Methods and compositions for treatment of leukemia

Abstract

Several embodiments of the methods and compositions disclosed herein relate to immune cells that are engineered to express chimeric receptors and various dosing regimens for administering such cells. In several embodiments, the immune cells express a chimeric receptor that targets ligands of NKG2D. In several embodiments, the cancer is a leukemia, such as acute myeloid leukemia (AML).

Description

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. In some embodiments, 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. In some embodiments, 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. For example, 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. Improved methods are needed, however, to increase the safety, efficacy, or both, of such methods. Provided are methods and uses that meet such needs. INCORPORATION BY REFERENCE OF MATERIAL IN SEQUENCE LISTING FILE

[0004] This application incorporates by reference the material contained in the Sequence Listing XML file being submitted concurrently herewith: File name: NKT.112WO_ST26.xml; created on November 11, 2024 and is 43, 246 bytes in size. SUMMARY

[0005] Provided herein are methods 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). In some embodiments, 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 afteradministration of the second dose of the genetically engineered NK cells, wherein: each dose of the genetically engineered NK cells comprises between about 1 x 109and about 2 x 109genetically 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).

[0006] Also provided herein is 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara-C), the overall survival rate (OS) of all subjects treated according to the method is at least about 25%. In some embodiments, 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.

[0007] Also provided herein is 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara-C), 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.

[0008] In some embodiments, 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 peripheralblood counts (CRh), or complete remission with incomplete hematologic recovery (CRi). 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. 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.

[0009] Also provided herein is 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara-C), 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). 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. 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.

[0010] Also provided herein is 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara-C), 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 isdetermined 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.

[0011] 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. 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.

[0012] Also provided herein is 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara-C), and 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). In some embodiments, 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.

[0013] Also provided herein is 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 109and about 2 x 109genetically engineeredNK cells, wherein: 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), and 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.

[0014] In some embodiments, 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. In some embodiments, 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.

[0015] In some embodiments, 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. In some embodiments, 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.

[0016] In some embodiments, 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. In some embodiments, 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. In some embodiments, each dose of the genetically engineered NK cells comprises about 1.5 x 109genetically engineered NK cells.

[0017] In some embodiments, the lymphodepleting therapy comprises administration of five doses of Flu. In some embodiments, each dose of Flu comprises between about 10 mg / m2and about 40 mg / m2. 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 / m2and about 4 g / m2. In some embodiments, 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-Care each given 3 days prior to initiation of the dosing cycle. In some embodiments, each dose of Flu comprises about 30 mg / m2. In some embodiments, each dose of Ara-C comprises about 2 g / m2.

[0018] In some embodiments, the AML is a relapsed / refractory AML (r / r AML).

[0019] Also provided herein is 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, the overall survival rate (OS) of all subjects treated according to the method is at least about 25%. In some embodiments, 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.

[0020] Also provided herein is 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

[0021] In some embodiments, 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 peripheralblood 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. In some embodiments, 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.

[0022] Also provided herein is 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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). 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. 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.

[0023] Also provided herein is 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

[0024] 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. 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.

[0025] Also provided herein is 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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). In some embodiments, 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.

[0026] Also provided herein is 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 cellgroup 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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.

[0027] In some embodiments, 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. In some embodiments, 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.

[0028] In some embodiments, 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. In some embodiments, 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.

[0029] In some embodiments, (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. In some embodiments, (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 at least about six months afteradministration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, (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 six months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0030] In some embodiments, (i) 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. In some embodiments, (i) 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. In some embodiments, (i) 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.

[0031] 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 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. 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.

[0032] In some embodiments, 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 cytokinerelease syndrome (CRS), immune cell associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GVHD).

[0033] In some embodiments, the dosing cycle is about 28 days.

[0034] In some embodiments, if the subject exhibits a clinical response following the dosing cycle, the method comprises administering an additional dosing cycle as a consolidation treatment. In some embodiments, the clinical response is a partial response (PR). In some embodiments, the clinical response is a complete response with incomplete hematologic recovery (CRi). In some embodiments, the clinical response is a complete response (CR). In some embodiments, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the method comprises administering an additional dosing cycle as retreatment.

[0035] In some embodiments, the method comprises administration of between one dosing cycle and five dosing cycles. In some embodiments, the subject is administered the lymphodepleting therapy prior to each dosing cycle.

[0036] In some embodiments, the subject has less than or equal to 5% peripheral blasts. In some embodiments, subject does not have evidence of extramedullary disease.

[0037] In some embodiments, 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. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a monoclonal antibody. In some embodiments, 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.

[0038] In some embodiments, 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. In some embodiments, 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.

[0040] In some embodiments, the chimeric receptor comprises an extracellular antigen-binding domain that binds a ligand of NKG2D, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the transmembrane domain comprises a CD8 transmembrane region. In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain and aCD3zeta. In some embodiments, the co-stimulatory domain comprises an OX40 domain. In some embodiments, the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 39.

[0041] In some embodiments, the genetically engineered NK cells express a membrane-bound interleukin 15 (mbIL15). In some embodiments, the mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0042] In some embodiments, the population of engineered NK cells are allogeneic to the subject.

[0043] In some embodiments, a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis. In some embodiments, each dose of the engineered NK cells is administered to the subject on an outpatient basis.

[0044] Also provided herein is use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara-C), 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. 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.

[0045] Also provided herein is use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara-C), 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 atime point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0046] Also provided herein is use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara-C), 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. 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.

[0047] Also provided herein is use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara-C), 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.

[0048] Also provided herein is use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara-C), and 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, the time point is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0049] Also provided herein is use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara-C), and 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.

[0050] Also provided herein is use of 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) 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 ofgenetically 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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. 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.

[0051] Also provided herein is use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

[0052] Also provided herein is use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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. 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.

[0053] Also provided herein is use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

[0054] Also provided herein is use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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 someembodiments, the time point that is about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0055] Also provided herein is use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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. BRIEF DESCRIPTION OF THE FIGURES

[0056] 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).

[0057] FIG.2 shows the pharmacokinetics of NKG2D NK cells in blood samples from patients treated according to the non-limiting dosing regimen shown in Figure 1 (n=5).

[0058] FIG. 3 shows clinical responses in patients treated according to the non-limiting dosing regimen. DETAILED DESCRIPTION

[0059] Some embodiments of the methods and compositions provided herein relate to immune cells engineered to express a chimeric receptor and compositions of the same for use in treating a leukemia, such as acute myeloid leukemia (AML). In several embodiments, the chimeric receptor binds a ligand of NKG2D. As used herein, the term “chimeric receptor” shall be given its ordinary meaning and shall also refer to (unless otherwise indicated), a chimeric antigen receptor (CAR).

[0060] AML is the most common acute leukemia in adults. It is primarily a disease of the elderly and has a poor prognosis. 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). Hypomethylating agents (HMAs) or low dose cytarabine (Ara-C) use alone results in responses of approximately 8% to 28% and median OS of five to 10 months in newly diagnosed, elderly, unfit patients (Kantarjian et al., J Clin Oncol (2012) 30(21):2670-77; Dombret et al., Blood (2015) 126(3):291-99). 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).

[0061] The most recent approvals have been for therapies targeting various mutationally selected AML subtypes, including gilteritinib (XOSPATA™) for FLT3 mutated disease, as well as ivosidenib (TIBSOVO™) and enasidenib (IDHIFA™), respectively, for isocitrate dehydrogenase (IDH) -1 or -2 mutated disease.

[0062] Even among those who respond to frontline therapy, most patients ultimately relapse. The median overall survival (OS) for relapsed and refractory AML patients is dismal, and there is no clear consensus on the management of relapsed / refractory AML (r / r AML). Prognosis at relapse or in refractory patients is poor; the median OS of r / r AML patients has been reported to be between four months (Zeicher et al., Blood (2015) 126(23):4955) and 5.3 months, and the five-year survival rate for r / r AML patients has been reported as 12.6% (Brandwein et al., Am J Blood Res (2020) 10(4):124–33). There is therefore an urgent need for new therapies, especially those with novel mechanisms of action and differentiated safety profiles, to provide additional treatment options for patients with r / r AML who have limited treatment options and poor prognoses.

[0063] As described herein, it was surprisingly observed that treatment of r / r AML patients with NKG2D CAR-expressing NK cells, preceded by a lymphodepletion regimen comprised of fludarabine and cytarabine (Flu / Ara-C), was both safe and efficacious. In particular, even among r / r AML patients having high-risk features (e.g., TP53 mutation and / or relapse within 6 months of HCT) and previous venetoclax exposure, 67% of patients achieved a best response of complete response (CR) or complete response with incomplete hematologic recovery (CRi). No patients experienced cytokine release syndrome (CRS), immune cell associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GVHD) of any grade, indicating that the exemplary treatment regimen can achieve significant clinical responses while maintaining a desirable safety profile. The methods described herein may thus provide a much-needed treatment option for r / r AML patients who otherwise have limited options.

[0064] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0065] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Engineered Cells

[0066] Provided herein are engineered immune cells and populations thereof. For example, an immune cell, such as an NK cell, may be engineered to express a chimeric receptor such as a NKG2D- based chimeric receptor, including as encoded by a nucleic acid as described herein.

[0067] Traditional anti-cancer therapies relied on a surgical approach, radiation therapy, chemotherapy, or combinations of these methods. As research led to a greater understanding of some of the mechanisms of certain cancers, this knowledge was leveraged to develop targeted cancer therapies. 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. More recently, genetic engineering has enabled approaches to be developed that harness certain aspects of the immune system to fight cancers. In some cases, 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.

[0068] To facilitate cancer immunotherapies, there are also provided for herein 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. For example, 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). Also provided are engineered immune cells (e.g., NK cells) expressing such chimeric receptors. There are also provided herein, in several embodiments, polynucleotides and vectors that encode such NKG2D chimeric receptors. 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] In several embodiments, 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. For example, a cell of the immune system may be engineered to include NKG2D- based chimeric receptor as described herein. In several embodiments, white blood cells or leukocytes, are used, since their native function is to defend the body against growth of abnormal cells and infectious disease. There are a variety of types of white bloods cells that serve specific roles in the human immune system, and are therefore a preferred starting point for the engineering of cells disclosed herein. Whiteblood 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. In several embodiments, the cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, 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. Thus, in several embodiments, the immune cells engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15. Monocytes for Immunotherapy

[0070] 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. Several embodiments of the methods and compositions disclosed herein relate to 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). In some embodiments, the monocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, 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. Thus, in some embodiments, the monocytes engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.

[0071] In several embodiments, 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. In several embodiments, the monocytes are derived from the subject. In some embodiments, the monocytes are allogeneic to the subject. Lymphocytes for Immunotherapy

[0072] Lymphocytes, 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). Several embodiments of the methods and compositions disclosed herein relate tolymphocytes 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). In some embodiments, the lymphocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, 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. Thus, in some embodiments, the lymphocytes engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.

[0073] In several embodiments, 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. In several embodiments, the lymphocytes are derived from the subject. In some embodiments, the lymphocytes are allogeneic to the subject. T Cells for Immunotherapy

[0074] 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. In some embodiments, CD4+ T cells are engineered. In some embodiments, CD8+ T cells are engineered. In some embodiments, regulatory T cells are engineered. In some embodiments, gamma delta T cells are engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. For example, in some embodiments, CD4+ and CD8+ T cells are engineered. In some embodiments, there is no specific selection of a type of T cells to be engineered to express the cytotoxic receptor complexes disclosed herein. In several embodiments, 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. In several embodiments, there is provided a method of treating or preventing cancer (e.g., AML), comprising administering T cells expressing the NKG2D-based chimeric receptor.

[0075] Several embodiments of the methods and compositions disclosed herein relate to 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). In some embodiments, the T cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, 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. Thus, insome embodiments, the T cells engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.

[0076] In several embodiments, the T cells are derived from a donor. In some embodiments, the donor does not have a cancer. In several embodiments, the T cells are autologous to the subject. In several embodiments, the T cells are derived from the subject. In some embodiments, 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.

[0078] Several embodiments of the methods and compositions disclosed herein relate to 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). In some embodiments, the NK cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, 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. Thus, in some embodiments, the NK cells engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.

[0079] In several embodiments, 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. In some embodiments, 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. Some embodiments of NK-92 cells described herein related to NK-92 cell engineered to silence certain additional inhibitory receptors, for example, SMAD3, allowing for upregulationof interferon- (IFN ), granzyme B, and / or perforin production. Additional information relating to the NK-92cell line is disclosed in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044 and incorporated in their entireties herein by reference. NK-92 cells are used, in several embodiments, in combination with one or more of the other cell types disclosed herein. For example, in one embodiment, NK-92 cells are used in combination with NK cells as disclosed herein. In an additional embodiment, NK- 92 cells are used in combination with T cells as disclosed herein.Hematopoietic Stem Cells for Cancer Immunotherapy

[0082] In some embodiments, the immune cells comprise hematopoietic stem cells (HSCs). Several embodiments of the methods and compositions disclosed herein relate to HSCs 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). In some embodiments, the HSCs are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, 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. Thus, in some embodiments, the HSCs engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain.

[0083] In several embodiments, the HSCs are derived from a donor. In some embodiments, the donor does not have a cancer. In several embodiments, the HSCs are autologous to the subject. In several embodiments, the HSCs are derived from the subject. In some embodiments, the HSCs are allogeneic to the subject. Induced Pluripotent Stem Cells

[0084] In some embodiments, 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. In several embodiments, the iPSCs are used to generate iPSC-derived NK or T cells.

[0085] Several embodiments of the methods and compositions disclosed herein relate to 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). In some embodiments, the iPSCs are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, 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. Thus, in some embodiments, the iPSCs engineered to express the NKG2D-based chimeric receptor are engineered to bicistronically express the mbIL15 domain. In some embodiments, the engineered iPSCs are differentiated into NK cells.

[0086] In several embodiments, the iPSCs are derived from a donor. In some embodiments, the donor does not have a cancer. In several embodiments, the iPSCs are autologous to the subject. In severalembodiments, 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. Thus, in some embodiments, the chimeric receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Also provided are 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.

[0089] In some embodiments, 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. In some embodiments, the tumor-binding domain contains more than one antigen-binding domain.

[0090] There are provided, in several embodiments, antigen-binding domain. As used herein, the term “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. In some embodiments, the antigen is a cancer antigen or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, 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.

[0091] In several embodiments, an antigen-binding domain comprises an antibody, antibody fragment (e.g., an antigen-binding fragment of an antibody), antibody derivative, and / or antibody analog. Further specific examples of antigen-binding domains include, but are not limited to, a single-chain variablefragment (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. These molecules can be derived from any mammalian source, such as human, mouse, rat, rabbit, or pig, dog, or camelid. 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. In addition, peptide antibody mimetics (“PAMs”) can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold.

[0092] In some embodiments, the antigen-binding domain comprises one or more antibody fragments incorporated into a single polypeptide chain or into multiple polypeptide chains. For instance, 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. IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0093] In some embodiments, the antigen-binding domain has the structure of an immunoglobulin. As used herein, the term “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). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0094] Within light and heavy chains, the variable (V) and constant regions (C) are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. The variable regions of each light / heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

[0095] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0096] Human light chains are classified as kappa and lambda light chains. An antibody “light chain”, refers to the smaller of the two types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations. Kappa (K) and lambda ( ) light chains refer to the two major antibodylight 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).

[0097] Heavy chains are classified as mu ( ), delta ( ), gamma ( ), alpha ( ), and epsilon ( ), anddefine 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. A heavy chain may include a polypeptide comprising, from amino terminus to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4).

[0098] The IgG-class is further divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4. The IgA-class is further divided into subclasses, namely IgA1 and IgA2. 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.

[0099] In some embodiments, the antigen-binding domain is or comprises an antibody. The term “antibody”, as used herein, 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. Unless otherwise indicated, 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. For example, 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 sameor similar binding and / or function as the antibodies comprising two full length heavy chains and two full length light chains. Examples of antibodies 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. As used herein, the term “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. As used herein, the term “monoclonal antibody” (“mAb”) shall be given its ordinary meaning, and shall also refer to one or more of a population of antibodies having identical sequences. Monoclonal antibodies bind to the antigen at a particular epitope on the antigen.

[0100] In some embodiments, the antigen-binding domain is or comprises a fragment or antigen- binding fragment of an antibody. The term “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. Examples of 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.

[0101] 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. In some embodiments, these antibody fragments can be incorporated into single domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. In some embodiments, the antibodies comprise at least one CDR as described herein.

[0102] There is also provided for herein, in several embodiments, single-chain variable fragments. As used herein, the term “single-chain variable fragment” (“scFv”) 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 syntheticsequence 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). For the sake of clarity, unless otherwise indicated as such, 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. According to several embodiments, if the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, 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.

[0103] In several embodiments, the antigen-binding domain comprises one or more CDRs. As used herein, the term “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. There are three heavy chain variable region CDRs (CDR-H1, CDR-H2 and CDR-H3) and three light chain variable region CDRs (CDR-L1, CDR-L2 and CDR-L3). 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. From N-terminus to C-terminus, 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. For heavy chain variable regions, 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. For light chain variable regions, the order is typically: 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. Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody may be identified using this system. 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. For any given embodiment containing more than one CDR, 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 asappropriate. One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen-binding protein.

[0104] In some embodiments, the antigen-binding domains provided herein comprise one or more CDR(s) as part of a larger polypeptide chain. In some embodiments, 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. 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. Depending on the embodiment, 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.

[0105] Depending on the embodiment, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. In some such embodiments, 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.

[0106] There is also provided, in some embodiments, antigen-binding domains with more than one binding site. In several embodiments, the binding sites are identical to one another while in some embodiments the binding sites are different from one another. For example, an antibody typically has two identical binding sites, while a “bispecific” or “bifunctional” antibody has two different binding sites. The two binding sites of a bispecific antigen-binding domain or antibody will bind to two different epitopes, which can reside on the same or different protein targets. In several embodiments, this is particularly advantageous, as a bispecific chimeric receptor (e.g., CAR) can impart to an engineered cell the ability to target multiple tumor markers. For example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others, and an additional tumor marker, such as CD70, CD123, CD19, Her2, mesothelin, Claudin 6, BCMA, EGFR, or any other marker disclosed herein or appreciated in the art as a tumor specific antigen or tumor associated antigen can be bound by a bispecific antibody.

[0107] Several embodiments of the 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.

[0108] Natural killer Group 2 member D (NKG2D) is an NK cell activating receptor that recognizes a variety of ligands expressed on cells. The surface expression of various NKG2D ligands is generally lowin 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.

[0109] In several embodiments, 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. When the balance between inhibitory and activating receptor activation is in favor of activating receptors, NK cell activation occurs and target (e.g., tumor) cells are lysed.

[0110] In several embodiments, 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. In one embodiment, full length NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the antigen-binding domain is encoded by SEQ ID NO: 27. In one embodiment, full length NKG2D comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 42.

[0111] In several embodiments, 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. In some embodiments, the fragment of NKG2D comprises all or a fragment of the extracellular domain of NKG2D. In one embodiment, the fragment of NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the antigen- binding domain is encoded by SEQ ID NO: 25. In one embodiment, the fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 26. In several embodiments, the full length NKG2D, or functional fragment thereof is human NKG2D. In several embodiments, full length NKG2D is human full length NKG2D. In several embodiments, 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.

[0112] In several embodiments, 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. In one embodiment, the fragment of NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 25. In several embodiments, 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. In several embodiments, 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. In several embodiments, the fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26.

[0113] In several embodiments, the NKG2D fragment is provided as a dimer, trimer, or other concatemeric format, such embodiments providing enhanced ligand-binding activity. In several embodiments, the sequence encoding the NKG2D fragment is fully or partially codon-optimized. In one embodiment, a polynucleotide sequence encoding a codon optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28.

[0114] Advantageously, according to several embodiments, 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. Thus, in several embodiments, the chimeric receptors provided herein do not comprise DAP10. In several embodiments, immune cells, such as 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. Such cells, in several embodiments, also express (e.g., bicistronically express) mbIL15.

[0115] In several embodiments, the chimeric receptors are configured to dimerize. Dimerization may comprise homodimers or heterodimers, depending on the embodiment. In several embodiments, 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. In several embodiments, the chimeric receptors employ internal dimers, or repeats of one or more component subunits. For example, in several embodiments, 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

[0116] Some embodiments of the compositions and methods described herein relate to chimeric receptors that comprise a transmembrane domain. 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. In some embodiments, the antigen-binding domain is linked to an intracellular signaling domain by a transmembrane domain. Thus, in some embodiments, the antigen-binding domain (e.g., fragment of NKG2D) is linked to a transmembrane domain and intracellular signaling domain.

[0117] In some embodiments, 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. Where the source is natural, 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. Alternatively, the transmembrane domain in some embodiments is synthetic.

[0118] In several embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells. In several embodiments, 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.

[0119] 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. In some embodiments, 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. In several embodiments, the CD8a hinge is encoded by the nucleic acid sequence of SEQ ID NO:1. In several embodiments, the CD8a hinge is truncated or modified. In some embodiments, 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. In several embodiments, the hinge of CD8a comprises the amino acid sequence of SEQ ID NO:2.

[0120] In several embodiments, the transmembrane domain comprises a CD8a transmembrane region. In several embodiments, the CD8a transmembrane region is truncated or modified. In some embodiments, 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. In several embodiments, the CD8a transmembrane region is encoded by a nucleic acid sequence of SEQ ID NO:3. In several embodiments, the CD8a transmembrane region is truncated or modified. In some embodiments, 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.

[0121] 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. In several embodiments, the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO:13. In some embodiments, 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. In several embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:14. Signaling Domains

[0122] The chimeric receptor, e.g., the CAR, generally includes an intracellular signaling domain comprising intracellular signaling components. In some embodiments, 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. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, upon ligation of the chimeric receptor, the cytoplasmic domainor 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. For example, in some contexts, 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. In some embodiments, 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. In some embodiments, 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.

[0123] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the 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). In some aspects, the receptor includes one or both of such signaling components.

[0124] In some aspects, 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. Examples of 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. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3zeta.

[0125] For example, 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). In several embodiments, the signaling domain comprises the CD3 zeta subunit. In several embodiments, the CD3 zeta can be truncated or modified. In some embodiments, 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. In several embodiments, the CD3zeta comprises the amino acid sequence of SEQ ID NO:8.

[0126] In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain, such as an intracellular signaling region of CD28, 4-1BB, OX40, DAP10, ICOS, or any combinationthereof. In some aspects, the same receptor includes both a CD3zeta and a co-stimulatory domain. Thus, in some embodiments, the intracellular signaling domain of the chimeric receptor comprises a CD3zeta and a co-stimulatory domain.

[0127] In several embodiments, the intracellular signaling domain comprises an OX40 co- stimulatory domain. In several embodiments, 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. In several embodiments, the OX40 co- stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO:5. In several embodiments, 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. In several embodiments, the OX40 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:6. In several embodiments, 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. For example, combinations of OX40 and CD3 zeta are used in some embodiments. In some embodiments, the intracellular signaling domain comprises an OX40 co-stimulatory domain linked to CD3 zeta. In some embodiments, the chimeric receptor (e.g., CAR) comprises the amino acid sequence set forth in SEQ ID NO:39.

[0128] By way of further example, combinations of CD28, OX40, 4-1BB and / or CD3 zeta are used in some embodiments.

[0129] In several embodiments, the intracellular signaling domain comprises a 4-1BB co- stimulatory domain. In several embodiments, 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. In several embodiments, the 4-1BB co- stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO:29. In several embodiments, 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. In several embodiments, 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. By way of further example, combinations of CD28, OX40, 4-1BB and / or CD3zeta are used in some embodiments.

[0130] In several embodiments, the intracellular signaling domain comprises a CD28 co- stimulatory domain. I In several embodiments, 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. In several embodiments, the 4-1BB co-stimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO:31. n several embodiments, 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. In several embodiments, 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. In some embodiments, the intracellular signaling domain comprises a CD28 co-stimulatory domain linked to CD3 zeta. By way of further example, combinations of CD28, OX40, 4-1BB and / or CD3zeta are used in some embodiments.

[0131] In any of the provided embodiments, the nucleic acid encoding the chimeric receptor, or a portion thereof, is codon-optimized. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some aspects, 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. Stimulatory Molecules

[0132] In several embodiments, 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. In some embodiments, the immune cells for therapy are engineered to express such molecules as a secreted form. In additional embodiments, such stimulatory molecules are engineered to be in membrane bound found, acting as autocrine stimulatory molecules and / or paracrine stimulators to neighboring cells.

[0133] In several embodiments, the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15). In some embodiments, the IL15 is expressed from a separate cassette on the construct comprising any one of the CARs disclosed herein. In some embodiments, the IL15 is expressed from the same cassette as any one of the CARs disclosed herein. In some embodiments, 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. In some embodiments, the nucleic acid sequence encoding the chimeric receptor and the nucleic acid sequence encoding IL15 are separated by a T2A sequence.

[0134] In some embodiments, the IL15 is membrane-bound IL15 (mbIL15). In some embodiments, the mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain. In some embodiments, 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. In some embodiments, the native IL15 sequence is encoded by SEQ ID NO: 11. In some embodiments, 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.

[0135] 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. In some embodiments, the leader sequence is a CD8 leader sequence. In some embodiments, the hinge sequence is a CD8 (e.g., CD8 alpha) hinge sequence. In some embodiments, the CD8 alpha comprises the sequence of SEQ ID NO: 2. In some embodiments, the transmembrane domain comprises a CD8 (e.g., CD8 alpha) hinge and a CD8 (e.g., CD8 alpha) transmembrane region. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 12.

[0136] In some embodiments, the mbIL15 comprises native IL15 fused to a CD8 (e.g., CD8 alpha) transmembrane domain. In some embodiments, 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.

[0137] In some embodiments, 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. In some embodiments, 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. As a result, depending on the embodiment, the chimeric receptor and the IL15 can be delivered to an immune cell in a single vector or by multiple vectors. Thus, 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. In several embodiments, a T2A cleavage site is used. In several embodiments, a T2A cleavage site is encoded by the nucleic acid sequence of SEQ ID NO: 9. In several embodiments, T2A cleavage site can be truncated or modified, such that it 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 with the sequence of SEQ ID NO: 9. In several embodiments, the T2A cleavage site comprises the amino acid sequence of SEQ ID NO: 10. In several embodiments, the T2A cleavage site is truncated or modified. In someembodiments, 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.

[0138] In several embodiments, NK cells are engineered to express membrane-bound interleukin 15 (mbIL15). In such embodiments, 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. In several embodiments, the mbIL15 is encoded by the same polynucleotide as the chimeric receptor. In some embodiments, 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. In several embodiments, the nucleic acid encoding mbIL15 can be truncated or modified. In some embodiments, 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. In several embodiments, the mbIL15 comprises the amino acid sequence of SEQ ID NO: 40. In several embodiments, 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. Membrane-bound IL15 sequences are explored in PCT publications WO 2018 / 183385 and WO 2020 / 056045, each of which is hereby expressly incorporated by reference in its entirety and pertaining to membrane-bound IL15 sequences.

[0139] In several embodiments, the domains or subdomains of the chimeric receptor are separated by a linker such as, a GS3 linker (SEQ ID NOs: 15 and 16, nucleotide and protein, respectively) is used (or a 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. In several embodiments, other linkers comprise the peptide sequence of one of SEQ ID NOs: 18, 20, 22, 24, or 43. In several embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 16. In several embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 18. In several embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 20. In several embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 22. In several embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 24. In several embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 43. This provides the potential to separate the various component parts of the receptor complex along the polynucleotide, which can enhance expression, stability, and / or functionality of the receptor complex. Chimeric Receptor Constructs

[0140] 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.

[0141] In several embodiments, there is provided 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. In some embodiments, the transmembrane domain comprises (e.g., from N- to C-terminus) a CD8 hinge and a CD8 transmembrane region. In some embodiments, the intracellular signaling domain comprises (e.g., from N- to C-terminus) an OX40 co-stimulatory domain and CD3 zeta. In several embodiments, there is provided 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. In several embodiments, 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. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 39.

[0142] In several embodiments, the polynucleotide encoding the chimeric receptor also encodes a mbIL15 domain as described herein. In some embodiments, the chimeric receptor and the mbIL15 domain are expressed bicistronically. In several embodiments, the polynucleotide further encodes a ribosomal skip element such as a 2A cleavage site, and an mbIL-15 domain as described herein. In some embodiments, 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. In several embodiments, 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.

[0143] In several embodiments, 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. In several embodiments, 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. It shall be appreciated that certain sequence variability, extensions, and / or truncations of the disclosed sequences may result when combining sequences, as a result of, for example, ease or efficiency in cloning (e.g., for creation of a restriction site). In several embodiments, 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 portion excluding the mbIL15 sequence and / or self-cleaving peptide sequence)

[0144] In several embodiments, there is provided 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. In several embodiments, there is provided 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. In several embodiments, this receptor complex is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 33. In yet another embodiment, this chimeric receptor comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, 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. In several embodiments, while 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. Additionally, in several embodiments, this construct can optionally be co-expressed with mbIL15, such as the mbIL15 encoded by SEQ ID NO: 35 or 37. In several embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0145] 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

[0146] Also provided are compositions including the chimeric receptors and engineered cells expressing the same, including pharmaceutical compositions and formulations. Also provided arecompositions comprising engineered NK cells that express a NKG2D-based chimeric receptor provided herein, such as CARs, including pharmaceutical compositions and formulations.

[0147] Provided are 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. In some embodiments, the composition includes at least one additional therapeutic agent.

[0148] The term “pharmaceutical formulation” 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.

[0149] 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.

[0150] In some aspects, 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. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, 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, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0151] In some aspects, 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. In some aspects, 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. Methods for preparing administrable pharmaceutical compositions are known.

[0152] 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. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, 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. In some embodiments, 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.

[0153] 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. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0154] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the agent or cell population is administered to the subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.

[0155] In some embodiments, 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. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, 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.

[0156] Sterile injectable solutions can be prepared by incorporating the agent or cell into a solvent, such as an admixture with a suitable carrier, diluent, or excipient (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. In some embodiments, the dose ofengineered cells administered is in a cryopreserved composition. In some aspects, the composition is administered after thawing the cryopreserved composition.

[0157] Also provided are 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. For example, in some embodiments, 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. In some embodiments, 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

[0158] Provided herein 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. Among such methods, such as 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. Also provided are methods of combination therapy and / or treatment.

[0159] Also provided are methods of administering and uses, such as therapeutic uses of the chimeric receptors, engineered cells expressing the chimeric receptors, plurality of engineered cells expressing the chimeric receptors, and / or compositions comprising the same. 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. In some embodiments, the chimeric receptor, cell, and / or composition is / are administered in an effective amount to effect treatment of the disease or disorder.

[0160] Provided herein are uses of the chimeric receptors and cells (e.g., engineered cells) in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, 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. Also provided herein are of use of any of the compositions, such as pharmaceuticalcompositions provided herein, for the treatment of a disease or disorder associated with NKG2D ligand expression, such as use in a treatment regimen.

[0161] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) 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.

[0162] As used herein, “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. In some embodiments, 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. For example, a late-stage cancer, such as development of metastasis, may be delayed.

[0163] “Preventing,” as used herein, 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.

[0164] As used herein, 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. For example, 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.

[0165] 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.

[0166] A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, cells, or composition 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. In some embodiments, the provided methods involve administering the molecules, antibodies, cells, and / or compositions at effective amounts, e.g., therapeutically effective amounts.

[0167] 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.

[0168] Methods for administration of cells for cell therapy are known and may be used in connection with the provided methods and compositions.

[0169] 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. In some embodiments, 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.

[0170] Disclosed herein are methods of treating a cancer (e.g., r / r / AML) in a subject. In some embodiments, 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. In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, or a combination thereof. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a monoclonal antibody. In some embodiments, the therapeutic agent is a NK cell engager. In some embodiments, the therapeutic agent increases expression of a NKG2D ligand in a subject.

[0171] In some embodiments, the therapeutic agent is administered prior to, concurrent with, and / or after administration of genetically engineered NK cells. In some embodiments, the therapeutic agent is administered prior to administration of genetically engineered NK cells. In some embodiments, the therapeutic agent is administered prior to treatment with a lymphodepleting therapy. In some embodiments, the therapeutic agent is administered after treatment with a lymphodepleting therapy and prior to administration of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered concurrently with administration of genetically engineered NK cells. In some embodiments, the therapeutic agent is administered after administration of genetically engineered NK cells.

[0172] In certain embodiments, 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. V. Subjects

[0173] Provided herein are methods for treating a subject having a cancer. As used herein, a “subject” or an “individual” is a mammal. In some embodiments, 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. In some embodiments, the subject is human.

[0174] In some embodiments, the cancer is a relapsed or refractory (r / r) cancer. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the subject has a r / r AML. In some embodiments, the subject has a r / r AML as determined by standard European LeukemiaNet (ELN) criteria (Döhner et al., Blood (2022) 140(12):1345-77). In some embodiments, the subject has minimum residual disease. In some embodiments, the subject has r / r AML with MRD. In some embodiments, the subject is in complete response (CR) with MRD. In some embodiments, the subject has r / r MDS as determined by standard International Working Group (IWG) criteria (Cheson et al., Blood (2006) 108(2):419-25). In some embodiments, 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).

[0175] 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. In some embodiments, if the subject has a FLT3-mutated cancer or a IHD 1 / 2-mutated cancer, the subject has received a prior line of therapy that targets the mutation.

[0176] In some embodiments, the subject has received a prior line of therapy for the AML. In some embodiments, the subject has received one, two, three, or four prior lines of therapy for the AML. In some embodiments, the subject has received one prior line of therapy for the AML. In some embodiments, the subject has received two prior lines of therapy for the AML. In some embodiments, the subject has received three prior lines of therapy for the AML. In some embodiments, the subject has received no more than three prior lines of therapy for the AML. In some embodiments, the subject has received four prior lines of therapy for the AML. In some embodiments, the prior line of therapy comprises venetoclax. In some embodiments, the subject has relapsed following HCT. In some embodiments, the subject has relapsed following HCT within about 6 months of administration of the genetically engineered NK cells to the subject. Thus, in some embodiments, the methods provided herein achieve particular clinical responses in subjects including those who relapsed following HCT.

[0177] In some embodiments, the subject has a white blood cell count of 25 × 109 / liter. In some embodiments, the subject has a platelet count of 30,000 / microliter. In

[0178] In some embodiments, the subject has marrow limited disease (also known as marrow localized disease). In some embodiments, 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.

[0179] 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.

[0180] 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. In some embodiments, the subject has an ECOG performance status of 1. In some embodiments, the subject has an ECOG performance status of 2.

[0181] 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. In some embodiments, the subject has not undergone a hematopoietic cell transplantation within 16 weeks prior to treatment. VI. Administration and Dosing

[0182] Further provided herein are methods of treating a subject having a cancer (e.g., r / r AML), comprising administering to the subject a composition comprising immune cells (e.g., natural killer cells) engineered to express a chimeric receptor as disclosed herein. Uses of such engineered immune cells for treating a cancer (e.g., r / r / AML) are also provided.

[0183] In certain embodiments, 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. For example, in some embodiments, 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. In some embodiments, 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 geneticallyengineered NK cells to the respective subject. In some embodiments, 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.

[0184] In several embodiments, 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. In some embodiments, 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).

[0185] 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 105cells per kg to about 1012cells per kg (e.g., 105-107, 107- 1010, 1010-1012and overlapping ranges therein). In one embodiment, a dose escalation regimen is used. In several embodiments, a range of immune cells such as NK cells is administered, for example between about 1 x 106cells / kg to about 1 x 108cells / kg.

[0186] In some embodiments, each dose of engineered NK cells comprises about 1 x 109genetically engineered NK cells. In some embodiments, each dose of engineered NK cells comprises about 1.5 x 109genetically engineered NK cells.

[0187] In several embodiments, 1 × 108NK cells are administered (2 × 106 / kg for a subject under 50 kg) three times over the cycle. In several embodiments, 1.5 × 108genetically engineered NK cells are administered (3 × 106 / kg for a subject under 50kg) three times over the cycle. In several embodiments, 3 × 108genetically engineered NK cells are administered three times over the cycle. In several embodiments, 4.5 × 108genetically engineered NK cells are administered three times over the cycle. In several embodiments, 1 × 109genetically engineered NK cells are administered three times over the cycle. In several embodiments, 1.5 × 109genetically engineered NK cells are administered three times over the cycle. In several embodiments, 3 × 109genetically engineered NK cells are administered three times over the cycle. In several embodiments, 1.5 × 1010genetically engineered NK cells are administered three times over the cycle. In several embodiments, at least 4.5 × 109genetically engineered NK cells are administered over the cycle. In some embodiments, 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.

[0188] In several embodiments, 1 × 108NK cells are administered (2 × 106 / kg for a subject under 50 kg) three times over a 28-day cycle. In several embodiments, 1.5 × 108genetically engineered NK cells are administered (3 × 106 / kg for a subject under 50kg) three times over a 28-day cycle. In severalembodiments, 3 × 108genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 4.5 × 108genetically engineered NK cells are administered three times over a 28- day cycle. In several embodiments, 1 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 3 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 × 1010genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, at least 4.5 × 109genetically engineered NK cells are administered over a 28-day cycle.

[0189] In several embodiments, 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. In some embodiments, 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.

[0190] 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. In some embodiments, 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. In some embodiments, 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 threedoses of the genetically engineered NK cells are administered within about 21 days of the conclusion of administration of the lymphodepleting therapy.

[0191] 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. In several embodiments, 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. In several embodiments, 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.

[0192] In several embodiments, 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. In several embodiments, 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. In several embodiments, 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. In several embodiments, 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. In some embodiments, 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 between days 5-10 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered between days 10-20 of the dosing cycle, wherein each dose is administered between about 5-10 days after the preceding dose.

[0193] 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, and 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 some embodiments, 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, and the third dose of the genetically engineered NK cells is administered on day 14 of the dosing cycle.

[0194] In several embodiments, each dose comprises between about 1 x 109genetically engineered NK cells and about 5 x 109genetically engineered NK cells. In several embodiments, each dose comprises between about 1.5 x 109genetically engineered NK cells and about 5 x 109genetically engineered NK cells. In several embodiments, each dose comprises between about 2 x 109genetically engineered NK cells and about 5 x 109genetically engineered NK cells. In several embodiments, each dose comprises between about 2 x 109genetically engineered NK cells and about 3 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 1 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 1.5 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 2 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 2.5 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 3 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 3.5 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 4 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 4.5 x 109genetically engineered NK cells. In several embodiments, each dose comprises about 5 x 109genetically engineered NK cells.

[0195] In several embodiments, 1 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 2 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, at least 6 × 109genetically engineered NK cells are administered over the cycle. In several embodiments, 2.5 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, at least 7.5 × 109genetically engineered NK cells are administered over the cycle. In several embodiments, 2.5 × 109genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, at least 9 × 109genetically engineered NK cells are administered over the cycle.

[0196] In several embodiments, 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 109NK cells. In several embodiments, a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-dayperiod), 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 109NK cells.

[0197] In several embodiments, 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. In several embodiments, 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.

[0198] 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.

[0199] In several embodiments, the administration of engineered NK cells is preceded by one or more preparatory treatments. In several embodiments, the administration of engineered NK cells is preceded by a lymphodepleting therapy (also referred to as “lymphodepletion”). In several embodiments, each dosing cycle is preceded by lymphodepletion. In several embodiments, a combination of chemotherapeutic agents is used for lymphodepletion. In several embodiments, a single chemotherapeutic agent is used for lymphodepletion. In several embodiments, wherein a combination of chemotherapeutic agents is used, agents with different mechanisms of actions are optionally used. In several embodiments, different classes of agents are optionally used. In several embodiments, an antimetabolic agent is used. In several embodiments, the antimetabolic agent inhibits and / or prevents cell replication. In several embodiments, the antimetabolic agent is an altered nucleotide that disrupts DNA replication, making it effective in targeting rapidly dividing tumor cells.

[0200] In some embodiments, a lymphodepleting therapy is administered to the subject prior to administration of the first dose of genetically engineered NK cells. In some embodiments, 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. In some embodiments, 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. In several embodiments, 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.

[0201] In several embodiments, the lymphodepleting therapy comprises cytosine arabinoside (also known as cytarabine; Ara-C). In several embodiments, a dose of between about 0.2 – about 10 g / m2Ara-C is administered, including doses of about 0.2 g / m2, about 0.5 g / m2, about 1.0 g / m2, about 1.5 g / m2, about 2.0 g / m2, about 2.5 g / m2, about 3.0 g / m2, about 3.5 g / m2, about 4.0 g / m2, about 5.0 g / m2, about 6.0 g / m2, about 7.0 g / m2, about 8.0 g / m2, about 9.0 g / m2, about 10.0 about 1.5 g / m2, or any dose between those listed. In several embodiments, a dose of about 2 g / m2of Ara-C is administered. In several embodiments, 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. In several embodiments, the dose of Ara-C is given daily for about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily. In several embodiments, an additional agent is used in combination with the Ara-C. In several embodiments, the additional agent is also an antimetabolite. In several embodiments, the additional agent inhibits one or more of DNA polymerase alpha, ribonucleotide reductase and / or DNA primase, thus inhibiting DNA synthesis.

[0202] In several embodiments, the lymphodepleting therapy comprises fludarabine (Flu). In several embodiments, a dose of between about 5.0 mg / m2– about 200 mg / m2fludarabine is administered, including doses of about 5.0 mg / m2, about 10.0 mg / m2, about 15.0 mg / m2, about 20.0 mg / m2, about 25.0 mg / m2, about 30.0 mg / m2, about 35.0 mg / m2, about 40.0 mg / m2, about 45.0 mg / m2, about 50.0 mg / m2, about 60.0 mg / m2, about 70.0 mg / m2, about 80.0 mg / m2, about 90.0 mg / m2, about 100.0 mg / m2, about 125.0 mg / m2, about 150.0 mg / m2, about 175.0 mg / m2, about 200.0 mg / m2, or any dose between those listed. In several embodiments, a dose of about 30 mg / m2of fludarabine is administered. In several embodiments, 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. In several embodiments, the dose of fludarabine is given daily for about 3 days. In several embodiments, the dose of fludarabine is given daily for about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[0203] In several embodiments, a combination of fludarabine and Ara-C is used with a daily dose of fludarabine of between about 20 mg / m2and 40 mg / m2and a daily dose of Ara-C of between about 1.5 g / m2and 2.5 g / m2. In several embodiments, a combination of fludarabine and Ara-C is used with a daily dose of fludarabine of about 30 mg / m2and a daily dose of Ara-C of about 2 g / m2. In several embodiments, the combination of fludarabine and Ara-C (or any other agent or agents as disclosed herein) is administered for about 5 days, with the administration started about 7 days prior to the first administration of the engineered NK cells (for example day -7 to day -3). In several embodiments, lymphodepletion is started at day -5 prior to administration of engineered NK cells. In several embodiments, this combination advantageously functions not only as a lymphodepletion regimen, but as an anti-cancer agent as well (in addition to the engineered NK cells). In several embodiments, the lymphodepletion regimen works synergistically with the engineered NK cells to provide effect reduction and / or elimination of cancerous cells.

[0204] In several embodiments, the lymphodepleting therapy comprises cyclophosphamide (Cy). In several embodiments, a dose of between about 100 mg / m2– about 100 mg / m2fludarabine isadministered, including doses of about 100.0 mg / m2, about 200 mg / m2, about 300 mg / m2, about 400 mg / m2, about 500 mg / m2, about 600 mg / m2, about 700 mg / m2, about 800 mg / m2, about 900 mg / m2, about 1000 mg / m2, or any dose between those listed. In several embodiments, a dose of about 300 mg / m2of cyclophosphamide is administered. In several embodiments, a dose of about 500 mg / m2of cyclophosphamide is administered. In several embodiments, 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. In several embodiments, the dose of cyclophosphamide is given daily for about 3 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[0205] In several embodiments, a combination of fludarabine and cyclophosphamide is used. In several embodiments, cyclophosphamide (300 mg / m2) and fludarabine (30mg / m2) are administered daily for 3 days. In several embodiments, cyclophosphamide (500 mg / m2) and fludarabine (30mg / m2) are administered daily for 3 days. In some embodiments, fludarabine and cyclophosphamide are each administered daily 5 days, 4 days, and 3 days prior to administration of the engineered NK cells.

[0206] In certain embodiments, 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. In other embodiments, 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. In some embodiments, 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. In certain embodiments, 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.

[0207] In some embodiments, if a subject exhibits a clinical response following a dosing cycle, an additional dosing cycle is administered to the subject as consolidation treatment. Thus, provided herein is a method of administering an additional dosing cycle as consolidation treatment to a subject who exhibited a clinical response following a previous dosing cycle. 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). In some embodiments, the clinical response is CR. In some embodiments, the clinical response is CRi. In 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).

[0208] 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. In someembodiments, if a subject exhibits a complete response with incomplete hematologic recovery (CRh) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject exhibits morphologic leukemia-free state (MLFS) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject exhibits a partial response (PR) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment.

[0209] In some embodiments, in a subject does not exhibit a clinical response from a dosing cycle, an additional dosing cycle is administered to the subject. Thus, provided herein is a method of administering an additional dosing cycle to a subject who did not exhibit a clinical response following a previous dosing cycle. In some embodiments, 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.

[0210] In some embodiments, 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. Thus, provided herein is 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. In some embodiments, if a subject exhibits a CR from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject exhibits a CRi from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject exhibits MLFS from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject exhibits a PR from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment.

[0211] In some embodiments, 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. In some embodiments, 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.

[0212] 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). For example, in several embodiments, 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 asecond lymphodepletion followed by a second dosing cycle. In such embodiments where multiple dosing cycles are used, 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). Depending on the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dosing cycles are performed. In some embodiments, no more than four dosing cycles are administered. In some embodiments, no more than five dosing cycles are administered.

[0213] Depending on the embodiment, various types of cancer can be treated. In several embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a leukemia or a lymphoma. In several embodiments, the cancer being treated is acute myeloid leukemia (AML). In several embodiments, the cancer being treated is relapsed / refractory acute myeloid leukemia (r / r AML). In some embodiments, the cancer is relapsed to HCT. In several embodiments, the cancer being treated is myelodysplastic syndrome (MDS).

[0214] In some embodiments, also provided herein are 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 chemokines (including, butnot limited to IFN , TNF , IL-22, CCL3, CCL4, and CCL5), and (vii) enhanced ability to stimulate furtherinnate and adaptive immune responses, and (viii) combinations thereof.

[0215] Additionally, in several embodiments, there are provided 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.

[0216] In several embodiments, polynucleotides encoding the disclosed chimeric receptors are mRNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is operably linked to at least one regulatory element for the expression of the chimeric receptor.

[0217] Additionally provided, according to several embodiments, is 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. In some embodiments, the polynucleotides are operatively linked to at least one regulatory element for expression of the chimericreceptor, mbIL15, or both. In several embodiments, the vector is a retroviral vector. In some embodiments, the vector is a gamma retroviral vector.

[0218] Further provided herein are engineered immune cells (such as NK cells) comprising the polynucleotide, vector, or chimeric receptor as disclosed herein. Further provided herein are 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

[0219] 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. In some embodiments, particular clinical outcomes are achieved in subjects treated according to the method. For example, in some embodiments, the provided methods achieve particular clinical outcomes at a time point 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 3, 4, 5, 6, or more months after subjects have been administered a first dose of the genetically engineered cells. In some embodiments, 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. In some embodiments, 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 geneticallyengineered cells. In some embodiments, the provided methods achieve particular clinical outcomes 24 months after subjects have been administered a first dose of the genetically engineered cells.

[0220] In some embodiments, the clinical outcome is overall survival (OS). 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. In some embodiments, 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.

[0221] 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%. 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. 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 at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0222] In some embodiments, 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%. In some embodiments, 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%.

[0223] 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%. 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 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.

[0224] In some embodiments, the OS rate of all subjects treated according to the method is about 25%. In some embodiments, 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%.

[0225] In some embodiments, the clinical outcome is best response. In some embodiments, the best response is CR, CRh, or CRi. 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. 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.

[0226] 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. 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, 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. 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, 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.

[0227] In some embodiments, at least about 25% of all subjects achieve a best response of CR. In some embodiments, at least about 30% of all subjects achieve a best response of CR. In some embodiments, at least about 35% of all subjects achieve a best response of CR. In some embodiments, 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.

[0228] 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. In some embodiments, 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 allsubjects 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.

[0229] 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. In some embodiments, 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.

[0230] 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. 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, 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. 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, 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.

[0231] In some embodiments, about 25% of all subjects achieve a best response of CR. In some embodiments, about 30% of all subjects achieve a best response of CR. In some embodiments, about 35% of all subjects achieve a best response of CR. In some embodiments, about 40% of all subjects achieve a best response of CR. In some embodiments, about 45% of all subjects achieve a best response of CR. In some embodiments, about 50% of all subjects achieve a best response of CR. In some embodiments, about 55% of all subjects achieve a best response of CR. In some embodiments, about 60% of all subjects achieve a best response of CR.

[0232] In some embodiments, about 25% of all subjects achieve a best response of CRh. In some embodiments, 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.

[0233] 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. In some embodiments, about 35% of all subjects achieve a best response of CRi. In some embodiments, about 40% of all subjects achieve abest response of CRi. In some embodiments, about 45% of all subjects achieve a best response of CRi. In some embodiments, about 50% of all subjects achieve a best response of CRi. In some embodiments, about 55% of all subjects achieve a best response of CRi. In some embodiments, about 60% of all subjects achieve a best response of CRi.

[0234] In some embodiments, 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. In some embodiments, 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.

[0235] 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. 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. 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 at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0236] In some embodiments, at least about 15% of all subjects are in CR at the time point. In some embodiments, at least about 16% of all subjects are in CR at the time point. In some embodiments, at least about 17% of all subjects are in CR at the time point. In some embodiments, at least about 18% of all subjects are in CR at the time point. In some embodiments, 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.

[0237] 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. 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 about six months after administration of the first dose of the genetically engineered NK cells to the respective subject. In some embodiments, atleast 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.

[0238] In some embodiments, 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.

[0239] 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. 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. 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 at least about six months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0240] In some embodiments, at least about 15% of all subjects are in CRi at the time point. In some embodiments, at least about 16% of all subjects are in CRi at the time point. In some embodiments, at least about 17% of all subjects are in CRi at the time point. In some embodiments, at least about 18% of all subjects are in CRi at the time point. In some embodiments, at least about 19% of all subjects are in CRi at the time point. In some embodiments, at least about 20% of all subjects are in CRi at the time point. In some embodiments, at least about 25% of all subjects are in CRi at the time point. In some embodiments, at least about 30% of all subjects are in CRi at the time point.

[0241] In some embodiments, 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. In some embodiments, the time point is about twenty-four months after administration of the first dose of the genetically engineered NK cells to the respective subject.

[0242] In some embodiments, 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 ormore. 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. In some embodiments, 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.

[0243] 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. 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 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.

[0244] 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 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. 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 6 months. NON-LIMITING EMBODIMENTS

[0245] 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara- C), 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. 2. 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara- C), the overall survival rate (OS) of all subjects treated according to the method is at least about 25%, andthe 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. 3. The method of embodiment 1 or embodiment 2, wherein 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. 4. The method of any one of embodiments 1-3, wherein 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. 5. 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), 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. 6. 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 achimeric 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), 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. 7. The method of any one of embodiments 1-6, wherein 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. 8. The method of any one of embodiments 1-7, wherein 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. 9. 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), and 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. 10. 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), and 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. 11. The method of embodiment 9 or embodiment 10, wherein 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 12. The method of any one of embodiments 9-11, wherein at least about 25% of all subjects treated according to the method achieve a best response of complete remission (CR), completeremission 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. 13. The method of any one of embodiments 6-12, wherein 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. 14 The method of any one of embodiments 6-13, wherein 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. 15 The method of any one of embodiments 1-14, wherein 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. 16. The method of any one of embodiments 1-15, wherein 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. 17. The method of any one of embodiments 1-16, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 109genetically engineered NK cells. 18. The method of any one of embodiments 1-17, wherein the lymphodepleting therapy comprises administration of five doses of Flu. 19. The method of embodiment 18, wherein each dose of Flu comprises between about 10 mg / m2and about 40 mg / m2. 20. The method of any one of embodiments 1-19, wherein 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 / m2and about 4 g / m2. 22. The method of embodiment 20 or embodiment 21, wherein 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. 23. The method of any one of embodiments 20-22, wherein each dose of Flu comprises about 30 mg / m2and / or each dose of Ara-C comprises about 2 g / m2.24. The method of any one of embodiments 1-23, wherein the AML is a relapsed / refractory AML (r / r AML). 25. 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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. 26. 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells,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. 27. The method of embodiment 25 or embodiment 26, wherein 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. 28. The method of any one of embodiments 25-27, wherein 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. 29. 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.30. 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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. 31. The method of any one of embodiments 25-30, wherein 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. 32. The method of any one of embodiments 25-31, wherein 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. 33. 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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. 34. 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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. 35. The method of embodiment 33 or embodiment 34, wherein 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.36. The method of any one of embodiments 33-35, wherein 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. 37. The method of any one of embodiments 20-23, wherein 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. 38. The method of any one of embodiments 29-37, wherein 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. 39. The method of any one of embodiments 1-38, wherein: (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. 40. The method of any one of embodiments 1-39, wherein: (i) 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. 41. The method of any one of embodiments 1-40, wherein at least about 17% 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. 42. The method of any one of embodiments 1-41, wherein 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). 43. The method of any one of embodiments 1-42, wherein the dosing cycle is about 28 days.44. The method of any one of embodiments 1-43, wherein, if the subject exhibits a clinical response, optionally a partial response (PR), a complete response with incomplete hematologic recovery (CRi), or a complete response (CR), following the dosing cycle, the method comprises administering an additional dosing cycle as a consolidation treatment. 45. The method of any one of embodiments 1-43, wherein, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the method comprises administering an additional dosing cycle as retreatment. 46. 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. The method of 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. 50. The method of embodiment 49, wherein administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells. 51. The method of any one of embodiments 1-50, wherein the subject has been treated with at least one prior line of therapy. 52. The method of any one of embodiments 1-51, wherein the subject has been treated with one, two, three, or four prior lines of therapy. 53. The method of any one of embodiments 1-52, wherein the subject has an ECOG of 0-2, optionally 0 or 1. 54. The method of any one of embodiments 1-53, wherein the subject is 18 years of age or older. 55. The method of any one of embodiments 1-54, wherein the chimeric receptor comprises an extracellular antigen-binding domain that binds a ligand of NKG2D, a transmembrane domain, and an intracellular signaling domain. 56. The method of embodiment 55, wherein the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 26. 57. The method of embodiment 55 or embodiment 56, wherein the transmembrane domain comprises a CD8 transmembrane region.58. The method of any one of embodiments 55-57, wherein the intracellular signaling domain comprises a co-stimulatory domain and a CD3zeta. 59. The method of embodiment 58, wherein the co-stimulatory domain comprises an OX40 domain. 60. The method of any one of embodiments 1-59, wherein the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 39. 61. The method of any one of embodiments 1-60, wherein the genetically engineered NK cells express a membrane-bound interleukin 15 (mbIL15). 62. The method of embodiment 61, wherein the 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. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara- C), 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. 66. Use of 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) for treating a subject havingacute myeloid leukemia (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 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara- C), 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. 67. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), 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.68. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), 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. 69. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), and 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 remissionwith 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. 70. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), and 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. 71. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells,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. 72. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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. 73. Use of 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) 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 109genetically engineered NK cells, wherein:the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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. 74. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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. 75. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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. 76. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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. EXAMPLES

[0246] The following are non-limiting descriptions of experimental methods and materials. Example 1 – Clinical Outcomes of Patients with Acute Myeloid Leukemia Treated with NKG2D CAR NK Cells Following Lymphodepletion with Fludarabine and Cytarabine

[0247] Patients having relapsed or refractory (r / r) acute myeloid leukemia (AML) were lymphodepleted with fludarabine and cytarabine (Flu / Ara-C) and subsequently administered allogeneic natural killer (NK) cells expressing a NKG2D ligand-directed chimeric receptor and a membrane-bound form of interleukin 15 (mbIL15) (NKG2D NK cells). Safety and anti-tumor activity were evaluated.

[0248] To generate NKG2D NK cells, primary 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). 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.

[0249] 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. In particular, patients were administered a dose of 1.5 × 109viable NKG2D receptor+ NK cells on each of days 0, 7, and 14. The dosing cycle was preceded by a lymphodepleting therapy of fludarabine (Flu; 30 mg / m2) and cytarabine (Ara-C; 2 g / m2) on each of Days -7, -6, -5, -4, and -3. 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).

[0250] At baseline, five out of six patients had high-risk genetic features, including TP53 mutation. Patients had received a median of two prior lines of therapy, with all patients having previous venetoclax exposure and three out of six patients having previous Ara-C exposure (Table 1). Two patients had prior hematopoietic cell transplant (HCT) with disease relapse within six months. Table 1. Baseline Characteristics

[0251] All patients received at least three doses of NKG2D NK cells. There were no cases of cytokine release syndrome (CRS), immune cell associated neurotoxicity syndrome (ICANS), or graft- versus-host disease of any grade in any patients (Table 2). Myelosuppression and infection were the most common grade 3 or higher toxicities. Table 2. Grade 3 or Higher Adverse Events Reported in >1 Patient* One patient had grade 5 sepsis not related to NKG2D NK cells prior to receiving a second cycle of NKG2D NK cells.

[0252] PK profiling showed that NKG2D NK cells were consistently detected with clearance after infusion as expected (FIG.2).

[0253] Four out of six patients had CR / CRi (67%), with three out of six achieving CR (FIG.3). Patients 1 and 6 had no detectable MRD by flow after one treatment cycle. Patient 3 had MRD of 0.18% after one cycle and was immediately taken to consolidative hematopoietic cell transplant. Patient 2 had three cycles of treatment with successive decreases in disease burden, resulting in CRi. Of those who achieved CR / CRi, three out of four remained in CR / CRi at four months. High-risk features, baseline marrow blast burden, and best response for all subjects is shown in Table 3. Table 3. Baseline High-Risk Features, Marrow Burden, and Best Response

[0254] Together, the data indicate that the dosing regimen yields clinical responses in patients with high-risk r / r AML and has a toxicity profile consistent with underlying AML and exposure to lymphodepleting therapy, without any events of CRS or ICANS.

[0255] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present inventions herein disclosed should not be limited by the particular disclosed embodiments described above. Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0256] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 90%” includes “90%.” In some embodiments, at least 95% sequence identity or homology includes 96%, 97%, 98%, 99%, and 100% sequence identity or homology to the reference sequence. In addition, when a sequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a reference shall also include, unless otherwise indicated, that the sequence “comprises”, “consists of” or “consists essentially of” the recited sequence. Any titles or subheadings used herein are for organization purposes and should not be used to limit the scope of embodiments disclosed herein.SEQUENCES

[0257] In several embodiments, there are provided 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. Furthermore, those sequences (whether nucleic acid or amino acid) 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.

[0258] In accordance with some embodiments described herein, 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.

Claims

WHAT IS CLAIMED IS:

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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara- C), 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.

2. The method of claim 1, wherein 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 genetically engineered NK cells to the respective subject.

3. The method of claim 1, wherein 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.

4. The method of claim 1, wherein 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.

5. 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 achimeric 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), 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.

6. The method of claim 5, wherein 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.

7. The method of claim 1, wherein 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.

8. The method of claim 1, wherein 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.

9. 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), and 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.

10. The method of claim 9, wherein 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.

11. The method of claim 9, wherein 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.

12. The method of claim 9, wherein 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. 13 The method of any one of claims 5-12, wherein 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.

14. The method of any one of claims 5-13, wherein the overall survival rate (OS) of all subjects treated according to the method is at least about 25%, and the survival of each subject isdetermined 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.

15. The method of any one of claims 1-14, wherein 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.

16. The method of any one of claims 1-15, wherein 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.

17. The method of any one of claims 1-16, wherein each dose of the genetically engineered NK cells comprises about 1.5 x 109genetically engineered NK cells.

18. The method of any one of claims 1-17, wherein the lymphodepleting therapy comprises administration of five doses of Flu.

19. The method of claim 18, wherein each dose of Flu comprises between about 10 mg / m2and about 40 mg / m2.

20. The method of any one of claims 1-19, wherein the lymphodepleting therapy comprises administration of five doses of Ara-C.

21. The method of claim 20, wherein each dose of Ara-C comprises between about 1 g / m2and about 4 g / m2.

22. The method of claim 20 or claim 21, wherein 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.

23. The method of any one of claims 20-22, wherein each dose of Flu comprises about 30 mg / m2and / or each dose of Ara-C comprises about 2 g / m2.

24. The method of any one of claims 1-23, wherein the AML is a relapsed / refractory AML (r / r AML).

25. 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

26. The method of claim 25, wherein 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.

27. The method of claim 25, wherein 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.

28. The method of claim 25, wherein 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.

29. 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 109genetically engineered NK cells, wherein:the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

30. The method of claim 29, wherein 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.

31. The method of any one of claims 25-30, wherein 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.

32. The method of any one of claims 25-31, wherein 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.

33. 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 109genetically engineered NK cells, wherein:the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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.

34. The method of claim 33, wherein 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.

35. The method of claim 33, wherein 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.

36. The method of claim 33, wherein 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.

37. The method of any one of claims 29-36, wherein 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.

38. The method of any one of claims 29-37, wherein 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.

39. The method of any one of claims 1-38, wherein: (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.

40. The method of any one of claims 1-39, wherein: (i) 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.

41. The method of any one of claims 1-40, wherein 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.

42. The method of any one of claims 1-41, wherein 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).

43. The method of any one of claims 1-42, wherein the dosing cycle is about 28 days.

44. The method of any one of claims 1-43, wherein, if the subject exhibits a clinical response, optionally a partial response (PR), a complete response with incomplete hematologic recovery (CRi), or a complete response (CR), following the dosing cycle, the method comprises administering an additional dosing cycle as a consolidation treatment.

45. The method of any one of claims 1-43, wherein, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the method comprises administering an additional dosing cycle as retreatment.

46. The method of any one of claims 1-45, wherein the method comprises administration of between one dosing cycle and five dosing cycles.

47. The method of any one of claims 44-46, wherein the subject is administered the lymphodepleting therapy prior to each dosing cycle.

48. The method of any one of claims 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. The method of any one of claims 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.

50. The method of claim 49, wherein administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells.

51. The method of any one of claims 1-50, wherein the subject has been treated with at least one prior line of therapy.

52. The method of any one of claims 1-51, wherein the subject has been treated with one, two, three, or four prior lines of therapy.

53. The method of any one of claims 1-52, wherein the subject has an ECOG of 0-2, optionally 0 or 1.

54. The method of any one of claims 1-53, wherein the subject is 18 years of age or older.

55. The method of any one of claims 1-54, wherein the chimeric receptor comprises an extracellular antigen-binding domain that binds a ligand of NKG2D, a transmembrane domain, and an intracellular signaling domain.

56. The method of claim 55, wherein the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:

26.

57. The method of claim 55 or claim 56, wherein the transmembrane domain comprises a CD8 transmembrane region.

58. The method of any one of claims 55-57, wherein the intracellular signaling domain comprises a co-stimulatory domain and a CD3zeta.

59. The method of claim 58, wherein the co-stimulatory domain comprises an OX40 domain.

60. The method of any one of claims 1-59, wherein the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO:

39.

61. The method of any one of claims 1-60, wherein the genetically engineered NK cells express a membrane-bound interleukin 15 (mbIL15).

62. The method of claim 61, wherein the mbIL15 comprises the amino acid sequence set forth in SEQ ID NO:

40.

63. The method of any one of claims 1-62, wherein the population of engineered NK cells are allogeneic to the subject.

64. The method of any one of claims 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. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 fludarabine (Flu) and cytarabine (Flu / Ara- C), 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.

66. The use of claim 65, wherein 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.

67. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), 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.

68. The use of claim 67, wherein 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.

69. Use of 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) for treating a subject having acute myeloid leukemia (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 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 109and about 2 x 109genetically 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 therapy comprising fludarabine (Flu) and cytarabine (Flu / Ara- C), and 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.

70. The method of claim 69, wherein 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.

71. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

72. The use of claim 71, wherein 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.

73. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, 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.

74. The use of claim 73, wherein 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.

75. Use of 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) 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 109genetically engineered NK cells, wherein: the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg / m2fludarabine (Flu) and about 2 g / m2cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells, and 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.

76. The use of claim 75, wherein 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.

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