are engineered to express a chimeric receptor comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain is a fragment of NKG2D.
  • the fragment of NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 25.
  • the fragment of NKG2D has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with full-length wild-type NKG2D.
  • the fragment is encoded by a polynucleotide that has one or more additional mutations from SEQ ID NO: 25, but retains, or in some embodiments, has enhanced, NKG2D ligand-binding function.
  • the fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26.
  • the NKG2D fragment is provided as a dimer, trimer, or other concatemeric format, such embodiments providing enhanced ligand-binding activity.
  • the sequence encoding the NKG2D fragment is fully or partially codon-optimized.
  • a polynucleotide sequence encoding a codon optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28.
  • the functional fragment lacks its native transmembrane domain, intracellular domain, or both, but retains its ability to bind ligands of NKG2D, as well as transduce activation signals upon ligand binding.
  • a further advantage of such NKG2D fragments is that expression of DAP10 to localize NKG2D to the cell membrane is not required.
  • the chimeric receptors provided herein do not comprise DAP10.
  • immune cells such as NK or T cells
  • NK or T cells are engineered to express an additional chimeric receptor that targets, for example CD70, CD19, CD123, Her2, mesothelin, Claudin 6, BCMA, EGFR, and/or a NKG2D ligand.
  • additional chimeric receptor that targets, for example CD70, CD19, CD123, Her2, mesothelin, Claudin 6, BCMA, EGFR, and/or a NKG2D ligand.
  • Such cells in several embodiments, also express (e.g., bicistronically express) mbIL15.
  • the chimeric receptors are configured to dimerize. Dimerization may comprise homodimers or heterodimers, depending on the embodiment.
  • dimerization results in improved ligand recognition by the chimeric receptors (and hence the NK cells expressing the receptor), resulting in a reduction in (or lack) of adverse toxic effects.
  • the chimeric receptors employ internal dimers, or repeats of one or more component subunits.
  • the chimeric receptors comprise a first NKG2D extracellular domain coupled to a second NKG2D extracellular domain, a transmembrane domain, and an intracellular signaling domain.
  • Transmembrane Domains [00116]
  • the antigen-binding domain generally is linked to an intracellular signaling domain comprising intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR.
  • the antigen-binding domain is linked to an intracellular signaling domain by a transmembrane domain.
  • the antigen-binding domain e.g., fragment of NKG2D
  • the transmembrane domain is fused to the extracellular antigen- binding domain.
  • the transmembrane domain in some embodiments is derived either from a natural or from a synthetic source.
  • the domain in some aspects is derived from any membrane- bound or transmembrane protein.
  • Transmembrane regions include those derived from (e.g., comprising at least the transmembrane region(s) of) CD3, CD4, CD5, CD8, CD9, CD 16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
  • the transmembrane domain in some embodiments is synthetic.
  • the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells.
  • the transmembrane domain comprises CD8alpha (CD8a). In several embodiments, the transmembrane domain comprises a CD8 (e.g., CD8a) hinge and a CD8 (e.g., CD8a) transmembrane region. [00119] In several embodiments, the transmembrane domain comprises a hinge, e.g. a CD8a hinge. In several embodiments, the sequence encoding the CD8a hinge is truncated or modified.
  • the CD8a hinge is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:1.
  • the CD8a hinge is encoded by the nucleic acid sequence of SEQ ID NO:1.
  • the CD8a hinge is truncated or modified.
  • the CD8a hinge has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • the hinge of CD8a comprises the amino acid sequence of SEQ ID NO:2.
  • the transmembrane domain comprises a CD8a transmembrane region.
  • the CD8a transmembrane region is truncated or modified.
  • the CD8a transmembrane region is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO:3.
  • the CD8a transmembrane region is encoded by a nucleic acid sequence of SEQ ID NO:3.
  • the CD8a transmembrane region is truncated or modified.
  • the CD8a transmembrane region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO:4. In several embodiments, the CD8a transmembrane region comprises the amino acid sequence of SEQ ID NO:5. [00121] Thus, in several embodiments, the CD8 transmembrane domain is truncated or modified and is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO:13.
  • the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO:13.
  • the CD8 transmembrane domain is truncated or modified and comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO:14.
  • the transmembrane domain comprises the amino acid sequence of SEQ ID NO:14.
  • Signaling Domains [00122]
  • the chimeric receptor e.g., the CAR, generally includes an intracellular signaling domain comprising intracellular signaling components.
  • the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain.
  • a TCR CD3 chain that mediates T-cell activation and cytotoxicity
  • the antigen-binding portion is linked to one or more cell signaling modules.
  • the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., NK cell engineered to express the chimeric receptor.
  • the chimeric receptor induces a function of an immune cell (e.g., NK cell) such as cytolytic activity and/or secretion of cytokines or other factors.
  • an immune cell e.g., NK cell
  • a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal.
  • the intracellular signaling domain includes the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects, also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.
  • TCR T cell receptor
  • T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen independent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences).
  • primary cytoplasmic signaling sequences those that initiate antigen-dependent primary activation through the TCR
  • secondary cytoplasmic signaling sequences those that act in an antigen independent manner to provide a secondary or co- stimulatory signal.
  • the receptor includes one or both of such signaling components.
  • the receptor includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d.
  • cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3zeta.
  • immune cells engineered according to several embodiments disclosed herein may comprise at least one subunit of the CD3 T cell receptor complex (or a fragment thereof).
  • the signaling domain comprises the CD3 zeta subunit.
  • the CD3 zeta can be truncated or modified.
  • the CD3zeta is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:7. In several embodiments, the CD3zeta is encoded by the nucleic acid sequence of SEQ ID NO:7. In several embodiments, the CD3zeta is truncated or modified. In some embodiments, the CD3zeta comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:8.
  • the CD3zeta comprises the amino acid sequence of SEQ ID NO:8.
  • the intracellular signaling domain comprises a co-stimulatory domain, such as an intracellular signaling region of CD28, 4-1BB, OX40, DAP10, ICOS, or any combination thereof.
  • the same receptor includes both a CD3zeta and a co-stimulatory domain.
  • the intracellular signaling domain of the chimeric receptor comprises a CD3zeta and a co-stimulatory domain.
  • the intracellular signaling domain comprises an OX40 co- stimulatory domain.
  • the OX40 co-stimulatory domain is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5.
  • the OX40 co- stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO:5.
  • the OX40 co-stimulatory domain comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6.
  • the OX40 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:6.
  • OX40 is used as the sole co-stimulatory domain in the construct, however, in several embodiments, OX40 can be used with one or more other components.
  • combinations of OX40 and CD3 zeta are used in some embodiments.
  • the intracellular signaling domain comprises an OX40 co-stimulatory domain linked to CD3 zeta.
  • the chimeric receptor (e.g., CAR) comprises the amino acid sequence set forth in SEQ ID NO:39.
  • the intracellular signaling domain comprises a 4-1BB co- stimulatory domain.
  • the 4-1BB co-stimulatory domain is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:29.
  • the 4-1BB co- stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO:29.
  • the 4-1BB co-stimulatory domain comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:30. In several embodiments, the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:30.
  • 4-1BB is used as the sole intracellular signaling component in the construct, however, in several embodiments, 4-1BB can be used with one or more other components. For example, combinations of 4-1BB and CD3 zeta are used in some embodiments. In some embodiments, the intracellular signaling domain comprises a 4-1BB co-stimulatory domain linked to CD3 zeta.
  • the intracellular signaling domain comprises a CD28 co- stimulatory domain.
  • the 4-1BB co-stimulatory domain is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:31.
  • the 4-1BB co- stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO:31.
  • the CD28 co-stimulatory domain comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:32. In several embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:32.
  • CD28 is used as the sole intracellular signaling component in the construct, however, in several embodiments, CD28 can be used with one or more other components. For example, combinations of CD28 and CD3 zeta are used in some embodiments.
  • the intracellular signaling domain comprises a CD28 co-stimulatory domain linked to CD3 zeta.
  • the nucleic acid encoding the chimeric receptor, or a portion thereof is codon-optimized.
  • the polynucleotides are optimized, or contain certain features designed for optimization, such as for codon usage, to reduce RNA heterogeneity and/or to modify, e.g., increase or render more consistent among cell product lots, expression, such as surface expression, of the encoded receptor.
  • polynucleotides, encoding chimeric receptors are modified as compared to a reference polynucleotide, such as to remove cryptic or hidden splice sites, to reduce RNA heterogeneity.
  • polynucleotides, encoding chimeric receptors are codon optimized, such as for expression in a mammalian, e.g., human, cell such as in a human NK cell.
  • the modified polynucleotides result in in improved, e.g., increased or more uniform or more consistent level of, expression, e.g., surface expression, when expressed in a cell.
  • constructs encoding a chimeric receptor can also encode a stimulatory molecule as described herein. These can be certain molecules that, for example, further enhance the activity of the immune cells. Cytokines may be used in some embodiments. For example, certain interleukins, such as IL-2 and/or IL-15 as non-limiting examples, are used.
  • the immune cells for therapy are engineered to express such molecules as a secreted form.
  • such stimulatory molecules are engineered to be in membrane bound found, acting as autocrine stimulatory molecules and/or paracrine stimulators to neighboring cells.
  • the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15).
  • the IL15 is expressed from a separate cassette on the construct comprising any one of the CARs disclosed herein.
  • the IL15 is expressed from the same cassette as any one of the CARs disclosed herein.
  • the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding IL15 are separated by a nucleic acid sequence encoding a cleavage site, for example, a proteolytic cleavage site or a T2A, P2A, E2A, or F2A self-cleaving peptide cleavage site.
  • the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding IL15 are separated by a T2A sequence.
  • the IL15 is membrane-bound IL15 (mbIL15).
  • the mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain.
  • the native IL15 sequence is encoded by a polynucleotide sequence having at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 11.
  • the native IL15 sequence is encoded by SEQ ID NO: 11.
  • the native IL15 sequence comprises an amino acid sequence having at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the native IL15 sequence comprises SEQ ID NO: 12. [00135] In some embodiments, IL15 is membrane-bound by virtue of its coupling to a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane region. In some embodiments, the CD8 (e.g., CD8 alpha) transmembrane region comprises the sequence of SEQ ID NO: 4. In some embodiments, the mbIL15 may comprise additional components, such as a leader sequence and/or a hinge sequence.
  • the leader sequence is a CD8 leader sequence.
  • the hinge sequence is a CD8 (e.g., CD8 alpha) hinge sequence.
  • the CD8 alpha comprises the sequence of SEQ ID NO: 2.
  • the transmembrane domain comprises a CD8 (e.g., CD8 alpha) hinge and a CD8 (e.g., CD8 alpha) transmembrane region.
  • the transmembrane domain comprises the sequence of SEQ ID NO: 12.
  • the mbIL15 comprises native IL15 fused to a CD8 (e.g., CD8 alpha) transmembrane domain.
  • the mbIL15 comprises an amino acid sequence having at least about 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 40. In some embodiments, the mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40.
  • the chimeric receptors are encoded by a polynucleotide that encodes for one or more cytosolic protease cleavage sites. Such sites are recognized and cleaved by a cytosolic protease, which can result in separation (and separate expression) of the various component parts of the receptor encoded by the polynucleotide.
  • the chimeric receptors are encoded by a polynucleotide that encodes for one or more self-cleaving peptides, for example a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and/or an F2A cleavage site.
  • the chimeric receptor and the IL15 can be delivered to an immune cell in a single vector or by multiple vectors.
  • the chimeric receptor and IL15 can be encoded by a single polynucleotide, but also include a cleavage site, such that the chimeric receptor and IL15 are expressed as separate proteins by the cell.
  • the T2A cleavage site has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 10.
  • NK cells are engineered to express membrane-bound interleukin 15 (mbIL15).
  • mbIL15 expression on the NK enhances the cytotoxic effects of the engineered NK cell by enhancing the proliferation and/or longevity of the NK cells.
  • the mbIL15 is encoded by the same polynucleotide as the chimeric receptor.
  • mbIL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 11. In some embodiments, mbIL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 11 and a sequence that encodes for a transmembrane domain. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12 functionally coupled to an amino acid sequence of a transmembrane domain. In several embodiments, mbIL15 is encoded by the nucleic acid sequence of SEQ ID NO: 35.
  • the nucleic acid encoding mbIL15 can be truncated or modified.
  • mbIL15 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 35.
  • the mbIL15 comprises the amino acid sequence of SEQ ID NO: 40.
  • the mbIL15 is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the mbIL15 having the sequence of SEQ ID NO: 40.
  • GS3 linker SEQ ID NOs: 15 and 16, nucleotide and protein, respectively
  • GSn linker Other linkers used according to various embodiments disclosed herein include, but are not limited to those encoded by SEQ ID NOs: 17, 19, 21 or 23.
  • linkers comprise the peptide sequence of one of SEQ ID NOs: 18, 20, 22, 24, or 43.
  • a linker comprises the amino acid sequence of SEQ ID NO: 16.
  • a linker comprises the amino acid sequence of SEQ ID NO: 18.
  • a linker comprises the amino acid sequence of SEQ ID NO: 20.
  • a linker comprises the amino acid sequence of SEQ ID NO: 22.
  • a linker comprises the amino acid sequence of SEQ ID NO: 24.
  • a linker comprises the amino acid sequence of SEQ ID NO: 43.
  • Chimeric Receptor Constructs [00140] Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as chimeric receptors that bind a ligand of NKG2D. The expression of these chimeric receptors in immune cells, such as NK cells, allows the targeting and destruction of particular target cells, such as cancerous cells. Non-limiting examples of such chimeric receptors are discussed in more detail below.
  • a polynucleotide encoding (e.g., from N- to C- terminus) an antigen-binding domain (e.g., a fragment of NKG2D); a transmembrane domain, and an intracellular signaling domain.
  • the transmembrane domain comprises (e.g., from N- to C-terminus) a CD8 hinge and a CD8 transmembrane region.
  • the intracellular signaling domain comprises (e.g., from N- to C-terminus) an OX40 co-stimulatory domain and CD3 zeta.
  • a polypeptide comprising (e.g., from N- to C-terminus) an antigen- binding domain (e.g., a fragment of NKG2D); a CD8 hinge; a CD8 transmembrane region; an OX40 co- stimulatory domain; and CD3 zeta.
  • the polynucleotide encodes, or the polypeptide comprises, an extracellular antigen-binding domain that binds a NKG2D ligand, a CD8 alpha hinge, a CD8 alpha transmembrane domain, an OX40 co-stimulatory domain, a CD3zeta domain.
  • the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 39.
  • the polynucleotide encoding the chimeric receptor also encodes a mbIL15 domain as described herein.
  • the chimeric receptor and the mbIL15 domain are expressed bicistronically.
  • the polynucleotide further encodes a ribosomal skip element such as a 2A cleavage site, and an mbIL-15 domain as described herein.
  • a polynucleotide provided herein comprises a nucleic acid sequence encoding the chimeric receptor and a nucleic acid sequence encoding the mbIL15 domain, separated by a nucleic acid sequence encoding a 2A (e.g., T2A) cleavage site.
  • the chimeric receptor is encoded by a polynucleotide comprising a combination of nucleic acid sequences disclosed herein or is a polypeptide comprising a combination of amino acid sequences disclosed herein.
  • the encoding nucleic acid sequence, or the amino acid sequence comprises a sequence in accordance with one or more SEQ ID NOS as described herein, such as those included herein as examples of constituent parts.
  • the encoding nucleic acid sequence, or the amino acid sequence comprises a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and/or functional equivalence with a sequence resulting from the combination one or more SEQ ID NOS as described herein.
  • the chimeric receptor comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a range defined by any two of the aforementioned percentages, identical to the sequence of one or more of the SEQ ID NOs provided for herein, or a portion thereof (e.g.
  • a polynucleotide encoding a chimeric receptor comprising (e.g., from N- to C-terminus) a NKG2D fragment/CD8a hinge/CD8a transmembrane region/OX40 co-stimulatory domain/CD3 zeta.
  • a polypeptide comprising (e.g., from N- to C-terminus) a NKG2D fragment/CD8a hinge/CD8a transmembrane region/OX40 co-stimulatory domain/CD3 zeta.
  • the polynucleotide or polypeptide comprises or is composed of a fragment of the NKG2D receptor capable of binding a ligand of the NKG2D receptor (e.g., an extracellular domain of NKG2D), a CD8alpha hinge, a CD8a transmembrane region, an OX40 co- stimulatory domain, and a CD3 zeta as described herein.
  • this receptor complex is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 33.
  • this chimeric receptor comprises the amino acid sequence of SEQ ID NO: 39.
  • the sequence of the chimeric receptor may be encoded by a nucleic acid sequence that varies from SEQ ID NO: 33, but remains expressed as a amino acid sequence, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 39.
  • the chimeric receptor amino acid sequence may vary from SEQ ID NO: 39
  • the chimeric receptor retains, or in some embodiments, has enhanced, NK cell activating and/or cytotoxic function.
  • this construct can optionally be co-expressed with mbIL15, such as the mbIL15 encoded by SEQ ID NO: 35 or 37.
  • the mbIL15 comprises the amino acid sequence of SEQ ID NO: 36, 38, or 40. In several embodiments, the mbIL15 is comprises the amino acid sequence of SEQ ID NO: 36. In several embodiments, the mbIL15 is comprises the amino acid sequence of SEQ ID NO: 38. In several embodiments, the mbIL15 is comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the amino acid sequence of the mbIL15 may vary from SEQ ID NO: 36, 38, or 40, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 36, 38, or 40.
  • the amino acid sequence of the mbIL15 may vary from SEQ ID NO: 36, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 36. In some embodiments, the amino acid sequence of the mbIL15 may vary from SEQ ID NO: 38, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 38.
  • the amino acid sequence of the mbIL15 may vary from SEQ ID NO: 40, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 40.
  • Additional information about chimeric receptors for use in the presently disclosed methods and compositions can be found in PCT Patent Publication No. WO 2018/183385, filed March 27, 2018, which is incorporated in its entirety by reference herein. III.
  • compositions and Formulations [00146] Also provided are compositions including the chimeric receptors and engineered cells expressing the same, including pharmaceutical compositions and formulations.
  • compositions comprising engineered NK cells that express a NKG2D-based chimeric receptor provided herein, such as CARs, including pharmaceutical compositions and formulations.
  • CARs a NKG2D-based chimeric receptor
  • pharmaceutical formulations comprising a chimeric receptor (e.g., CAR), engineered cells expressing a chimeric receptor, a plurality of engineered cells expressing a chimeric receptor, and/or additional agents for combination treatment or therapy.
  • the pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient.
  • the composition includes at least one additional therapeutic agent.
  • composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • the choice of carrier is determined in part by the particular cell, binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • the pharmaceutical composition can contain preservatives.
  • Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride.
  • a mixture of two or more preservatives is used.
  • the preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • a buffer is included in the composition.
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts.
  • a mixture of two or more buffers is used.
  • the buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the total composition.
  • Formulations or compositions may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
  • the pharmaceutical composition in some embodiments contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
  • Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the agent or cell population is administered to the subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.
  • the compositions are provided as sterile liquid formulations (e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions), which in some aspects may be buffered to a selected pH.
  • sterile liquid formulations e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions
  • Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions.
  • liquid compositions are somewhat more convenient to administer, particularly by injection.
  • the liquid composition can comprise a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.
  • a carrier such as an admixture with a suitable carrier, diluent, or excipient (e.g., sterile water, saline, glucose, dextrose, and the like).
  • a suitable carrier e.g., sterile water, saline, glucose, dextrose, and the like.
  • Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.
  • the dose of engineered cells administered is in a cryopreserved composition. In some aspects, the composition is administered after thawing the cryopreserved composition.
  • pharmaceutical compositions for combination therapy Any of the additional agents for combination therapy described herein can be prepared and administered as one or more pharmaceutical compositions, with the chimeric receptor and/or engineered cells expressing the chimeric receptor as described herein.
  • the combination therapy can be administered in one or more pharmaceutical compositions, e.g., where the chimeric receptors and/or cells are in the same pharmaceutical composition as the additional agent, or in separate pharmaceutical compositions.
  • the additional agent is an additional engineered cell, e.g., cell engineered to express a different chimeric receptor that targets an antigen other than a NKG2D ligand, and is administered in the same composition or in a separate composition.
  • each of the pharmaceutical composition is formulated in a suitable formulation according to the particular binding molecule, recombinant receptor, cell, e.g., engineered cell, and/or additional agent, and the particular dosage regimen and/or method of delivery. IV.
  • Methods and Uses are methods of using and uses of chimeric receptors, engineered cells, and pharmaceutical compositions and formulations thereof, such as in the treatment of diseases, conditions, and disorders in which a NKG2D ligand is expressed, and/or in detection, diagnostic, and prognostic methods.
  • methods of treatment, and uses are those that involve administering to a subject engineered NK cells, such as a plurality of engineered NK cells, expressing the provided chimeric receptors.
  • methods of combination therapy and/or treatment are also provided.
  • Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules (e.g., chimeric receptors), cells (e.g., engineered cells), or compositions containing the same, to a subject having a disease, condition, or disorder associated with NKG2D ligands such as a disease, condition, or disorder associated with NKG2D ligand expression, and/or in which cells or tissues express, e.g., specifically express a NKG2D ligand.
  • the chimeric receptor, cell, and/or composition is/are administered in an effective amount to effect treatment of the disease or disorder.
  • the methods are carried out by administering the chimeric receptors or cells, or compositions comprising the same, to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.
  • treatment refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith.
  • Desirable effects of treatment include, but are not limited to, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
  • “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated.
  • the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.
  • a sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease.
  • a late-stage cancer such as development of metastasis, may be delayed.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • an antibody or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody or composition or cell.
  • An “effective amount” of an agent e.g., a pharmaceutical formulation, binding molecule, antibody, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
  • a “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment.
  • the therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
  • the provided methods involve administering the molecules, antibodies, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • Methods for administration of cells for cell therapy are known and may be used in connection with the provided methods and compositions.
  • Some embodiments relate to a method of treating a cancer (e.g., r/r AML) with immune cells (e.g., NK cells) genetically engineered to express a chimeric receptor, as disclosed herein.
  • Some embodiments relate to a method of treating a cancer (e.g., r/r AML) with immune cells (e.g., NK cells) genetically engineered to express a chimeric receptor as provided herein, in combination with a therapeutic agent.
  • the method includes administering a therapeutically effective amount of immune cells (e.g., NK cells) engineered to express a NKG2D-based chimeric receptor to a subject having a cancer such as r/r AML, as described herein.
  • a cancer e.g., r/r/ AML
  • the methods comprise administering to the subject any one of the chimeric receptors disclosed herein, engineered NK cells expressing the same, or a composition comprising a plurality of engineered NK cells expressing any one of the chimeric receptors disclosed herein, and a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, or a combination thereof.
  • the therapeutic agent is a chemotherapeutic agent.
  • the therapeutic agent is a monoclonal antibody.
  • the therapeutic agent is a NK cell engager.
  • the therapeutic agent increases expression of a NKG2D ligand in a subject.
  • the therapeutic agent is administered prior to, concurrent with, and/or after administration of genetically engineered NK cells.
  • the therapeutic agent is administered prior to administration of genetically engineered NK cells.
  • the therapeutic agent is administered prior to treatment with a lymphodepleting therapy.
  • the therapeutic agent is administered after treatment with a lymphodepleting therapy and prior to administration of the genetically engineered NK cells.
  • the therapeutic agent is administered concurrently with administration of genetically engineered NK cells.
  • the therapeutic agent is administered after administration of genetically engineered NK cells.
  • treatment of a subject with genetically engineered NK cell(s) and a therapeutic agent as described herein achieves an effect comprising: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; and/or (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.
  • a “subject” or an “individual” is a mammal.
  • a “mammal” includes humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc.
  • the subject is human.
  • the cancer is a relapsed or refractory (r/r) cancer.
  • the cancer is a leukemia.
  • the cancer is acute myeloid leukemia (AML).
  • the subject has a r/r AML.
  • the subject has a r/r AML as determined by standard European LeukemiaNet (ELN) criteria (Döhner et al., Blood (2022) 140(12):1345-77).
  • ENN European LeukemiaNet
  • the subject has minimum residual disease.
  • the subject has r/r AML with MRD.
  • the subject is in complete response (CR) with MRD.
  • the subject has r/r MDS as determined by standard International Working Group (IWG) criteria (Cheson et al., Blood (2006) 108(2):419-25).
  • IWG International Working Group
  • the subject has a high-risk genetic mutation (e.g., TP53 or monosomy 7). In some embodiments, a high-risk genetic mutation as defined by 2022 European LeukemiaNet (Dohner 2022). [00175] In some embodiments, the subject has a FLT3-mutated cancer or a IHD 1/2-mutated cancer. In some embodiments, the subject has a FLT3-mutated cancer. In some embodiments, the subject has a IHD 1/2-mutated cancer. In some embodiments, the subject has a FLT3-mutated cancer or a IHD 1/2-mutated cancer, the subject has received four prior lines of therapy.
  • TP53 TP53 or monosomy 7
  • a high-risk genetic mutation as defined by 2022 European LeukemiaNet (Dohner 2022).
  • the subject has a FLT3-mutated cancer or a IHD 1/2-mutated cancer. In some embodiments, the subject has a FLT3-mutated cancer. In some embodiments, the subject has a FLT
  • the subject has received a prior line of therapy that targets the mutation.
  • the subject has received a prior line of therapy for the AML.
  • the subject has received one, two, three, or four prior lines of therapy for the AML.
  • the subject has received one prior line of therapy for the AML.
  • the subject has received two prior lines of therapy for the AML.
  • the subject has received three prior lines of therapy for the AML.
  • the subject has received no more than three prior lines of therapy for the AML.
  • the subject has received four prior lines of therapy for the AML.
  • the prior line of therapy comprises venetoclax.
  • the subject has relapsed following HCT.
  • the subject has relapsed following HCT within about 6 months of administration of the genetically engineered NK cells to the subject.
  • the methods provided herein achieve particular clinical responses in subjects including those who relapsed following HCT.
  • the subject has a white blood cell count of 25 ⁇ 10 9 /liter.
  • the subject has a platelet count of 30,000/microliter.
  • the subject has marrow limited disease (also known as marrow localized disease).
  • the subject has less than or equal to 5% peripheral blasts. In some embodiments, the subject has less than 5% peripheral blasts. In some embodiments, the subject does not have evidence of extramedullary disease. [00179] In some embodiments, the subject is at least 18 years of age. In some embodiments, the subject is less than 75 years of age. In some embodiments, the subject is between about 18 years of age and about 75 years of age. [00180] In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. In some embodiments, the subject has an ECOG performance status of 0.
  • ECOG Eastern Cooperative Oncology Group
  • the subject has an ECOG performance status of 1. In some embodiments, the subject has an ECOG performance status of 2. [00181] In some embodiments, the subject does not have acute promyelocytic leukemia with t(15;17) (q22;q12) or abnormal promyelocytic leukemia/retinoic acid receptor alpha (APML-RARA) and AML arising from chronic myelomonocytic leukemia (CMML). In some embodiments, the subject does not have leukemic meningitis or known active central nervous system disease. In some embodiments, the subject does not have peripheral leukocytosis with 20,000 blasts/microliter.
  • the subject has not undergone a hematopoietic cell transplantation within 16 weeks prior to treatment.
  • a cancer e.g., r/r AML
  • methods of treating a subject having a cancer comprising administering to the subject a composition comprising immune cells (e.g., natural killer cells) engineered to express a chimeric receptor as disclosed herein.
  • immune cells e.g., natural killer cells
  • Uses of such engineered immune cells for treating a cancer are also provided.
  • treatment of a subject in accord with the methods provided herein may achieve desirable safety and/or efficacy outcomes, including at time points such as about six months or more after administration of a first dose of the genetically engineered cells to a subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 25% (e.g., at least about 50%), and the survival of each subject is determined at a time point that is about or at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • At least about 25% (e.g., at least about 50% or at least about 60%) of all subjects treated according to the method achieve a best response of complete remission (CR), complete remission with partial recovery of peripheral blood counts (CRh), or complete remission with incomplete hematologic recovery (CRi), and the best response of each subject is determined at a time point that is about or at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • at least about 15% of all subjects treated according to the method are in complete remission (CR) at a time point that is about or at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • genetically engineered NK cells are administered to a subject in a dosing cycle comprising (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells.
  • a lymphodepleting therapy comprising fludarabine (Flu) and cytarabine (Flu/Ara-C).
  • Flu fludarabine
  • cytarabine Flu/Ara-C
  • Doses of immune cells such as NK cells can be readily determined for a given subject based on their body mass, disease type and state, and desired aggressiveness of treatment, but range, depending on the embodiments, from about 10 5 cells per kg to about 10 12 cells per kg (e.g., 10 5 -10 7 , 10 7 - 10 10 , 10 10 -10 12 and overlapping ranges therein).
  • a dose escalation regimen is used.
  • a range of immune cells such as NK cells is administered, for example between about 1 x 10 6 cells/kg to about 1 x 10 8 cells/kg.
  • each dose of engineered NK cells comprises about 1 x 10 9 genetically engineered NK cells.
  • each dose of engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells.
  • 1 ⁇ 10 8 NK cells are administered (2 ⁇ 10 6 /kg for a subject under 50 kg) three times over the cycle.
  • 1.5 ⁇ 10 8 genetically engineered NK cells are administered (3 ⁇ 10 6 /kg for a subject under 50kg) three times over the cycle.
  • 3 ⁇ 10 8 genetically engineered NK cells are administered three times over the cycle. In several embodiments, 4.5 ⁇ 10 8 genetically engineered NK cells are administered three times over the cycle. In several embodiments, 1 ⁇ 10 9 genetically engineered NK cells are administered three times over the cycle. In several embodiments, 1.5 ⁇ 10 9 genetically engineered NK cells are administered three times over the cycle. In several embodiments, 3 ⁇ 10 9 genetically engineered NK cells are administered three times over the cycle. In several embodiments, 1.5 ⁇ 10 10 genetically engineered NK cells are administered three times over the cycle. In several embodiments, at least 4.5 ⁇ 10 9 genetically engineered NK cells are administered over the cycle.
  • the cycle is between about 14 days and about 35 days. In some embodiments, the cycle is about 14 days. In some embodiments, the cycle is about 21 days. In some embodiments, the cycle is about 28 days. In some embodiments, the cycle is about 35 days. [00188] In several embodiments, 1 ⁇ 10 8 NK cells are administered (2 ⁇ 10 6 /kg for a subject under 50 kg) three times over a 28-day cycle. In several embodiments, 1.5 ⁇ 10 8 genetically engineered NK cells are administered (3 ⁇ 10 6 /kg for a subject under 50kg) three times over a 28-day cycle. In several embodiments, 3 ⁇ 10 8 genetically engineered NK cells are administered three times over a 28-day cycle.
  • 4.5 ⁇ 10 8 genetically engineered NK cells are administered three times over a 28- day cycle.
  • 1 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle.
  • 1.5 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle.
  • 3 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle.
  • 1.5 ⁇ 10 10 genetically engineered NK cells are administered three times over a 28-day cycle.
  • at least 4.5 ⁇ 10 9 genetically engineered NK cells are administered over a 28-day cycle.
  • three doses of the genetically engineered NK cells are administered within about 10 days, within about 11 days, without about 12 days, without about 13 days, within about 14 days, within about 15 days, within about 16 days, within about 17 days, or within about 17 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 10 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 11 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 12 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 13 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 14 days.
  • three doses of the genetically engineered NK cells are administered within about 15 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 16 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 17 days. [00190] In several embodiments, all three doses of the genetically engineered NK cells are administered within about 14 days, within about 15 days, within about 16 days, within about 17 days, within about 18 days, within about 19 days, within about 20 days, or within about 21 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 14 days of the conclusion of administration of the lymphodepleting therapy.
  • all three doses of the genetically engineered NK cells are administered within about 15 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 16 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 17 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 18 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 19 days of the conclusion of administration of the lymphodepleting therapy.
  • all three doses of the genetically engineered NK cells are administered within about 20 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 21 days of the conclusion of administration of the lymphodepleting therapy. [00191] In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 5-10 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 5 days after the first dose of the genetically engineered NK cells is administered to the subject.
  • the second dose of the genetically engineered NK cells is administered to the subject about 6 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 7 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 8 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 9 days after the first dose of the genetically engineered NK cells is administered to the subject.
  • the second dose of the genetically engineered NK cells is administered to the subject about 10 days after the first dose of the genetically engineered NK cells is administered to the subject.
  • the third dose of the genetically engineered NK cells is administered to the subject about 5-10 days after the second dose of the genetically engineered NK cells is administered to the subject.
  • the third dose of the genetically engineered NK cells is administered to the subject about 5 days after the second dose of the genetically engineered NK cells is administered to the subject.
  • the third dose of the genetically engineered NK cells is administered to the subject about 6 days after the second dose of the genetically engineered NK cells is administered to the subject.
  • the third dose of the genetically engineered NK cells is administered to the subject about 7 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the third dose of the genetically engineered NK cells is administered to the subject about 8 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the third dose of the genetically engineered NK cells is administered to the subject about 9 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the third dose of the genetically engineered NK cells is administered to the subject about 10 days after the second dose of the genetically engineered NK cells is administered to the subject.
  • a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle
  • the second dose of the genetically engineered NK cells is administered on day 7 of the dosing cycle
  • the third dose of the genetically engineered NK cells is administered on day 14 of the dosing cycle.
  • each dose comprises between about 1 x 10 9 genetically engineered NK cells and about 5 x 10 9 genetically engineered NK cells.
  • each dose comprises between about 1.5 x 10 9 genetically engineered NK cells and about 5 x 10 9 genetically engineered NK cells.
  • each dose comprises between about 2 x 10 9 genetically engineered NK cells and about 5 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises between about 2 x 10 9 genetically engineered NK cells and about 3 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 1 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 1.5 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 2 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 2.5 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 3 x 10 9 genetically engineered NK cells.
  • each dose comprises about 3.5 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 4 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 4.5 x 10 9 genetically engineered NK cells. In several embodiments, each dose comprises about 5 x 10 9 genetically engineered NK cells. [00195] In several embodiments, 1 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 2 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle.
  • At least 6 ⁇ 10 9 genetically engineered NK cells are administered over the cycle.
  • 2.5 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle.
  • at least 7.5 ⁇ 10 9 genetically engineered NK cells are administered over the cycle.
  • 2.5 ⁇ 10 9 genetically engineered NK cells are administered three times over a 28-day cycle.
  • at least 9 ⁇ 10 9 genetically engineered NK cells are administered over the cycle.
  • a dosing cycle comprises administration of three doses of genetically engineered NK cells (e.g., over a 28-day period), wherein the second dose is administered about 5-10 days after administration of the first dose, the third dose is administered about 5-10 days after administration of the second dose, and each dose comprises at least about 1.5 x 10 9 NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 7 days after administration of the first dose, the third dose is administered about 7 days after administration of the second dose, and each dose comprises about 1.5 x 10 9 NK cells.
  • subjects exhibiting at least a partial response will receive at least one additional dosing cycle. Dosing cycles may continue, depending on the embodiment as long as the subject is exhibiting an anti-tumor response and tolerating the engineering NK cells. In several embodiments, a subject will not receive an additional dosing cycle if they are not responding (e.g., no tumor response) and/or if the therapy is not tolerated. However, as discussed herein, in several embodiments the disclosed dosing regimens have limited, or no, adverse impacts or toxicities.
  • a determination about receiving/administering an additional dosing cycle is made at an evaluation about 28 days after the inception of a dosing cycle (whether that be the first dosing cycle, or a subsequent cycle). In several embodiments, not more than 4 additional cycles are given to a subject. In several embodiments, not more than 5 additional cycles are given to a subject. [00198] In some embodiments, a dose of NK cells of the dosing cycle is administered on an outpatient basis. In some embodiments, two doses of NK cells of the dosing cycle are administered on an outpatient basis. In some embodiments, each dose of NK cells of the dosing cycle is administered on an outpatient basis.
  • the administration of engineered NK cells is preceded by one or more preparatory treatments.
  • the administration of engineered NK cells is preceded by a lymphodepleting therapy (also referred to as “lymphodepletion”).
  • each dosing cycle is preceded by lymphodepletion.
  • a combination of chemotherapeutic agents is used for lymphodepletion.
  • a single chemotherapeutic agent is used for lymphodepletion.
  • agents with different mechanisms of actions are optionally used.
  • different classes of agents are optionally used.
  • an antimetabolic agent is used.
  • the antimetabolic agent inhibits and/or prevents cell replication.
  • the antimetabolic agent is an altered nucleotide that disrupts DNA replication, making it effective in targeting rapidly dividing tumor cells.
  • a lymphodepleting therapy is administered to the subject prior to administration of the first dose of genetically engineered NK cells.
  • a lymphodepleting therapy is administered to the subject on each of 5 days, 4 days, and 3 days prior to administration of the first dose of genetically engineered NK cells.
  • a lymphodepleting therapy is administered to the subject on each of 7 days, 6 days, 5 days, 4 days, and 3 days prior to administration of the first dose of genetically engineered NK cells.
  • the first dose of the genetically engineered NK cells is administered to the subject about 3 days after administration of a lymphodepleting therapy to the subject has concluded.
  • the lymphodepleting therapy comprises cytosine arabinoside (also known as cytarabine; Ara-C).
  • a dose of between about 0.2 – about 10 g/m 2 Ara-C is administered, including doses of about 0.2 g/m 2 , about 0.5 g/m 2 , about 1.0 g/m 2 , about 1.5 g/m 2 , about 2.0 g/m 2 , about 2.5 g/m 2 , about 3.0 g/m 2 , about 3.5 g/m 2 , about 4.0 g/m 2 , about 5.0 g/m 2 , about 6.0 g/m 2 , about 7.0 g/m 2 , about 8.0 g/m 2 , about 9.0 g/m 2 , about 10.0 about 1.5 g/m 2 , or any dose between those listed.
  • a dose of about 2 g/m 2 of Ara-C is administered.
  • the dose of Ara-C is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.
  • the dose of Ara-C is given daily for about 5 days.
  • the dose can be split and given, for example, twice daily.
  • an additional agent is used in combination with the Ara-C.
  • the additional agent is also an antimetabolite.
  • the additional agent inhibits one or more of DNA polymerase alpha, ribonucleotide reductase and/or DNA primase, thus inhibiting DNA synthesis.
  • the lymphodepleting therapy comprises fludarabine (Flu).
  • a dose of between about 5.0 mg/m 2 – about 200 mg/m 2 fludarabine is administered, including doses of about 5.0 mg/m 2 , about 10.0 mg/m 2 , about 15.0 mg/m 2 , about 20.0 mg/m 2 , about 25.0 mg/m 2 , about 30.0 mg/m 2 , about 35.0 mg/m 2 , about 40.0 mg/m 2 , about 45.0 mg/m 2 , about 50.0 mg/m 2 , about 60.0 mg/m 2 , about 70.0 mg/m 2 , about 80.0 mg/m 2 , about 90.0 mg/m 2 , about 100.0 mg/m 2 , about 125.0 mg/m 2 , about 150.0 mg/m 2 , about 175.0 mg/m 2 , about 200.0 mg/m 2 , or any dose between those listed.
  • a dose of about 30 mg/m 2 of fludarabine is administered.
  • the dose of fludarabine is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.
  • the dose of fludarabine is given daily for about 3 days.
  • the dose of fludarabine is given daily for about 5 days.
  • the dose can be split and given, for example, twice daily.
  • a combination of fludarabine and Ara-C is used with a daily dose of fludarabine of between about 20 mg/m 2 and 40 mg/m 2 and a daily dose of Ara-C of between about 1.5 g/m 2 and 2.5 g/m 2 .
  • a combination of fludarabine and Ara-C is used with a daily dose of fludarabine of about 30 mg/m 2 and a daily dose of Ara-C of about 2 g/m 2 .
  • the lymphodepleting therapy comprises cyclophosphamide (Cy).
  • a dose of between about 100 mg/m 2 – about 100 mg/m 2 fludarabine is administered, including doses of about 100.0 mg/m 2 , about 200 mg/m 2 , about 300 mg/m 2 , about 400 mg/m 2 , about 500 mg/m 2 , about 600 mg/m 2 , about 700 mg/m 2 , about 800 mg/m 2 , about 900 mg/m 2 , about 1000 mg/m 2 , or any dose between those listed.
  • a dose of about 300 mg/m 2 of cyclophosphamide is administered.
  • a dose of about 500 mg/m 2 of cyclophosphamide is administered.
  • the dose of cyclophosphamide is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.
  • the dose of cyclophosphamide is given daily for about 3 days.
  • the dose can be split and given, for example, twice daily.
  • a combination of fludarabine and cyclophosphamide is used.
  • cyclophosphamide (300 mg/m 2 ) and fludarabine (30mg/m 2 ) are administered daily for 3 days.
  • cyclophosphamide 500 mg/m 2
  • fludarabine 30mg/m 2
  • fludarabine and cyclophosphamide are each administered daily 5 days, 4 days, and 3 days prior to administration of the engineered NK cells.
  • a dose of a genetically engineered cell(s) described herein or composition thereof is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks.
  • two, three or four doses of a genetically engineered cell(s) described herein or composition thereof is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks.
  • a dose(s) of a genetically engineered cell(s) described herein or composition thereof is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days.
  • a dose of a genetically engineered cell(s) described herein or composition thereof is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.
  • an additional dosing cycle is administered to the subject as consolidation treatment.
  • Clinical responses may include complete response (CR; e.g., complete remission), complete response with incomplete hematologic recovery (CRi; e.g., CR with residual thrombocytopenia), CR with partial hematologic recovery (CRh), morphologic leukemia-free state (MLFS), and partial response (PR; e.g., partial remission).
  • CR complete response
  • CRi complete hematologic recovery
  • CRh CR with residual thrombocytopenia
  • CRh CR with partial hematologic recovery
  • MLFS morphologic leukemia-free state
  • PR partial response
  • the clinical response is CR.
  • the clinical response is CRi. In some embodiments, the clinical response is MLFS. In some embodiments, the clinical response is CRh. In some embodiments, the clinical response is PR. Methods for assessing clinical response are known in the art (Döhner et al., Blood (2017) 129(4):424-47). [00208] For example, in some embodiments, if a subject exhibits a complete response (CR) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject exhibits a complete response with incomplete hematologic recovery (CRi) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment.
  • CRi complete hematologic recovery
  • a subsequent dosing cycle is administered as consolidation treatment.
  • CRh incomplete hematologic recovery
  • a subsequent dosing cycle is administered as consolidation treatment.
  • MLFS morphologic leukemia-free state
  • PR partial response
  • a subsequent dosing cycle is administered as consolidation treatment.
  • a method of administering an additional dosing cycle to a subject who did not exhibit a clinical response following a previous dosing cycle if a subject does not exhibit a CR following a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if a subject does not exhibit a CRi following a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if a subject does not exhibit MLFS following a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if a subject does not exhibit a PR following a dosing cycle, a subsequent dosing cycle is administered.
  • a subject if a subject exhibits a clinical response from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment.
  • a method of administering an additional dosing cycle as retreatment to a subject who exhibited a clinical response following a previous dosing cycle and subsequently exhibited disease progression.
  • the subject if a subject exhibits a CR from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment.
  • the subject if a subject exhibits a CRi from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment.
  • the dosing regimen comprises between one dosing cycle and five dosing cycles. In some embodiments, the dosing regimen consists of between one dosing cycle and five dosing cycles. In some embodiments, the dosing regimen consists of between one dosing cycle and five dosing cycles. In some embodiments, the dosing regimen consists of one dosing cycle.
  • the dosing regimen consists of two dosing cycles. In some embodiments, the dosing regimen consists of three dosing cycles. In some embodiments, the dosing regimen consists of four dosing cycles. In some embodiments, the dosing regimen consists of five dosing cycles. In some embodiments, the subject is administered a lymphodepleting therapy prior to each dosing cycle. [00212] In several embodiments, lymphodepletion is performed prior to the inception of each dosing cycle, if subsequent dosing cycles are required (e.g., the subject requires further treatment).
  • a subject undergoes lymphodepletion, receives a plurality of doses of engineered cells according to a cycle, is evaluated at the end of the cycle time and, if deemed necessary undergoes a second lymphodepletion followed by a second dosing cycle.
  • a first and a second dosing cycle need not be the same (e.g., a first cycle may have 2 doses, while a second uses three doses).
  • a first cycle may have 2 doses, while a second uses three doses).
  • dosing cycles are performed.
  • no more than four dosing cycles are administered.
  • no more than five dosing cycles are administered.
  • the cancer is a hematologic malignancy.
  • the cancer is a leukemia or a lymphoma.
  • the cancer being treated is acute myeloid leukemia (AML).
  • the cancer being treated is relapsed/refractory acute myeloid leukemia (r/r AML).
  • the cancer is relapsed to HCT.
  • the cancer being treated is myelodysplastic syndrome (MDS).
  • nucleic acid and amino acid sequences that have sequence identity and/or homology of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges therein) as compared with the respective nucleic acid or amino acid sequences of SEQ ID NOS.1- 44 (or combinations of two or more of SEQ ID NOS: 1-44) and that also exhibit one or more of the functions as compared with the respective SEQ ID NOS.1-44 (or combinations of two or more of SEQ ID NOS: 1- 44) including but not limited to, (i) enhanced proliferation, (ii) enhanced activation, (iii) enhanced cytotoxic activity against cells presenting ligands to which NK cells harboring receptors encoded by the nucleic acid and amino acid sequences bind, (iv) enhanced homing to tumor or infected sites, (v) reduced off target cytotoxic effects, (vi) enhanced secretion of immunostimulatory cytokines and
  • amino acid sequences that correspond to any of the nucleic acids disclosed herein, while accounting for degeneracy of the nucleic acid code. Furthermore, those sequences (whether nucleic acid or amino acid) that vary from those expressly disclosed herein, but have functional similarity or equivalency are also contemplated within the scope of the present disclosure. The foregoing includes mutants, truncations, substitutions, or other types of modifications.
  • polynucleotides encoding the disclosed chimeric receptors are mRNA. In some embodiments, the polynucleotide is DNA.
  • the polynucleotide is operably linked to at least one regulatory element for the expression of the chimeric receptor.
  • a vector comprising the polynucleotide encoding any of the chimeric receptors provided for herein. Additionally provided, according to several embodiments, is a vector comprising the polynucleotide encoding mbIL15 as provided for herein. Additionally provided, according to several embodiments, is a vector comprising the polynucleotide encoding a chimeric receptor and mbIL15 as provided for herein.
  • the polynucleotides are operatively linked to at least one regulatory element for expression of the chimeric receptor, mbIL15, or both.
  • the vector is a retroviral vector.
  • the vector is a gamma retroviral vector.
  • engineered immune cells such as NK cells
  • compositions comprising a plurality of engineered immune cells (such as NK cells) comprising the polynucleotide, vector, or chimeric receptor as disclosed herein. VII.
  • Non-Limiting Outcomes [00219] Provided herein are methods for treating AML in a subject comprising administering to a subject having a cancer a population of NK cells genetically engineered to express a chimeric receptor that binds a ligand of NKG2D.
  • particular clinical outcomes are achieved in subjects treated according to the method.
  • the provided methods achieve particular clinical outcomes at a time point after subjects have been administered a first dose of the genetically engineered cells.
  • the provided methods achieve particular clinical outcomes at least 3, 4, 5, 6, or more months after subjects have been administered a first dose of the genetically engineered cells.
  • the provided methods achieve particular clinical outcomes 3 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 4 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 5 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes at least 6 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 6 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 7 months after subjects have been administered a first dose of the genetically engineered cells.
  • the provided methods achieve particular clinical outcomes 8 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 9 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 10 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 11 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 12 months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 18 months after subjects have been administered a first dose of the genetically engineered cells.
  • the provided methods achieve particular clinical outcomes 24 months after subjects have been administered a first dose of the genetically engineered cells.
  • the clinical outcome is overall survival (OS).
  • OS overall survival
  • the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the OS rate of all subjects treated according to the method is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%. In some embodiments, the OS rate of all subjects treated according to the method is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%, and the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the OS rate of all subjects treated according to the method is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%, and the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the OS rate of all subjects treated according to the method is at least about 25%. In some embodiments, the OS rate of all subjects treated according to the method is at least about 30%. In some embodiments, the OS rate of all subjects treated according to the method is at least about 35%.
  • the OS rate of all subjects treated according to the method is at least about 40%. In some embodiments, the OS rate of all subjects treated according to the method is at least about 45%. In some embodiments, the OS rate of all subjects treated according to the method is at least about 55%. In some embodiments, the OS rate of all subjects treated according to the method is at least about 55%. In some embodiments, the OS rate of all subjects treated according to the method is at least about 60%. [00223] In some embodiments, the OS rate of all subjects treated according to the method is about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%.
  • the OS rate of all subjects treated according to the method is about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%, and the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the OS rate of all subjects treated according to the method is about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%, and the survival of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. [00224] In some embodiments, the OS rate of all subjects treated according to the method is about 25%.
  • the OS rate of all subjects treated according to the method is about 30%. In some embodiments, the OS rate of all subjects treated according to the method is about 35%. In some embodiments, the OS rate of all subjects treated according to the method is about 40%. In some embodiments, the OS rate of all subjects treated according to the method is about 45%. In some embodiments, the OS rate of all subjects treated according to the method is about 55%. In some embodiments, the OS rate of all subjects treated according to the method is about 55%. In some embodiments, the OS rate of all subjects treated according to the method is about 60%. [00225] In some embodiments, the clinical outcome is best response. In some embodiments, the best response is CR, CRh, or CRi.
  • the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. [00226] In some embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% of all subjects achieve a best response of CR, CRh, or CRi.
  • At least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% of all subjects achieve a best response of CR, CRh, or CRi, and the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • At least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% of all subjects achieve a best response of CR, CRh, or CRi, and the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • at least about 25% of all subjects achieve a best response of CR.
  • at least about 30% of all subjects achieve a best response of CR.
  • at least about 35% of all subjects achieve a best response of CR.
  • At least about 40% of all subjects achieve a best response of CR. In some embodiments, at least about 45% of all subjects achieve a best response of CR. In some embodiments, at least about 50% of all subjects achieve a best response of CR. In some embodiments, at least about 55% of all subjects achieve a best response of CR. In some embodiments, at least about 60% of all subjects achieve a best response of CR. [00228] In some embodiments, at least about 25% of all subjects achieve a best response of CRh. In some embodiments, at least about 30% of all subjects achieve a best response of CRh. In some embodiments, at least about 35% of all subjects achieve a best response of CRh.
  • At least about 40% of all subjects achieve a best response of CRh. In some embodiments, at least about 45% of all subjects achieve a best response of CRh. In some embodiments, at least about 50% of all subjects achieve a best response of CRh. In some embodiments, at least about 55% of all subjects achieve a best response of CRh. In some embodiments, at least about 60% of all subjects achieve a best response of CRh. [00229] In some embodiments, at least about 25% of all subjects achieve a best response of CRi. In some embodiments, at least about 30% of all subjects achieve a best response of CRi. In some embodiments, at least about 35% of all subjects achieve a best response of CRi.
  • At least about 40% of all subjects achieve a best response of CRi. In some embodiments, at least about 45% of all subjects achieve a best response of CRi. In some embodiments, at least about 50% of all subjects achieve a best response of CRi. In some embodiments, at least about 55% of all subjects achieve a best response of CRi. In some embodiments, at least about 60% of all subjects achieve a best response of CRi. [00230] In some embodiments, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of all subjects achieve a best response of CR, CRh, or CRi.
  • about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of all subjects achieve a best response of CR, CRh, or CRI
  • the best response of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of all subjects achieve a best response of CR, CRh, or CRI
  • the best response of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • about 25% of all subjects achieve a best response of CR.
  • about 30% of all subjects achieve a best response of CR.
  • about 35% of all subjects achieve a best response of CR.
  • about 40% of all subjects achieve a best response of CR.
  • about 45% of all subjects achieve a best response of CR.
  • about 50% of all subjects achieve a best response of CR.
  • about 55% of all subjects achieve a best response of CR.
  • about 60% of all subjects achieve a best response of CR.
  • about 25% of all subjects achieve a best response of CRh.
  • about 30% of all subjects achieve a best response of CRh. In some embodiments, about 35% of all subjects achieve a best response of CRh. In some embodiments, about 40% of all subjects achieve a best response of CRh. In some embodiments, about 45% of all subjects achieve a best response of CRh. In some embodiments, about 50% of all subjects achieve a best response of CRh. In some embodiments, about 55% of all subjects achieve a best response of CRh. In some embodiments, about 60% of all subjects achieve a best response of CRh. [00233] In some embodiments, about 25% of all subjects achieve a best response of CRi. In some embodiments, about 30% of all subjects achieve a best response of CRi.
  • the clinical outcome is complete remission (CR) at a particular time point. In some embodiments, the clinical outcome is CRh at a particular time point. In some embodiments, the clinical outcome is CRi at a particular time point.
  • the clinical outcome of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the time point is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the clinical outcome of each subject is determined at a time point that is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the time point is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • At least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CR at the time point. In some embodiments, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CR at the time point, and the time point is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • At least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CR at the time point, and the time point is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • at least about 15% of all subjects are in CR at the time point.
  • at least about 16% of all subjects are in CR at the time point.
  • at least about 17% of all subjects are in CR at the time point.
  • at least about 18% of all subjects are in CR at the time point.
  • At least about 19% of all subjects are in CR at the time point. In some embodiments, at least about 20% of all subjects are in CR at the time point. In some embodiments, at least about 25% of all subjects are in CR at the time point. In some embodiments, at least about 30% of all subjects are in CR at the time point. [00237] In some embodiments, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CRh at the time point.
  • At least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CRh at the time point, and the time point is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CRh at the time point, and the time point is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • At least about 15% of all subjects are in CRh at the time point. In some embodiments, at least about 16% of all subjects are in CRh at the time point. In some embodiments, at least about 17% of all subjects are in CRh at the time point. In some embodiments, at least about 18% of all subjects are in CRh at the time point. In some embodiments, at least about 19% of all subjects are in CRh at the time point. In some embodiments, at least about 20% of all subjects are in CRh at the time point. In some embodiments, at least about 25% of all subjects are in CRh at the time point. In some embodiments, at least about 30% of all subjects are in CRh at the time point.
  • At least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CRi at the time point. In some embodiments, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CRi at the time point, and the time point is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • At least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, or at least about 30% of all subjects are in CRi at the time point, and the time point is at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • at least about 15% of all subjects are in CRi at the time point.
  • at least about 16% of all subjects are in CRi at the time point.
  • at least about 17% of all subjects are in CRi at the time point.
  • at least about 18% of all subjects are in CRi at the time point.
  • the time point is about nine months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the time point is about twelve months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, the time point is about eighteen months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the time point is about twenty-four months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • subjects treated according to any of the methods provided herein are able to maintain their best clinical response for a particular period of time, such as three months or more. In some embodiments, subjects treated according to any of the methods provided herein maintain their best clinical response for about three months. In some embodiments, subjects treated according to any of the methods provided herein maintain their best clinical response for at least about three months. In some embodiments, subjects treated according to any of the methods provided herein maintain their best clinical response for about six months. In some embodiments, subjects treated according to any of the methods provided herein maintain their best clinical response for at least about six months.
  • subjects treated according to any of the methods provided herein maintain their best clinical response for about nine months. In some embodiments, subjects treated according to any of the methods provided herein maintain their best clinical response for at least about nine months. In some embodiments, subjects treated according to any of the methods provided herein maintain their best clinical response for about twelve months. In some embodiments, subjects treated according to any of the methods provided herein maintain their best clinical response for at least about twelve months. [00243] In some embodiments, for any of the provided methods herein, among all subjects who were treated according to the method and achieved a best response of CR, CRi, or CRh, at least about 50% of the subjects maintain their best response for about or at least about 3 months.
  • any of the provided methods herein among all subjects who were treated according to the method and achieved a best response of CR, CRi, or CRh, at least about 50% of the subjects maintain their best response for about or at least about 4 months In some embodiments, for any of the provided methods herein, among all subjects who were treated according to the method and achieved a best response of CR, CRi, or CRh, at least about 50% of the subjects maintain their best response for about or at least about 5 months In some embodiments, for any of the provided methods herein, among all subjects who were treated according to the method and achieved a best response of CR, CRi, or CRh, at least about 50% of the subjects maintain their best response for about or at least about 6 months.
  • any of the provided methods herein among all subjects who were treated according to the method and achieved a best response of CR or CRi, at least about 50% of the subjects maintain their best response for about or at least about 3 months. In some embodiments, for any of the provided methods herein, among all subjects who were treated according to the method and achieved a best response of CR or CRi, at least about 50% of the subjects maintain their best response for about or at least about 4 months. In some embodiments, for any of the provided methods herein, among all subjects who were treated according to the method and achieved a best response of CR or CRi, at least about 50% of the subjects maintain their best response for about or at least about 5 months.
  • NON-LIMITING EMBODIMENTS [00245] Among the embodiments provided herein are: 1.
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject
  • a method of treating acute myeloid leukemia (AML) in a subject comprising administering to a subject having AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject
  • each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells. 18.
  • the lymphodepleting therapy comprises administration of five doses of Flu. 19.
  • each dose of Flu comprises between about 10 mg/m 2 and about 40 mg/m 2 .
  • the lymphodepleting therapy comprises administration of five doses of Ara-C. 21.
  • the method of embodiment 20, wherein each dose of Ara-C comprises between about 1 g/m 2 and about 4 g/m 2 . 22.
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engine
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r AML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of
  • a method of treating relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having r/r RML a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 25%, and the survival of each subject is determined at a time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • the overall survival rate (OS) of all subjects treated according to the method is at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%; and (ii) the survival of each subject is determined at a time point that is about or at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.
  • PR partial response
  • CRi complete response with incomplete hematologic recovery
  • CR complete response
  • the method of any one of embodiments 1-45 wherein the method comprises administration of between one dosing cycle and five dosing cycles. 47. The method of any one of embodiments 44-46, wherein the subject is administered the lymphodepleting therapy prior to each dosing cycle. 48. The method of any one of embodiments 1-47, wherein the subject has less than or equal to 5% peripheral blasts and/or wherein the subject does not have evidence of extramedullary disease. 49.
  • any one of embodiments 1-48 wherein the method further comprises administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, a therapeutic agent that increases expression of a NKG2D ligand in the subject, and any combination thereof, optionally wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, and a NK cell engager.
  • a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, and a NK cell engager.
  • transmembrane domain comprises a CD8 transmembrane region.
  • the intracellular signaling domain comprises a co-stimulatory domain and a CD3zeta.
  • the co-stimulatory domain comprises an OX40 domain.
  • the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 39.
  • the genetically engineered NK cells express a membrane-bound interleukin 15 (mbIL15).
  • mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40.
  • 63. The method of any one of embodiments 1-62, wherein the population of engineered NK cells are allogeneic to the subject. 64. The method of any one of embodiments 1-63, wherein a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis, optionally wherein each dose of the engineered NK cells is administered to the subject on an outpatient basis. 65.
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • AML acute myeloid leukemia
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy comprising flu
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • AML acute myeloid leukemia
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • AML acute myeloid leukemia
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodeple
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • AML acute myeloid leukemia
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodeple
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • AML acute myeloid leukemia
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodeple
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • AML acute myeloid leukemia
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 5-10 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 5-10 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10 9 and about 2 x 10 9 genetically engineered NK cells, wherein: prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m 2 fludarabine (
  • NK cells Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed or refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m 2 fludarabine (F
  • NK cells Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed or refractory acute myeloid leukemia (r/r/ AML), wherein the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m 2 fludarabine
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m 2 fludarabine
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m 2 fludarabine (F
  • NK Natural Killer
  • NSG2D natural killer cell group 2D
  • the genetically engineered NK cells are for administration in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 7 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m 2 fludarabine (
  • Example 1 Clinical Outcomes of Patients with Acute Myeloid Leukemia Treated with NKG2D CAR NK Cells Following Lymphodepletion with Fludarabine and Cytarabine
  • AML acute myeloid leukemia
  • Flu/Ara-C fludarabine and cytarabine
  • NK allogeneic natural killer
  • NKG2D NK cells were isolated by immunoaffinity-based selection from leukapheresis samples from healthy donors, transduced with a viral vector encoding a NKG2D chimeric receptor, expanded in culture, and cryopreserved.
  • the NKG2D chimeric receptor contains a NKG2D-based extracellular domain (e.g., SEQ ID NO:26), a CD8alpha hinge (e.g., SEQ ID NO:2) and transmembrane region (e.g., SEQ ID NO:4), and an intracellular signaling domain having an OX40 co- stimulatory domain (e.g., SEQ ID NO:6) and a CD3 zeta (e.g., SEQ ID NO:8).
  • SEQ ID NO:26 NKG2D-based extracellular domain
  • a CD8alpha hinge e.g., SEQ ID NO:2
  • transmembrane region e.g., SEQ ID NO:4
  • an intracellular signaling domain having an OX40 co- stimulatory domain e.g., SEQ ID NO:6
  • CD3 zeta e.g., SEQ ID NO:8
  • Polynucleotides encoding the NKG2D chimeric receptor construct were engineered to bicistronically express mbIL15 (e.g., SEQ ID NO: 40); the sequences encoding the chimeric receptor and mbIL15 were separated by a sequence encoding a T2A ribosomal skip sequence (e.g., SEQ ID NO:10). Cryopreserved NKG2D NK cells were thawed prior to administration to subjects. [00249] Six patients with r/r AML were administered the NKG2D NK cells in a dosing regimen in which a dose of NKG2D chimeric receptor+ NK cells was administered three times in a 28-day dosing cycle.
  • mbIL15 e.g., SEQ ID NO: 40
  • T2A ribosomal skip sequence e.g., SEQ ID NO:10
  • FIG.1 depicts the non-limiting example of a treatment regimen. Additional treatment cycles were given to some patients to deepen or consolidate responses, with each cycle preceded by the lymphodepleting therapy. Pharmacokinetic and cytokine sampling were performed throughout the treatment cycle(s).
  • amino acid sequences that correspond to any of the nucleic acids disclosed herein (and/or included in the accompanying sequence listing), while accounting for degeneracy of the nucleic acid code.
  • those sequences that vary from those expressly disclosed herein (and/or included in the accompanying sequence listing), but have functional similarity or equivalency are also contemplated within the scope of the present disclosure.
  • the foregoing includes mutants, truncations, substitutions, codon optimization, or other types of modifications.
  • any of the sequences may be used, or a truncated or mutated form of any of the sequences disclosed herein (and/or included in the accompanying sequence listing) may be used and in any combination.

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Abstract

Plusieurs modes de réalisation des procédés et des compositions de l'invention concernent des cellules immunitaires qui sont modifiées pour exprimer des récepteurs chimériques et divers schémas posologiques pour administrer de telles cellules. Dans plusieurs modes de réalisation, les cellules immunitaires expriment un récepteur chimérique qui cible des ligands de NKG2D. Dans plusieurs modes de réalisation, le cancer est une leucémie, telle qu'une leucémie aiguë myéloïde (LMA).
PCT/US2024/055742 2023-11-14 2024-11-13 Méthodes et compositions pour le traitement de la leucémie Pending WO2025106552A1 (fr)

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WO2022241036A1 (fr) * 2021-05-13 2022-11-17 Nkarta, Inc. Schémas posologiques pour l'immunothérapie anticancéreuse
US20230011889A1 (en) * 2019-12-06 2023-01-12 University Health Network Genetically engineered double negative t cells as an adoptive cellular therapy

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230011889A1 (en) * 2019-12-06 2023-01-12 University Health Network Genetically engineered double negative t cells as an adoptive cellular therapy
WO2022241036A1 (fr) * 2021-05-13 2022-11-17 Nkarta, Inc. Schémas posologiques pour l'immunothérapie anticancéreuse

Non-Patent Citations (1)

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Title
PARISI SARAH, RUGGERI LOREDANA, DAN ELISA, RIZZI SIMONETTA, SINIGAGLIA BARBARA, OCADLIKOVA DARINA, BONTADINI ANDREA, GIUDICE VALER: "Long-Term Outcome After Adoptive Immunotherapy With Natural Killer Cells: Alloreactive NK Cell Dose Still Matters", FRONTIERS IN IMMUNOLOGY, FRONTIERS MEDIA, LAUSANNE, CH, vol. 12, Lausanne, CH , XP093316712, ISSN: 1664-3224, DOI: 10.3389/fimmu.2021.804988 *

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