Dosing regimens for combination therapies

Abstract

Several embodiments of the methods and compositions disclosed herein relate to immune cells that are engineered to express cytotoxic 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 on tumor cells. In several embodiments, the cancer is a blood cancer, for example, acute myeloid leukemia (e.g., relapsed / refractory acute myeloid leukemia) or myelodysplastic syndrome. In several embodiments, the tumor is a solid tumor, for example, breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancers, hepatocellular carcinoma, lung cancer, melanoma, intrahepatic cholangiocarcinoma or other liver tumor, for example, secondary metastases from colorectal cancer. In several embodiments, the immune cells are administered in conjunction with a therapeutic agent, such as an additional anti-cancer agent.

Description

DOSING REGIMENS FOR COMBINATION THERAPIESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent Application Nos. 63 / 384,073, filed November 16, 2022; 63 / 489,946 filed March 13, 2023; 63 / 499,470, filed May 1, 2023; and 63 / 523,333, filed June 26, 2023, the entire contents of each of which are incorporated by reference herein.FIELD

[0002] Several embodiments disclosed herein relate to methods and compositions comprising genetically engineered cells for cancer immunotherapy. In several embodiments, the present disclosure relates to cells engineered to express cytotoxic receptor complexes and administration of such cells in accordance with certain dosing regimens to achieve successful cancer immunotherapy.BACKGROUND

[0003] As further knowledge is gained about various cancers and what characteristics a cancerous cell has that can be used to specifically distinguish that cell from a healthy cell, therapeutics are under development that leverage the distinct features of a cancerous cell. Immunotherapies that employ engineered immune cells are one approach to treating cancers.INCORPORATION BY REFERENCE OF MATERIAL IN SEQUENCE LISTING FILE

[0004] This application incorporates by reference the material in the Sequence Listing contained in the following XML file being submitted concurrently herewith: File name: NKT096WO_ST26.xmlcreated November 15, 2023, which is 46,520 bytes in size.SUMMARY

[0005] Immunotherapy presents a new technological advancement in the treatment of disease, wherein immune cells are engineered to express certain targeting and / or effector molecules that specifically identify and react to diseased or damaged cells. This represents a promising advance due, at least in part, to the potential for specifically targeting diseased or damaged cells, as opposed to more traditional approaches, such as chemotherapy, where all cells are impacted, and the desired outcome is that sufficient healthy cells survive to allow the patient to live. One immunotherapy approach is the recombinant expression of chimeric receptors in immune cells to achieve the targeted recognition and destruction of aberrant cells of interest.

[0006] Provided herein, in several embodiments, is a method of treating a cancer in a subject, comprising (a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells. In several embodiments, the method comprises administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0007] In several embodiments, the cancer is a solid tumor. In several embodiments, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0008] Provided herein, in several embodiments, is a method of treating a cancer in a subject, comprising (a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells, wherein the cancer is a solid tumor and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy. In several embodiments, the method comprises administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0009] In several embodiments, the therapeutic agent, when administered, is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof.

[0010] In several embodiments, the subject has less than or equal to 5% peripheral blasts. In several embodiments, the subject has less than 5% peripheral blasts. In several embodiments, prior to administering the genetically engineered NK cells to the subject, the percentage of peripheral blasts is determined in the subject, and if the subject has less than or equal to 5% peripheral blasts, the subject is selected for treatment.

[0011] In several embodiments, prior to administering the genetically engineered NK cells to the subject, additional evaluation of the subject is performed and comprises (a) determining thepercentage of peripheral blasts in the subject and (b) if the subject has less than or equal to 5% peripheral blasts, selecting the subject for treatment.

[0012] In several embodiments, the subject does not have evidence of extramedullary disease.

[0013] In several embodiments, administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells. In several embodiments, administration of the therapeutic agent is concurrent with administration of a dose of the genetically engineered NK cells, optionally concurrent with administration of the first dose of the genetically engineered NK cells. In several embodiments, administration of the therapeutic agent is after administration of a dose of the genetically engineered NK cells, optionally after administration of the third dose of the genetically engineered NK cells. In several embodiments, the therapeutic is administered before, concurrently with, and / or after administration of a dose of the genetically engineered NK cells.

[0014] In several embodiments, the lymphodepleting therapy comprises administration of fludarabine (Flu). In several embodiments, the lymphodepleting therapy comprises administration of three doses of Flu. In several embodiments, each dose of Flu comprises between about 10 mg / m2and about 60 mg / m2. In several embodiments, the lymphodepleting therapy comprises administration of Flu and cyclophosphamide (Cy). In several embodiments, the lymphodepleting therapy comprises administration of three doses of Cy. In several embodiments, the first doses of Flu and Cy are each given 5 days prior to initiation of the dosing cycle, the second doses of Flu and Cy are each given 4 days prior to initiation of the dosing cycle, and the third doses of Flu and Cy are each given 3 days prior to initiation of the dosing cycle. In several embodiments, each dose of Cy comprises between about 200 mg / m2and about 600 mg / m2. In several embodiments, each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 300 mg / m2. In additional embodiments, each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 500 mg / m2.

[0015] In several embodiments, the lymphodepleting therapy comprises administration of five doses of Flu. In several embodiments, the lymphodepleting therapy comprises administration of Flu and cytosine arabinoside (Ara-C). In several embodiments, the lymphodepleting therapy comprises administration of five doses of Ara-C. In several 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 3 days prior to initiation of the dosing cycle, and the fifth doses of Flu and Ara-C are each given 4 days prior to initiation of the dosing cycle. In several embodiments, each dose of Ara-C comprises between about 1 g / m2and about 4 g / m2. In several embodiments, each dose of Flu comprises about 30 mg / m2and each dose of Ara-C comprises about 2 g / m2.

[0016] In several embodiments, there is provided a method of selecting a subject having a cancer for treatment with a population of Natural Killer (NK) cells genetically engineered to express achimeric receptor that binds ligands of natural killer cell group 2D (NKG2D), the method comprising (a) assessing the level or amount of a NKG2D ligand in a biological sample from a subject having a cancer, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or a polynucleotide encoded by the NKG2D ligand gene, (b) selecting the subject for treatment with a population of NK cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D if the level or amount of the NKG2D ligand is above a reference value, and (c) administering the genetically engineered NK cells to the subject. In several embodiments, the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells.

[0017] In several embodiments, the genetically engineered NK cells are administered to the 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells.

[0018] In several embodiments, the biological sample is obtained from the subject within about 14 days, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administration of the genetically engineered NK cells to the subject.

[0019] In several embodiments, the percentage of blasts in the subject’s bone marrow is between about 0% and about 60%, between about 0% and about 50%, between about 0% and about 40%, between about 0% and about 30%, or between about 0% and about 20%.

[0020] In several embodiments, the NKG2D ligand comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof. In several embodiments, the NKG2D ligand comprises MICA and / or MICB. In several embodiments, the NKG2D ligand comprises ULBP1 and / or ULBP3.

[0021] In several embodiments, each of the first, second, and third doses of the genetically engineered NK cells comprises about 1 x 109genetically engineered NK cells or about 1.5 x 109genetically engineered NK cells.

[0022] In several embodiments, the dosing cycle is between about 14 days and about 35 days. In several embodiments, the dosing cycle is about 21 days. In several embodiments, the dosing cycle is about 28 days. In some embodiments, the method comprises administering an additional dosing cycle.

[0023] In several embodiments, 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.

[0024] In several 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.

[0025] In several embodiments, the method comprises administration of between one dosing cycle and five dosing cycles. In several embodiments, the subject is administered a lymphodepleting therapy prior to each cycle.

[0026] In several embodiments, the second dose of the genetically engineered cells is administered to the subject about 7 days after administration of the first dose of the genetically engineered cells. In several embodiments, the third dose of the genetically engineered cells is administered to the subject about 7 days after administration of the second dose of the genetically engineered cells.

[0027] In several embodiments, the cancer is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), optionally wherein the cancer is relapsed / refractory (r / r) AML.

[0028] In several embodiments, the therapeutic agent comprises a chemotherapeutic agent and the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly (ADP-ribose) polymerase (PARP), or any combination thereof.

[0029] In several embodiments, the therapeutic agent comprises a monoclonal antibody and the monoclonal antibody comprises an anti-CTLA4 antibody, an anti-EGFR antibody, an anti- HER2 / neu antibody, an anti-PDl antibody, an anti-PD-Ll antibody, an anti-VEGF antibody, or any combination thereof.

[0030] In several embodiments, the therapeutic agent comprises an NK cell engager and the NK cell engager binds to an activating receptor on an NK cell and an antigen expressed by cells of the cancer, optionally wherein the activating receptor is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof.

[0031] In several embodiments, the cancer to be treated is a carcinoma, a sarcoma, or a melanoma. In several embodiments, the cancer to be treated is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

[0032] In several embodiments, the cancer to be treated comprises liver cancer or colorectal cancer. In several embodiments, the therapeutic agent comprises capecitabine, cisplatin, doxorubicin, regorafenib, or sorafenib.

[0033] In several embodiments, the cancer comprises bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, or uterine cancer. In several embodiments, the therapeutic agent comprises doxorubicin or gemcitabine.

[0034] In several embodiments, the cancer comprises esophageal cancer, head and neck cancer, or lung cancer. In several embodiments, the therapeutic agent comprises gemcitabine, irinotecan, or vinorelbine.

[0035] In several embodiments, the cancer comprises melanoma. In several embodiments, the therapeutic agent comprises a MEK inhibitor, a BRAF inhibitor, an anti-CTLA4 antibody, an anti- LAG3 antibody, an anti-PDl antibody, or an anti-PD-Ll antibody.

[0036] In several embodiments, the cancer is a relapsed / refractory (R / R) cancer.

[0037] In several embodiments, the subject has been treated with one prior line of therapy. In several embodiments, the subject has been treated with two prior lines of therapy. In several embodiments, the has been treated with three (or more) prior lines of therapy. In several embodiments, the subject has an ECOG of 0-2, optionally 0 or 1. In several embodiments, the subject is not a minor (e.g., is 18 years of age or older).

[0038] In several embodiments, the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling region. In several embodiments, extracellular binding domain has at least 90%, 91%, 92%, 93%, 94%, 95%, or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 42. In several embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In several embodiments, the intracellular signaling region comprises a co-stimulatory domain and a CD3zeta. In several embodiments, the co-stimulatory domain comprises an 0X40 domain. In several embodiments, the chimeric receptor has at least 90%, 91%, 92%, 93%, 94%, 95%, or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 39. In several embodiments, the genetically engineered NK cells express a membrane -bound interleukin 15 (mbIL15). In several embodiments, the mbIL15 has at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 40.

[0039] In several embodiments, the population of engineered NK cells are allogeneic to the subject. In several embodiments, the population of engineered NK cells are derived from a subject without cancer.

[0040] In several embodiments, at least one 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.

[0041] Also provided for herein is a 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 cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 subjectbetween about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells. In several embodiments, the cancer is a solid tumor. In several embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells. In several embodiments, the subject is administered a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof. In several embodiments, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0042] Also provided for herein is the 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 cancer in a subject, wherein (a) the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells; and (b) the subject is administered a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0043] In several embodiments, the therapeutic agent increases expression of a NKG2D ligand in the subject.

[0044] Provided for 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 cancer in a subject, wherein (a) the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells, and (b) the subject is administered a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0045] Provided for herein is also the 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 cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells. In several embodiments, the subject is administered a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0046] Provided for herein is also the 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 cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells, and wherein the subject has less than or equal to 5% peripheral blasts.

[0047] In several embodiments, the population of genetically engineered NK cells is for use with 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.

[0048] In several embodiments, the subject has less than or equal to 5% peripheral blasts. In several embodiments, the subject has less than 5% peripheral blasts. In several embodiments, the subject does not have evidence of extramedullary disease.

[0049] In several embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof.

[0050] In several embodiments, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0051] Provided for herein is also a 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 cancer in a subject, wherein the genetically engineered NK cells are administered in adosing 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 about 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 about 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, and wherein the subject has less than or equal to 5% peripheral blasts and / or does not have evidence of extramedullary disease.

[0052] Provided for is also a 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 cancer in a subject, 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 about 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 about 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, and wherein the subject has been administered a dose of about 20 mg / m2 decitabine daily for five days prior to administration of the first dose of the genetically engineered NK cells. In several embodiments, the subject has less than or equal to 5% peripheral blasts, optionally wherein the subject does not have evidence of extramedullary disease.

[0053] In several embodiments of the uses provided, the subject is administered an additional dosing cycle. In several embodiments of the uses provided, 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, an additional dosing cycle is administered to the subject as a consolidation treatment. In several embodiments of the uses provided, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the subject is administered an additional dosing cycle as retreatment.

[0054] In several embodiments of the uses provided, the subject is administered between one dosing cycle and five dosing cycles. In several embodiments of the uses provided, the subject is administered a lymphodepleting therapy prior to each cycle. In several embodiments, the cancer is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), optionally wherein the cancer is relapsed / refractory (r / r) AML or very high-risk MDS. In several embodiments, 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.

[0055] Also provided for herein is a method of treating a cancer in a subject, comprising (a) administering to a subject having a cancer 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 firstdose of the genetically engineered NK cells, (ii) a second dose of the genetically engineered NK cells administered to the subject between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells, and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0056] In several embodiments, there is also provided a method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells, and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy. In several embodiments, the therapeutic agent increases expression of a NKG2D ligand in the subject.

[0057] In several embodiments, the method of treating a cancer in a subject, comprising (a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells, and (b) administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0058] Additionally, provided for herein is a method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells, and wherein the subject has less than or equal to 5% peripheral blasts.

[0059] Additionally provide for herein is a method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 about 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 about 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, and wherein the subject has less than or equal to 5% peripheral blasts and does not have evidence of extramedullary disease.

[0060] Also provided for herein is a method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 about 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 about 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 has less than or equal to 5% peripheral blasts, and wherein the subject has been administered a dose of about 20 mg / m2 decitabine daily for five days prior to administration of the first dose of the genetically engineered NK cells.

[0061] Further provided herein is a method of treating a cancer in a subject, comprising: (a) administering to the subject 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 geneticallyengineered NK cells are administered in a dosing cycle; and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof; wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0062] Also provided herein is a method of treating a cancer in a subject, comprising administering to the subject 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; wherein the subject has been treated with, or is a candidate for treatment with, a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof; and, wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0063] Also provided herein is a method of treating a cancer in a subject, comprising administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof; wherein the subject has been treated with, or is a candidate for treatment with, 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; and, wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0064] In some embodiments, the dosing cycle comprises: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject between about 5 days and about 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 between about 5 days and about 10 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 between about 1 x 108NK cells and about 1 x 1010NK cells. In some embodiments, each of the first, second, and third doses of the genetically engineered NK cells comprises about 1 x 109NK cells or about 1.5 x 109NK cells. In some embodiments, each of the first, second, and third doses of the genetically engineered NK cells comprises about 1 x 109NK cells. In some embodiments, each of the first, second, and third doses of the genetically engineered NK cells comprises about 1.5 x 109NK cells.

[0065] In some embodiments, the therapeutic agent increases expression of a NKG2D ligand in the subject.

[0066] Also provided herein is a method of treating a cancer in a subject, comprising: (a) administering to the subject 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 geneticallyengineered NK cells are administered in a dosing cycle; and (b) administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0067] Also provided herein is a method of treating a cancer in a subject, comprising administering to the subject 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; and wherein the subject has been treated with, or is a candidate for treatment with, a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0068] Also provided herein is a method of treating a cancer in a subject, comprising administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject, wherein the subject has been treated with, or is a candidate for treatment with, 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); and wherein the genetically engineered NK cells are administered in a dosing cycle comprising.

[0069] Also provided herein is a method of treating a cancer in a subject, comprising: administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells, and wherein the subject has less than or equal to 5% peripheral blasts.

[0070] In some embodiments, the method 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.

[0071] 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. In some embodiments, prior to administering the population of NK cells to the subject, the percentage of peripheral blasts is determined in the subject. In some embodiments, prior to administering the population of NK cells to the subject, if the subject is determined to have less than or equal to 5% peripheral blasts, the subject is selected for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the percentage of peripheral blasts is determined in the subject, and if the subject is determined to have less than or equalto 5% peripheral blasts, the subject is selected for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the method comprises determining the percentage of peripheral blasts in the subject. In some embodiments, prior to administering the population of NK cells to the subject, if the subject is determined to have less than or equal to 5% peripheral blasts, the method comprises selecting the subject for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the method comprises: (a) determining the percentage of peripheral blasts in the subject; and (b) if the subject is determined to have less than or equal to 5% peripheral blasts, selecting the subject for treatment.

[0072] In some embodiments, the dosing cycle comprises: (i) a first dose of the genetically engineered NK cells; fii) a second dose of the genetically engineered NK cells administered to the subject between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells.

[0073] Also provided herein is a method of selecting a subject having a cancer for treatment with a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds ligands of natural killer cell group 2D (NKG2D), the method comprising: (a) assessing the level or amount of a NKG2D ligand in a biological sample from a subject having a cancer, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or a polynucleotide encoded by the NKG2D ligand gene; (b) selecting the subject for treatment with a population of NK cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D if the level or amount of the NKG2D ligand is above a reference value; and (c) administering the genetically engineered NK cells to the subject, wherein the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells.

[0074] Also provided herein is a method of selecting a subject having a cancer for treatment with a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds ligands of natural killer cell group 2D (NKG2D), the method comprising administering the genetically engineered NK cells to a subject having a cancer, wherein: (a) the level or amount of a NKG2D ligand in a biological sample from the subject has been assessed prior to administration of the population of genetically engineered NK cells, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or a polynucleotide encoded by the NKG2D ligand gene; (b) the subject was selected for treatment with the population of NK cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D based on the level or amount of the NKG2D ligand being above a reference value; and (c) the biological sample was obtained from the subject prior to the administration of the population of genetically engineered NK cells.

[0075] In some embodiments, the biological sample comprises a bone marrow sample. In some embodiments, the biological sample is a bone marrow sample. In some embodiments, the cancer isacute myeloid leukemia (AML), optionally relapsed / refractory AML (r / r AML). In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is relapsed / refractory AML (r / r AML).

[0076] In some embodiments, the genetically engineered NK cells are administered to the 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells.

[0077] In some embodiments, the reference value is within 25%, within 20%, within 15%, within 10%, or within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the genetically engineered NK cells who did not exhibit a clinical response following administration of the genetically engineered NK cells. In some embodiments, the reference value is the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the genetically engineered NK cells who did not exhibit a clinical response following administration of the genetically engineered NK cells.

[0078] In some embodiments, the biological sample is obtained from the subject within about 14 days, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 14 days, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administration of the lymphodepleting therapy to the subject.

[0079] In some embodiments, the percentage of blasts in the subject’s bone marrow is between about 0% and about 60%, between about 0% and about 50%, between about 0% and about 40%, between about 0% and about 30%, or between about 0% and about 20%. In some embodiments, the percentage of blasts in the subject’s peripheral blood is between about 0% and about 5%, between about 0% and about 10%, between about 0% and about 20%, between about 0% and about 30%, between about 0% and about 40%, between about 0% and about 50%, between about 0% and about 60%, between about 0% and about 70%, between about 0% and about 80%, or between about 0% and about 90%.

[0080] In some embodiments, the NKG2D ligand comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA. In some embodiments, the NKG2D ligand comprises MICB. In some embodiments, the NKG2D ligand comprises MICA and MICB. In some embodiments, the NKG2Dligand comprises ULBP1. In some embodiments, the NKG2D ligand comprises ULBP2. In some embodiments, the NKG2D ligand comprises ULBP3. In some embodiments, the NKG2D ligand comprises ULBP4. In some embodiments, the NKG2D ligand comprises ULBP5. In some embodiments, the NKG2D ligand comprises ULBP6. In some embodiments, the NKG2D ligand comprises ULBP1 and ULBP3.

[0081] In some embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 108NK cells and about 1 x IO10NK cells. In some embodiments, each of the first, second, and third doses of the genetically engineered NK cells comprises about 1 x 109NK cells or about 1.5 x 109NK cells. In some embodiments, each of the first, second, and third doses of the genetically engineered NK cells comprises about 1 x 109NK cells. In some embodiments, each of the first, second, and third doses of the genetically engineered NK cells comprises about 1.5 x 109NK cells. In some embodiments, each of the first, second, and third dose each of the genetically engineered NK cells comprises 2 x 109NK cells, 3 x 109NK cells, 4 x 109NK cells, or 5 x 109NK cells. In some embodiments, each of the first, second, and third dose each of the genetically engineered NK cells comprises 2 x 109NK cells. In some embodiments, each of the first, second, and third dose each of the genetically engineered NK cells comprises 3 x 109NK cells. In some embodiments, each of the first, second, and third dose each of the genetically engineered NK cells comprises 4 x 109NK cells. In some embodiments, each of the first, second, and third dose each of the genetically engineered NK cells comprises 5 x 109NK cells. In some embodiments, the NK cells are genetically engineered NK cells.

[0082] In some embodiments, therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof.

[0083] 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.

[0084] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is acute myeloid leukemia (AML).

[0085] In some embodiments, the second dose is administered to the subject between 6-8 days after the first dose. In some embodiments, the third dose is administered to the subject between 6-8 days after the second dose. In some embodiments, the second dose of the genetically engineered 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 the genetically engineered cells is administered to the subject about 7 days after administration of the second dose of the genetically engineered cells.

[0086] In some embodiments, the dosing cycle is between about 14 days and about 35 days. In some embodiments, the dosing cycle is about 21 days. In some embodiments, the dosing cycle is about 28 days. In several embodiments, the first, second, and third doses of engineered NK cells are administered to the subject within about 21 days of the first time point. In several embodiments, thefirst, second, and third doses of engineered NK cells are administered to the subject within about 14 days after the first time point.

[0087] Also provided herein is a method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 about 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 about 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; and wherein the subject has less than or equal to 5% peripheral blasts and does not have evidence of extramedullary disease.

[0088] In some embodiments, the method comprises administering an additional dosing cycle. In some embodiments, if the subject exhibits a clinical response following the dosing cycle, the method comprises administering an additional dosing cycle. 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, if the subject exhibits a complete response (CR) following the dosing cycle, the method comprises administering an additional dosing cycle. In some embodiments, if the subject exhibits a complete response (CR) following the dosing cycle, the method comprises administering an additional dosing cycle as a consolidation treatment. In some embodiments, if the subject exhibits a complete response with incomplete hematologic recovery (CRi) following the dosing cycle, the method comprises administering an additional dosing cycle. In some embodiments, if the subject exhibits a CRi following the dosing cycle, the method comprises administering an additional dosing cycle as a consolidation treatment.

[0089] 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. 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. In some embodiments, the method comprises administration of between one dosing cycle and five dosing cycles. In some embodiments, the method consists of administration of one dosing cycle. In some embodiments, the method consists of administration of two dosing cycles. In some embodiments, the method consists of administration of three dosing cycles. In some embodiments, the method consists of administration of four dosing cycles. In some embodiments, the method consists of administration of five dosing cycles. In some embodiments, the method consists of administration of no more than five dosing cycles. In some embodiments, the subject is administered a lymphodepleting therapy prior to each cycle.

[0090] In some embodiments, the therapeutic agent is administered prior to administration of the first dose of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered prior to administration of the lymphodepleting therapy. In some embodiments, the therapeutic agent is administered after administration of the lymphodepleting therapy and until administration of a dose of genetically engineered NK cells. In some embodiments, the therapeutic agent is administered concurrent with administration of a dose of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered concurrent with administration of the first dose of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered after administration of a dose of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered after administration of the third dose of the genetically engineered NK cells.

[0091] In some embodiments, the first dosing cycle is initiated after the subject is administered a lymphodepleting therapy. In some embodiments, the first dosing cycle is followed by an additional dosing cycle. In some embodiments, the first dosing cycle is followed by two, three, four or more additional dosing cycles. In some embodiments, the additional cycle(s) is administered depending on the state of the cancer in a subject, e.g., in the event of progression or development of an additional cancer. In some embodiments, the additional cycle(s) is administered as retreatment in the event of disease progression. In some embodiments, if the subject exhibits a clinical response to a dosing cycle and subsequently exhibits disease progression, the additional cycle(s) is administered as retreatment. In some embodiments, the additional cycle(s) is administered as a consolidating treatment after the subject exhibits a clinical response to the previous cycle. In several embodiments, an additional cycle is not needed when a subject exhibits a response (e.g., a complete response).

[0092] In some embodiments, the lymphodepleting therapy comprises administration of fludarabine (Flu). In some embodiments, the lymphodepleting therapy comprises administration of three doses of Flu. In some embodiments, each dose of Flu comprises between about 10 mg / m2and about 60 mg / m2. In some embodiments, each dose of Flu comprises about 30 mg / m2. In some embodiments, the lymphodepleting therapy comprises administration of Flu and cyclophosphamide (Cy). In some embodiments, the lymphodepleting therapy comprises administration of three doses of Cy. In some embodiments, the first doses of Flu and Cy are each given 5 days prior to initiation of the dosing cycle; the second doses of Flu and Cy are each given 4 days prior to initiation of the dosing cycle; and the third doses of Flu and Cy are each given 3 days prior to initiation of the dosing cycle. In some embodiments, each dose of Cy comprises between about 200 mg / m2and about 600 mg / m2. In some embodiments, each dose of Cy comprises about 300 mg / m2. In some embodiments, each dose of Cy comprises about 500 mg / m2. In some embodiments, each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 300 mg / m2. In some embodiments, each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 500 mg / m2. In some embodiments, 30 mg / m2of Flu and 500 mg / m2of Cy are each given five days, four days, and three days prior to initiation of thedosing cycle. In several embodiments, about two days are allowed to lapse between the third dose of cyclophosphamide and fludarabine and initiation of the dosing cycle.

[0093] In some embodiments, the lymphodepleting therapy comprises administration of fludarabine (Flu). In some embodiments, the lymphodepleting therapy comprises administration of three five doses of Flu. In some embodiments, each dose of Flu comprises between about 10 mg / m2and about 60 mg / m2. In some embodiments, each dose of Flu comprises about 30 mg / m2. In some embodiments, the lymphodepleting therapy comprises administration of Flu and cytosine arabinoside (Ara-C). In some embodiments, the lymphodepleting therapy comprises five doses of Ara-C. In some embodiments, the lymphodepleting therapy comprises 5 daily doses of Ara-C and 5 daily doses of fludarabine, wherein the first of the doses of Ara-C and fludarabine are administered 7 days prior to the initiation of the dosing cycle. 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-C are each given 3 days prior to initiation of the dosing cycle. In some embodiments, about two days are allowed to lapse between the fifth doses of Ara-C and fludarabine and initiation of the dosing cycle. In some embodiments, each dose of Ara-C comprises between about 1 g / m2and about 4 g / m2. In some embodiments, each dose of Ara-C comprises about 2 g / m2. In some embodiments, each dose of Flu comprises about 30 mg / m2and each dose of Ara- C comprises about 2 g / m2. In some embodiments, about 30 mg / m2of Flu and about 2 g / m2of Ara-C are each given seven days, six days, five days, four days, and three days prior to initiation of the dosing cycle.

[0094] Depending on the embodiment, other lymphodepletion agents may be used in addition to, or in place of cyclophosphamide, Ara-C and / or fludarabine, such as for example, daunorubicin (daunomycin) or idarubicin, mycophenolate mofetil, and / or bendamustine.

[0095] In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly (ADP-ribose) polymerase (PARP), or any combination thereof.

[0096] In some embodiments, the chemotherapeutic agent comprises an antimetabolite. In some embodiments, the antimetabolite comprises methotrexate, pemetrexed, cytarabine, 5-fluorouracil (5-FU), capecitabine, gemcitabine, 6-mercaptopurine (6-MP), azathioprine, fludarabine, cladribine, hydroxyurea, or any combination thereof. In some embodiments, the therapeutic agent comprises gemcitabine.

[0097] In some embodiments, the chemotherapeutic agent comprises an alkylating agent. In some embodiments, the alkylating agent comprises cyclophosphamide, ifosfamide, chlorambucil, melphalan, temozolomide, carmustine, lomustine, streptozocin, busulfan, procarbazine, cisplatin,carboplatin, oxaliplatin, or any combination thereof. In some embodiments, the therapeutic agent comprises cisplatin. In some embodiments, the therapeutic agent comprises bendamustine. In some embodiments, the therapeutic agent comprises cyclophosphamide. In some embodiments, the therapeutic agent comprises dacarbazine. In some embodiments, the therapeutic agent comprises procarbazine. In some embodiments, the alkylating agent comprises ifosfamide. In some embodiments, the alkylating agent comprises chlorambucil. In some embodiments, the alkylating agent comprises melphalan. In some embodiments, the alkylating agent comprises temozolomide. In some embodiments, the alkylating agent comprises carmustine. In some embodiments, the alkylating agent comprises lomustine. In some embodiments, the alkylating agent comprises streptozocin. In some embodiments, the alkylating agent comprises busulfan. In some embodiments, the alkylating agent comprises carboplatin. In some embodiments, the alkylating agent comprises oxaliplatin.L0098J In some embodiments, the therapeutic agent comprises a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor comprises irinotecan, topotecan, etoposide, or any combination thereof. In some embodiments, the therapeutic agent comprises etoposide.

[0099] In some embodiments, the therapeutic agent comprises a mitotic inhibitor. In some embodiments, the mitotic inhibitor comprises vincristine, vinblastine, vinorelbine, docetaxel, paclitaxel, eribulin, izabepilone, epothilone, or any combination thereof. In some embodiments, the therapeutic agent comprises vincristine. In some embodiments, the therapeutic agent comprises vinblastine. In some embodiments, the therapeutic agent comprises paclitaxel. In some embodiments, the therapeutic agent comprises docetaxel.

[0100] In some embodiments, the therapeutic agent comprises an antibiotic. In some embodiments, the antibiotic comprises bleomycin; actinomycin D; an anthracycline, optionally doxorubicin, daunorubicin, or idarubicin, mitomycin, or any combination thereof. In some embodiments, the antibiotic comprises bleomycin; actinomycin D; an anthracycline. In some embodiments, the antibiotic comprises doxorubicin, daunorubicin, or idarubicin, mitomycin, or any combination thereof. In some embodiments, the therapeutic agent comprises bleomycin. In some embodiments, the therapeutic agent comprises an anthracycline. In some embodiments, the therapeutic agent comprises doxorubicin. In some embodiments, the therapeutic agent comprises daunorubicin.

[0101] In some embodiments, the therapeutic agent comprises a protein kinase inhibitor. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BCR-ABL, c-KIT, EGFR, VEGF, ALK, BRAF, MEK, BTK, JAK, CDK, or any combination thereof. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BCR-ABL. In some embodiments, the protein kinase inhibitor comprises an inhibitor of c-Kit. In some embodiments, the protein kinase inhibitor comprises an inhibitor of EGFR. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BRAF. In some embodiments, the protein kinase inhibitor comprises an inhibitor of MEK. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BTK (e.g., ibrutinib). In someembodiments, the protein kinase inhibitor comprises an inhibitor of JAK. In some embodiments, the protein kinase inhibitor comprises an inhibitor of CDK.

[0102] In some embodiments, the therapeutic agent comprises an inhibitor of BCL2 (e.g., venetoclax). In some embodiments, the therapeutic agent comprises a glucocorticoid (e.g., prednisone).

[0103] In some embodiments, the therapeutic agent comprises a proteasome inhibitor. In some embodiments, the proteasome inhibitor comprises bortezomib, carfilzomib, ixazomib, or any combination thereof. In some embodiments, the therapeutic agent comprises a PARP inhibitor. In some embodiments, the PARP inhibitor comprises olaparib.

[0104] In some embodiments, the therapeutic agent comprises a monoclonal antibody. In some embodiments, the monoclonal antibody comprises an anti-CD20 antibody, an anti-CTLA4 antibody, an anti-EGFR antibody, an anti-HER2 / neu antibody, an anti-PDl antibody, an anti-PD-Ll antibody, an anti-VEGF antibody, or any combination thereof. In some embodiments, the monoclonal antibody comprises an anti-CD20 antibody (e.g., rituximab). In some embodiments, the monoclonal antibody comprises an anti-EGFR antibody (e.g., cetuximab). In some embodiments, the monoclonal antibody comprises an anti-PDl antibody. In some embodiments, the monoclonal antibody comprises an anti- PD-Ll antibody.

[0105] In some embodiments, the NK cell engager binds to an activating receptor on an NK cell and an antigen expressed by cells of the cancer. In some embodiments, the activating receptor is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof.

[0106] In some embodiments, the therapeutic agent is decitabine. In some embodiments, a dose of decitabine is administered to the subject daily for five days prior to initiation of the dosing cycle. In some embodiments, a dose of decitabine comprises about 20 mg / m2.

[0107] Also provided herein is a method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 about 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 about 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; and wherein the subject has been administered a dose of about 20 mg / m2decitabine daily for five days prior to administration of the first dose of the genetically engineered NK cells.

[0108] In some embodiments, the subject has less than or equal to 5% peripheral blasts. In some embodiments, the subject does not have evidence of extramedullary disease.

[0109] In some embodiments, prior to administering the population of NK cells to the subject, the percentage of peripheral blasts is determined in the subject. In some embodiments, prior toadministering the population of NK cells to the subject, if the subject is determined to have less than or equal to 5% peripheral blasts, the subject is selected for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the percentage of peripheral blasts is determined in the subject, and if the subject is determined to have less than or equal to 5% peripheral blasts, the subject is selected for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the method comprises determining the percentage of peripheral blasts in the subject. In some embodiments, prior to administering the population of NK cells to the subject, if the subject is determined to have less than or equal to 5% peripheral blasts, the method comprises selecting the subject for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the method comprises: (a) determining the percentage of peripheral blasts in the subject; and (b) if the subject is determined to have less than or equal to 5% peripheral blasts, selecting the subject for treatment.

[0110] In some embodiments, the cancer is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). In some embodiments, the cancer is AML. In some embodiments, the cancer is MDS. In some embodiments, the cancer is relapsed / refractory (r / r) AML or very high-risk MDS. In some embodiments, the cancer is r / r AML. In some embodiments, the cancer is very high-risk MDS.

[0111] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a leukemia or a lymphoma. In some embodiments, the cancer is relapsed / refractory (R / R). In some embodiments, the cancer is R / R Acute Myeloid Leukemia (AML). In some embodiments, the cancer is Myelodysplastic Syndrome (MDS).

[0112] In some embodiments, the cancer is a carcinoma, a sarcoma, or a melanoma. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is a sarcoma.

[0113] In some embodiments, the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer. In some embodiments, the cancer is a bladder cancer. In some embodiments, the cancer is a bone cancer. In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a cervical cancer. In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is an endometrial cancer. In some embodiments, the cancer is an esophageal cancer. In some embodiments, the cancer is a gastric cancer. In some embodiments, the cancer is a head and neck cancer. In some embodiments, the cancer is a kidney cancer. In some embodiments, the cancer is a liver cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is a mesothelioma. In some embodiments, the cancer is an ovarian cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is a prostate cancer. In some embodiments, the cancer is a thyroid cancer. In some embodiments, the cancer is a uterine cancer.

[0114] In some embodiments, the cancer comprises liver cancer or colorectal cancer. In some embodiments, the therapeutic agent comprises capecitabine, cisplatin, doxorubicin, regorafenib, or sorafenib. In some embodiments, the therapeutic agent comprises capecitabine. In some embodiments, the therapeutic agent comprises cisplatin. In some embodiments, the therapeutic agent comprises doxorubicin. In some embodiments, the therapeutic agent comprises regorafenib. In some embodiments, the therapeutic agent comprises sorafenib.

[0115] In some embodiments, the cancer comprises bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, or uterine cancer. In some embodiments, the therapeutic agent comprises doxorubicin or gemcitabine. In some embodiments, the therapeutic agent comprises doxorubicin. In some embodiments, the therapeutic agent comprises gemcitabine.

[0116] In some embodiments, the cancer comprises esophageal cancer, head and neck cancer, or lung cancer. In some embodiments, the therapeutic agent comprises gemcitabine, irinotecan, or vinorelbine. In some embodiments, the therapeutic agent comprises gemcitabine. In some embodiments, the therapeutic agent comprises irinotecan. In some embodiments, the therapeutic agent comprises vinorelbine.

[0117] In some embodiments, the cancer comprises melanoma. In some embodiments, the therapeutic agent comprises a MEK inhibitor, a BRAF inhibitor, an anti-CTLA4 antibody, an anti- LAG3 antibody, an anti-PDl antibody, or an anti-PD-Ll antibody. In some embodiments, the therapeutic agent comprises a MEK inhibitor. In some embodiments, the therapeutic agent comprises a BRAF inhibitor.

[0118] In some embodiments, the cancer is a relapsed / refractory (R / R) cancer. In some embodiments, the cancer is relapsed after hematopoietic cell transplantation (HCT). In some embodiments, cells of the cancer have a fms-like tyrosine kinase 3 (FLT3) mutation or a isocitrate dehydrogenase (IDH)l / 2 mutation.

[0119] 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. In some embodiments, if the subject has a targetable FLT3- mutated cancer or a targetable IHD 1 / 2-mutated cancer, the subject has been treated with four prior lines of therapy. In some embodiments, the subject has a targetable FLT3-mutated cancer. In some embodiments, the subject has a targetable IHD 1 / 2-mutated cancer.

[0120] In some embodiments, the subject has an ECOG of 0-2. In some embodiments, the subject has an ECOG of 0 or 1. 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.

[0121] In some embodiments, the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the extracellular binding domain has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the intracellular signaling region comprises a costimulatory domain and a CD3zeta. In some embodiments, the co-stimulatory domain comprises an 0X40 domain. In some embodiments, the chimeric receptor is encoded by a polynucleotide having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 33. In several embodiments, the chimeric receptor has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39. In several embodiments, the chimeric receptor has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39. In several embodiments, the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 39.

[0122] In some embodiments, the genetically engineered NK cells express a membrane -bound interleukin 15 (mbIL15). In several embodiments, the mbIL15 has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36. In several embodiments, the mbIL15 has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38. In several embodiments, the mbIL15 has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In several embodiments, the mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the population of genetically engineered NK cells are allogeneic to the subject. In some embodiments, the population of genetically engineered cells are derived from a subject without cancer.

[0123] 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.

[0124] Also provided herein is use of a combination for treating a cancer in a subject, the combination comprising: (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) and (b) a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof; wherein the genetically engineered NK cells are administered to the subject in a dosing cycle; and wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0125] 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 cancer in a subject, wherein the genetically engineered NK cells are administered to thesubject in a dosing cycle; wherein the subject has been treated with, or is a candidate for treatment with, a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof; and wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0126] Also provided herein is use of a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof for treating a cancer in the subject; wherein the subject has been treated with, or is a candidate for treatment with, 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 to the subject in a dosing cycle; and wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0127] In some embodiments, the therapeutic agent increases expression of a NKG2D ligand in the subject.

[0128] Also provided herein is a combination for treating a cancer in a subject, the combination comprising: (a) 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) and (b) a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0129] 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 cancer in a subject; wherein the subject has been treated with, or is a candidate for treatment with, a therapeutic agent that increases expression of a NKG2D ligand in the subject.

[0130] Also provided herein is use of a therapeutic agent that increases expression of a NKG2D ligand in a subject for treating a cancer in the subject; wherein the subject has been treated with, or is a candidate for treatment with, 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).

[0131] In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, 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, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0132] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is acute myeloid leukemia (AML).

[0133] In some embodiments, the dosing cycle comprises: (i) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject between about 5 days and about 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 between about 5 days and about 10 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 between about 1 x 108NK cells and about 1 x IO10NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 109NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises between about 1.5 x 109NK cells.

[0134] In some embodiments, the first dosing cycle is initiated after the subject is administered a lymphodepleting therapy. In some embodiments, the first dosing cycle is followed by an additional dosing cycle. In some embodiments, the first dosing cycle is followed by two, three, four or more additional dosing cycles. In some embodiments, the additional cycle(s) is be administered depending on the state of the cancer in a subject, e.g., in the event of progression or development of an additional cancer. In several embodiments, an additional cycle is not needed when a subject exhibits a response (e.g., a complete response).BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figures 1A-1B depict non-limiting schematics of polynucleotides encoding cytotoxic receptor constructs comprising a binding moiety for ligands of NKG2D and either encoding mbIL15 (Receptor B) or not (Receptor A).

[0136] Figures 2A-2B depict non-limiting schematics of polynucleotides encoding cytotoxic receptor constructs comprising an NKG2D receptor domain (e.g., fragment) and either encoding mb IL 15 (Receptor B) or not (Receptor A).

[0137] Figures 3A-3B depict non- limiting schematics of dosing cycles according to embodiments disclosed herein. Figure 3A shows a 28-day cycle comprising three dosing events. Figure 3B shows a 28-day cycle comprising two dosing events.

[0138] Figure 4A depicts specific killing of A-431 cells by NKG2D chimeric receptorexpressing NK cells (NKG2D NK) or untransduced NK cells (UT NK) when combined with 10 ug / mL cetuximab or an isotype control antibody.

[0139] Figure 4B depicts specific killing of A-431 cells by NKG2D chimeric receptorexpressing NK cells (NKG2D NK) and cetuximab (cet) in the presence of 20 ug / mL of an anti-CD16 neutralizing antibody (anti-CD16) or an isotype control antibody (iso).

[0140] Figure 5 A shows IC50 curves from specific killing of A-431 cells treated with NKG2D chimeric receptor-expressing NK cells derived from donors having the indicated CD 16 phenotypes, in combination with 10 ug / mL cetuximab or an isotype control antibody.

[0141] Figures 5B-5C shows the percent killing potency increase of NKG2D chimeric receptor-expressing cells in combination with cetuximab when co-cultured with A-431 (5B) or NCI- 112228 cells (5C).

[0142] Figures 6A-6B depict HSA and Loewe synergy scores, respectively for the combination of NKG2D chimeric receptor-expressing NK cells and cetuximab for all six donors.

[0143] Figure 7 shows expression of NucRed™ or NKG2D ligands by NucRed™-labeled UMUC3 cells.

[0144] Figures 8A, 8B, and 8C show expression of NKG2D ligands by UMUC3 cells following 24-hour, 48-hour, or 72-hour treatment, respectively, with gemcitabine at the indicated concentrations.

[0145] Figure 9 shows the IC50 curve of gemcitabine against UMUC3 cells.

[0146] Figures 10A, 10B, and 10C show the cytotoxicity of NKG2D chimeric receptorexpressing natural killer (NKG2D NK) cells against UMUC3 cells at various effector-to-target ratios (E:T), in the absence (- gem) or presence (+ gem) of 24-hour, 48-hour, or 72-hour treatment, respectively, with gemcitabine.DETAILED DESCRIPTION

[0147] Some embodiments of the methods and compositions provided herein relate to engineered immune cells, therapeutic agents, and combinations of the same for use in immunotherapy. In several embodiments, the engineered cells are engineered in multiple ways, for example, to express a cytotoxicity-inducing receptor complex. As used herein, the term “cytotoxic receptor complexes” shall be given its ordinary meaning and shall also refer to (unless otherwise indicated), Chimeric Antigen Receptors (CAR), chimeric receptors (also called activating chimeric receptors in the case of NKG2D chimeric receptors). In several embodiments, the cells are further engineered to achieve a modification of the reactivity of the cells against non-tumor tissue and / or other therapeutic cells.I. Cell Types

[0148] Some embodiments of the methods and compositions provided herein relate to a cell such as an immune cell. For example, an immune cell, such as an NK cell or a T cell, may be engineered to include a chimeric receptor such as a NKG2D-ligand-directed chimeric receptor, or engineered to include a nucleic acid encoding said chimeric receptor as described herein. Additional embodiments relate to engineering a second set of cells to express another cytotoxic receptor complex, such as an NKG2D chimeric receptor complex as disclosed herein.

[0149] 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 genesor 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. In some embodiments, the immune cells comprise T cells. In some embodiments, the immune cells comprise NK cells. In some embodiments, the immune cells comprise T cells and NK cells.

[0150] 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 extracellular domain that is directed against a tumor marker, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others, to facilitate targeting of an immune cell to a cancer and exerting cytotoxic effects on the cancer cell. Also provided are engineered immune cells (e.g., NK cells and / or T cells) expressing such chimeric receptors. There are also provided herein, in several embodiments, polynucleotides, polypeptides, and vectors that encode a construct comprising an extracellular domain comprising two or more subdomains, e.g., first and second ligand binding receptor and a cytotoxic signaling complex. Also provided are engineered immune cells (e.g., NK cells and / or T cells) expressing such bi-specific constructs (in some embodiments the first and second ligand binding domain target the same ligand). Methods of treating cancer and other uses of such cells for cancer immunotherapy are also provided for herein.Engineered Cells for Immunotherapy

[0151] In several embodiments, cells of the immune system are engineered to have enhanced cytotoxic effects against target cells, such as tumor cells. For example, a cell of the immune system may be engineered to include a tumor-directed chimeric receptor and / or a tumor-directed CAR 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. White blood 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 antigen receptor, such as a NKG2D ligand-directed chimeric receptor, or a nucleic acidencoding the chimeric receptor. In several embodiments, the immune cells engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain. As discussed in more detail below, in several embodiments, the therapeutic cells, are further genetically modified enhance the cytotoxicity and / or persistence of the cells. In several embodiments, the genetic modification enhances the ability of the cell to resist signals emanating from the tumor microenvironment that would otherwise cause a reduced efficacy or shortened lifespan of the therapeutic cells.Monocytes for Immunotherapy

[0152] In some embodiments, the immune cells comprise monocytes. 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 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 an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, the monocytes engineered to express a chimeric receptor are also engineered to also express (e.g., bicistronically express) a membrane- bound interleukin 15 (mbIL15) domain. In several embodiments, the monocytes are autologous cells. In some embodiments, the monocytes are allogeneic cells.Lymphocytes for Immunotherapy

[0153] In some embodiments, the immune cells comprise lymphocytes. 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). Thus, in some embodiments, the immune cells comprise T cells. Thus, in some embodiments, the immune cells comprise NK cells. In some embodiments, the immune cells comprise B cells. While B cells are engineered according to several embodiments, disclosed herein, several embodiments also relate to engineered T cells or engineered NK cells (mixtures of T cells and NK cells are used in some embodiments, either from the same donor, or different donors). Several embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, the lymphocytes engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express)a membrane -bound interleukin 15 (mbIL15) domain. In several embodiments, the lymphocytes are autologous cells. In some embodiments, the lymphocytes are allogeneic cells.T Cells for Immunotherapy

[0154] In some embodiments, the immune cells are T cells. 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, 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 or an infectious disease, comprising administering a therapeutically effective amount of T cells expressing the cytotoxic receptor complex and / or a homing moiety as described herein. In several embodiments, the T cells are autologous cells. In some embodiments, the T cells are allogeneic cells. Several embodiments of the methods and compositions disclosed herein relate to T cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, T cells engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (tnbIL15) co-stimulatory domain.NK Cells for Immunotherapy

[0155] In some embodiments, the immune cells comprise NK cells. Thus, in several embodiments, there is provided a method of treating or preventing cancer or an infectious disease, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing the cytotoxic receptor complex and / or a homing moiety as described herein. In several embodiments, there is provided a method of treating a cancer comprising administering a therapeutically effective amount of natural killer (NK) cells expressing the cytotoxic receptor complex. In several embodiments, the NK cells are autologous cells. In some embodiments, the NK cells are allogeneic cells. In severalembodiments, 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). Several embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, NK cells engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane -bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, NK cells are engineered to express (e.g., bicistronically express) a chimeric receptor (e.g., a NKG2D ACR) and mbIL15.

[0156] 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 upregulation of interferon-y (IFNy), granzyme B, and / or perforin production. Additional information relating to the NK-92 cell 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

[0157] In some embodiments, the immune cells are hematopoietic stem cells (HSCs). Thus, in some embodiments, HSCs are used in the methods of immunotherapy disclosed herein. In several embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. HSCs are used, in several embodiments, to leverage their ability to engraft for long-term blood cell production, which could result in a sustained source of targeted anti-cancer effector cells, for example to combat cancer remissions. In several embodiments, this ongoing production helps to offset anergy or exhaustion of other cell types, for example due to the tumor microenvironment. In several embodiments allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In several embodiments, HSCs are used in combination with one or more additional engineered cell type disclosed herein. Several embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6(among others). In some embodiments, the HSCs engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane -bound interleukin 15 (mbIL15) domain.Induced Pluripotent Stem Cells

[0158] In some embodiments, NK, T, or other immune cells derived from pluripotent stem cells (iPSCs) are used in the method of immunotherapy disclosed herein. In some embodiments, induced pluripotent stem cells (iPSCs) are used in the method of immunotherapy disclosed herein. iPSCs are used, in several embodiments, to leverage their ability to differentiate and derive into non-pluripotcnt 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. In several embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. In several embodiments, iPSCs are used in combination with one or more additional engineered cell type disclosed herein. Several embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, the iPSCs engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane -bound interleukin 15 (mbIL15) co-stimulatory domain. In several embodiments, the engineered iPSCs are differentiated into NK, T, or other immune cells, such as for use in a composition or method provided herein.II. Extracellular domains (Tumor binder)

[0159] Some embodiments of the compositions and methods described herein relate to a chimeric receptor that includes an extracellular domain that comprises a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain) as described herein. Several embodiments of the compositions and methods described herein relate to a chimeric receptor that includes an extracellular domain that comprises a ligand binding domain that binds a ligand expressed by a tumor cell (also referred to as an activating chimeric receptor) as described herein. In some embodiments, the ligand binding domain binds to a ligand of NKG2D. The ligand binding domain, depending on the embodiment, targets for example MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others).

[0160] In some embodiments, the antigen-binding domain is derived from or comprises wildtype 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.Antigen-Binding Proteins

[0161] There are provided, in several embodiments, antigen-binding proteins. As used herein, the term “antigen-binding protein” 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 antigenbinding 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 antigenbinding fragment in some embodiments is an antibody fragment.

[0162] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., an antigen-binding fragment of an antibody), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, a single-chain variable fragment (scFv), a nanobody (e.g. VH domain of camelid heavy chain antibodies; VHH fragment,), a Fab fragment, a Fab' fragment, a F(ab')j 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 protein 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 aswell 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.

[0163] In some embodiments, the antigen-binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or into multiple polypeptide chains. For instance, antigen-binding proteins 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-lgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0164] In some embodiments, the antigen-binding protein 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.

[0165] 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.

[0166] 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.

[0167] 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 their naturally occurring conformations. Kappa (K) and lambda ( / -) light chains refer to the two major antibody light chain isotypes. A light chain may include a polypeptide comprising, from amino terminus to carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL).

[0168] Heavy chains are classified as mu (p), delta (A), gamma (y), alpha (a), and epsilon (e), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. An antibody “heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibodybelongs. 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 (CHI), 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).

[0169] The IgG-class is further divided into subclasses, namely, IgGl, IgG2, IgG3, and IgG4. The IgA-class is further divided into subclasses, namely IgAl and IgA2. The IgM has subclasses including, but not limited to, IgMl and IgM2. The heavy chains in IgG, IgA, and IgD antibodies have three domains (CHI, CH2, and CH3), whereas the heavy chains in IgM and IgE antibodies have four domains (CHI, 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 interpolypeptide disulfide bonds between the CL domain and the CHI domain (e.g., between the light and heavy chain) and between the hinge regions of the antibody heavy chains.

[0170] In some embodiments, the antigen-binding protein is 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 same or 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.

[0171] In some embodiments, the antigen-binding protein is 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’)z, 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 VE 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’)z, 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.

[0172] In some embodiments, Fab fragments are provided. A Fab fragment is a monovalent fragment having the VE, VH, CL and CHI 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 CHI 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, singlechain 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.

[0173] 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 synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site). 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 areidentical, 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.

[0174] In several embodiments, the antigen-binding protein 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 as appropriate. One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen-binding protein.

[0175] In some embodiments, the antigen-binding proteins provided herein comprise one or more CDR(s) as part of a larger polypeptide chain. In some embodiments, the antigen-binding proteins covalently link the one or more CDR(s) to another polypeptide chain. In some embodiments, the antigen- binding proteins incorporate the one or more CDR(s) noncovalently. In some embodiments, the antigen-binding proteins may comprise at least one of the CDRs described herein incorporated into abiocompatible 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 nonhuman primate or other mammal, other vertebrate, invertebrate, plant, bacteria or virus.

[0176] 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, CPI zinc finger, PST1, coiled coil, LACI-D1, Z domain and / or tendamistat domains.

[0177] There is also provided, in some embodiments, antigen-binding proteins 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 protein 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 antigen receptor can impart to an engineered cell the ability to target multiple tumor markers. For example, MICA, MICE, 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.Natural Killer Group Domains that Bind Tumor Ligands

[0178] 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.

[0179] 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 low in healthy cells but is upregulated upon, for example, malignant transformation. Non-limiting examples of ligands recognized by NKG2D include, but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, as well as other molecules expressed on target cells that control the cytolytic or cytotoxic function of NK cells. In several embodiments, T cells are engineered to express an extracellular domain to binds to one or more tumor ligands and activates the T cell. For example, in several embodiments, T cells are engineered to express an NKG2D receptor as the binder / activation moiety. In several embodiments, NK cells are engineered to express an extracellular domain to binds to one or more tumor ligands and activates the NK cell. For example, in several embodiments, NK cells are engineered to express an NKG2D receptor as the binder / activation moiety. In several embodiments, engineered cells as disclosed herein are engineered to express another member of the NKG2 family, e.g., NKG2A, NKG2C, and / or NKG2E. Combinations of such receptors are engineered in some embodiments. Moreover, in several embodiments, other receptors are expressed, such as the Killer-cell immunoglobulin-like receptors (KIRs).

[0180] In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a full length NKG2D as an extracellular component to recognize ligands on the surface of tumor cells (e.g., liver cells). In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a functional fragment of NKG2D (e.g., human NKG2D) as an extracellular component to recognize ligands on the surface of tumor cells (e.g., liver cells). In one embodiment, full length NKG2D (e.g., full length human NKG2D) has the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, full length NKG2D (e.g., full length human NKG2D) has the amino acid sequence of SEQ ID NO: 43. In several embodiments, the full length NKG2D, or functional fragment thereof is 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.

[0181] In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a functional fragment of NKG2D as an extracellular component to recognize ligands on the surface of tumor cells or other diseased cells. In one embodiment, the functional 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-encoding nucleic acid sequence may have one or more additional mutations from SEQ ID NO: 25, but retains, or in some embodiments, has enhanced, ligand-binding function. In several embodiments, the functional fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26. 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 optionally fully or partially codon optimized. In one embodiment, a sequence encoding a codon optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28. Advantageously, according to several embodiments, the functional fragment lacks its nativetransmembrane or intracellular domains 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 DAP 10 to localize NKG2D to the cell membrane is not required. Thus, in several embodiments, the cytotoxic receptor complex encoded by the polypeptides disclosed herein does not comprise DAP10. In several embodiments, immune cells, such as NK or T cells (e.g., non-alloreactive T cells engineered according to embodiments disclosed herein), are engineered to express one or more chimeric receptors that target, for example CD70, CD19, CD123, Her2, mesothelin, Claudin 6, BCMA, EGFR, and an NKG2D ligand, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. Such cells, in several embodiments, also express (e.g., bicistronically express) mbIL15.

[0182] In several embodiments, the cytotoxic receptor complexes 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 cytotoxic receptor complexes (and hence the NK cells expressing the receptor), resulting in a reduction in (or lack) of adverse toxic effects. In several embodiments, the cytotoxic receptor complexes employ internal dimers, or repeats of one or more component subunits. For example, in several embodiments, the cytotoxic receptor complexes may optionally comprise a first NKG2D extracellular domain coupled to a second NKG2D extracellular domain, and a transmembrane / signaling region (or a separate transmembrane region along with a separate signaling region).

[0183] In several embodiments, the various domains / subdomains 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). In several embodiments, the various domains / subdomains are separated by a linker comprising the sequence of SEQ ID NO: 44. 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. 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.III. Cytotoxic Signaling Complex

[0184] Some embodiments of the compositions and methods described herein relate to a chimeric receptor, such as a chimeric receptor directed against an NKG2D ligand, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6) that includes a cytotoxic signaling complex. As disclosed herein, according to several embodiments, the provided chimeric receptors comprise one or more transmembrane and / or intracellular domains that initiate cytotoxic signaling cascades upon the extracellular domain(s) binding to ligands on the surface of target cells. Thus, in some embodiments, the chimeric receptor comprises an extracellular binding domain, a transmembranedomain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain (e.g., CD3zeta) and a co-stimulatory signaling domain.

[0185] In several embodiments, the chimeric receptor comprises at least one transmembrane domain, at least one co-stimulatory domain, and / or at least one signaling domain. In some embodiments, more than one component part makes up a given domain - e.g., a co-stimulatory domain may comprise two subdomains. Moreover, in some embodiments, a domain may serve multiple functions, for example, a transmembrane domain may also serve to provide signaling function.IV. Transmembrane Domains

[0186] Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or ligand-directed chimeric receptors) that comprise a transmembrane domain. Some embodiments include a transmembrane domain from NKG2D or another transmembrane protein. In several embodiments in which a transmembrane domain is employed, the portion of the transmembrane protein employed retains at least a portion of its normal transmembrane domain.

[0187] In several embodiments, however, 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 CD8a. In several embodiments, the transmembrane domain comprises a CD8 (e.g., CD8 a) hinge and a CD8 (e.g., CD8 a) transmembrane region.

[0188] In several embodiments, the transmembrane domain comprises a “hinge,” e.g., a CD8(X hinge. In several embodiments, the hinge of CD8a has the nucleic acid sequence of SEQ ID NO: 1. In several embodiments, the CD8a hinge 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 CD8a hinge having the sequence of SEQ ID NO: 1. In several embodiments, the hinge of CD8a comprises the amino acid sequence of SEQ ID NO: 2. In several embodiments, the CD8a hinge can be truncated or modified, such that it 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: 2.

[0189] In several embodiments, the transmembrane domain comprises a CD8a transmembrane region. In several embodiments, the CD8a transmembrane region is encoded by a nucleic acid sequence comprises the sequence of SEQ ID NO: 3. In several embodiments, the CD8a transmembrane region 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 with the sequence of SEQ ID NO: 3. In several embodiments, the CD8a transmembrane region comprises the amino acid sequence of SEQ ID NO: 4. In several embodiments, the CD8a transmembrane region is truncated or modifiedand has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD8a having the sequence of SEQ ID NO: 4.

[0190] Taken together in several embodiments, the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 13. In several embodiments, the CD8 transmembrane domain 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 CD8 transmembrane domain sequence of SEQ ID NO: 13. In several embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 14. In several embodiments, the CD8 transmembrane domain 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 to the sequence of SEQ ID NO: 14.

[0191] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof. In several embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 30. In several embodiments, the CD28 transmembrane domain 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 to the sequence of SEQ ID NO: 30.V. Stimulatory Molecules

[0192] In some embodiments, the intracellular signaling domain of a chimeric receptor provided herein comprises a stimulatory molecule. Thus, some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors) that comprise a stimulatory molecule. In addition to the various transmembrane domains and signaling domains (and the combination transmembrane / signaling domains), additional stimulatory molecules can be provided, in several embodiments. These can be certain molecules that, for example, further enhance 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 membrane bound, acting as autocrine stimulatory molecules (or even as paracrine stimulators to neighboring cells).

[0193] 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 chimeric receptor and IL15 arc 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 chimeric receptor and IL15 are separated by a T2A sequence. In someembodiments, the T2A sequence comprises SEQ ID NO: 10. In some embodiments, the T2A sequence comprises SEQ ID NO: 45.

[0194] In some embodiments, the IL15 is a 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 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 comprises a peptide 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.

[0195] In some embodiments, IL15 is membrane-bound by virtue of its coupling to at least one transmembrane domain. In some embodiments, the at least one transmembrane domain comprises a CD8 transmembrane domain (e.g., 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 hinge sequence (e.g., SEQ ID NO: 14). In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain (e.g., SEQ ID NO: 4) and a CD8 hinge (e.g., SEQ ID NO: 2). In some embodiments, the mhIL15 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.

[0196] In some embodiments, the tumor antigen-directed CARs and / or tumor ligand-directed 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 tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptor 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 various constituent parts of an engineered cytotoxic receptor complex can be delivered to an NK cell or T cell in a single vector or by multiple vectors. Thus, as shown schematically, in the Figures, a construct can be encoded by a single polynucleotide, but also include a cleavage site, such that downstream elements of the constructs are expressed by the cells as a separate protein (as is the case in some embodiments with IL-15). 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 comprises the amino acid sequence of SEQ ID NO: 45. In severalembodiments, the T2A cleavage site is truncated or modified. In some embodiments, 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. In some embodiments, 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: 45.

[0197] 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 CAR. 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. Membranebound 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 IL 15 sequences.VI. Signaling Domains

[0198] Some embodiments of the compositions and methods described herein relate to a chimeric receptor (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors) that includes an intracellular signaling domain. 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 intracellular signaling domain comprises the CD3zeta subunit. In several embodiments, the CD3zeta is encoded by the nucleic acid sequence of SEQ ID NO: 7. In several embodiments, the CD3zeta 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 to SEQ ID NO: 7. In several embodiments, the CD3zeta domain comprises the amino acid sequence of SEQ ID NO: 8. In several embodiments, the CD3zetadomain is truncated or modified. In some embodiments, the CD3zeta domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD3zeta domain having the sequence of SEQ ID NO: 8.

[0199] In several embodiments, unexpectedly enhanced signaling is achieved through the use of multiple signaling domains whose activities act synergistically. For example, in several embodiments, the intracellular signaling domain further comprises an 0X40 domain. In several embodiments, the 0X40 domain is an intracellular signaling domain. In several embodiments, the 0X40 intracellular signaling domain is encoded by a nucleic acid that comprises the sequence of SEQ ID NO: 5. In several embodiments, the 0X40 intracellular signaling domain 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 to SEQ ID NO: 5. In several embodiments, the 0X40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 6. In several embodiments, the 0X40 intracellular signaling domain is truncated or modified. In some embodiments, the 0X40 intracellular signaling domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 6. In several embodiments, 0X40 is used as the sole signaling domain in the chimeric receptor construct, however, in several embodiments, 0X40 can be used with one or more other domains. For example, combinations of 0X40 and CD3zeta are used in some embodiments. For example, in some embodiments, the intracellular signaling domain comprises CD3zeta and 0X40, or a signaling portion thereof. In some embodiments, the intracellular signaling domain comprises CD3zeta and 0X40. By way of further example, combinations of CD28, 0X40, 4-1BB, and / or CD3zeta are used in some embodiments.

[0200] In several embodiments, the signaling domain comprises a 4- IBB domain. In several embodiments, the 4-1BB domain is an intracellular signaling domain. In several embodiments, the 4- 1BB domain is encoded by the nucleic acid sequence of SEQ ID NO: 29. In several embodiments, the 4- IBB domain 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 to SEQ ID NO: 29. In several embodiments, the 4- IBB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 30. In several embodiments, the 4- IBB intracellular signaling domain is truncated or modified. In several embodiments, the 4- IBB intracellular signaling domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the 4- IBB intracellular signaling domain having the sequence of SEQ ID NO: 30. In several embodiments, 4- IBB is used as the sole signaling domain in the chimeric receptor construct, however, in several embodiments, 4-1BB can be used with one or more other domains. For example, combinations of 4- IBB andCD3zeta are used in some embodiments. For example, in some embodiments, the intracellular signaling domain comprises CD3zeta and 4- IBB, or a signaling portion thereof. In some embodiments, the intracellular signaling domain comprises CD3zeta and 4- IBB. Byway of further example, combinations of CD28, 0X40, 4-1BB, and / or CD3zeta are used in some embodiments.

[0201] In several embodiments, the signaling domain comprises a CD28 domain. In several embodiments the CD28 domain is an intracellular signaling domain. In several embodiments, the CD28 intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 31. In several embodiments, the CD28 intracellular signaling domain 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 to SEQ ID NO: 32. In several embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 32. In several embodiments, the CD28 intracellular signaling domain is truncated or modified. In several embodiments, the CD28 intracellular signaling domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 32. In several embodiments, CD28 is used as the sole signaling domain in the construct, however, in several embodiments, CD28 can be used with one or more other domains. For example, combinations of CD28 andCD3zeta are used in some embodiments. For example, in some embodiments, the intracellular signaling domain comprises CD3zeta and CD28, or a signaling portion thereof. In some embodiments, the intracellular signaling domain comprises CD3zeta and CD28. By way of further example, combinations of CD28, 0X40, 4-1BB, and / or CD3zeta are used in some embodiments.VII. Cytotoxic Receptor Complex Constructs[00202J Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as an activating chimeric receptor (ACR) that targets ligands of NKG2D. The expression of these cytotoxic receptor complexes in immune cells, such as genetically modified non- alloreactive T cells and / or NK cells, allows the targeting and destruction of particular target cells, such as cancerous cells. Non-limiting examples of such cytotoxic receptor complexes are discussed in more detail below.

[0203] In several embodiments, there is provided a polynucleotide encoding a tumor binder / CD8hinge-CD8TM / OX40 / CD3zeta chimeric receptor complex (see Figure 2A, Chimeric Receptor A). The polynucleotide comprises or is composed of a NKG2D ligand binding moiety, a CD8a hinge, a CD8a transmembrane domain, an 0X40 domain, and a CD3zeta domain. In several embodiments, the polynucleotide further encodes a 2A cleavage site, and an mbIL-15 domain as described herein (see Figure 2A, Chimeric Receptor B, representing the polynucleotide structure where a single polynucleotide encodes both the receptor and the mbIL15). In several embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In several embodiments, the encoding nucleic acidsequence, 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 IDs provided for herein, or a portion thereof (e.g. a portion excluding the mbIL15 sequence and / or self-cleaving peptide sequence).

[0204] In several embodiments, there is provided a polynucleotide encoding an NKG2D / CD8a hinge / CD8a transmembrane domain / QX40 / CD3zeta activating chimeric receptor complex (see Figure 2B, NKG2D ACR A). The polynucleotide comprises or is composed of a fragment of the NKG2D receptor capable of binding a ligand of the NKG2D receptor, a CD8alpha hinge, a CD8a transmembrane domain, an 0X40 domain, and a CD3zeta domain 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: 40. 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: 40. In several embodiments, while the chimeric receptor amino acid sequence may vary from SEQ ID NO: 40, 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 (Figure 2B, NKG2D ACR B, representing the polynucleotide structure where a single polynucleotide encodes both the receptor and the mbIL15). 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 least85%, 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.

[0205] 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.VIII. Methods of Treatment

[0206] Some embodiments relate to a method of treating, ameliorating, inhibiting, or preventing cancer with a cell or immune cell comprising a chimeric antigen receptor and / or an activating chimeric receptor, as disclosed herein. Some embodiments relate to a method of treating, ameliorating, inhibiting, or preventing cancer with a cell or immune cell comprising a chimeric receptor provided herein, in combination with a therapeutic agent. In some embodiments, the method includes treating or preventing cancer. In some embodiments, the method includes administering a therapeutically effective amount of immune cells expressing a tumor-directed chimeric antigen receptor and / or tumor-directed chimeric receptor as described herein. Examples of types of cancer that may be treated as such are described herein.

[0207] Disclosed herein are methods of treating cancer in a subject. In some embodiments, the methods comprise administering to the subject any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, or any one of the cells disclosed herein, or any combination thereof, and a therapeutic agent.

[0208] 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.

[0209] In some embodiments, the therapeutic agent is administered prior to, concurrent with, and / or after administration of genetically engineered cells.

[0210] In some embodiments, the therapeutic agent is administered prior to administration of genetically engineered cells. In some embodiments, the therapeutic agent is administered prior to treatment with a lymphodcplcting therapy. In some embodiments, the therapeutic agent is administered after treatment with a lymphodepleting therapy and prior to administration of the genetically engineered cells. In some embodiments, the therapeutic agent is administered concurrently with administration ofgenetically engineered cells. In some embodiments, the therapeutic agent is administered after administration of genetically engineered cells.

[0211] In certain embodiments, treatment of a subject with genetically engineered cell(s) and a therapeutic agent as described herein achieves one, two, three, four, or more of the following effects, including, for example: (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 (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy. Advantageously, the non-alloreactive engineered T cells disclosed herein further enhance one or more of the above.

[0212] Administration of the engineered cells can be by a variety of routes, including, without limitation, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to an affected tissue. Administration of the therapeutic agent can be by a variety of routes, including, without limitation, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to an affected tissue.

[0213] Also disclosed herein are uses of any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, any one of the cells disclosed herein, and a therapeutic agent for the treatment of cancer.

[0214] Also disclosed herein are uses of any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, any one of the cells disclosed herein, and a therapeutic agent in the manufacture of a medicament for the treatment of cancer.IX. Selection of Subjects

[0215] Provided herein are methods for selecting subjects for treatment. In some aspects, the methods for selecting subjects are used to identify subjects who are likely to exhibit a clinical response (e.g., a partial response (PR), a complete response with incomplete hematologic recovery (CRi) or a complete response (CR)) to the treatment. In some embodiments, identified subjects are selected for treatment and / or administered the treatment.NKG2D Ligand( s )

[0216] In some embodiments, the methods comprise selecting a subject having a level or amount of a NKG2D ligand above a reference value. For example, in some cases, a subject having a level or amount of a NKG2D ligand above the reference value is predicted to exhibit a clinical responseto treatment. Conversely, a subject having a level or amount of expression of a NKG2D ligand below the reference value would not be predicted to exhibit a clinical response to treatment. In some embodiments, the subject has acute myeloid leukemia (AML), optionally relapsed / refractory AML (r / r AML). In some embodiments, the treatment comprises administration of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D.

[0217] In some embodiments, the comparison of a level or amount of a NKG2D ligand to a reference value of the NKG2D ligand allows for the assessment, measurement, and / or determination of the probability and / or likelihood of a clinical outcome (e.g. CR, PR, or PD) following administration of and / or associated with a treatment. In some embodiments, the level or amount of a NKG2D ligand in a biological sample is compared to a reference value, e.g., a NKG2D ligand reference value. In some embodiments, the reference value is a value of a level, amount, or concentration of the NKG2D ligand. In some embodiments, the reference value is or is derived from an amount or level of an RNA gene product or a protein gene product. In particular embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, that indicate a likelihood of a clinical response (e.g. CR or PR) and / or an increased, elevated, or high probability of a clinical response (e.g. CR or PR), following administration of a treatment and values or measurements of the NKG2D ligand gene or protein product that indicate an absent or low likelihood and / or a decreased, reduced, or low probability of a clinical response (e.g. CR or PR), following administration of a treatment. In some embodiments, the reference value is a boundary, divide, and / or threshold value between the amounts or levels of the NKG2D ligand gene or protein product where a majority of one or more clinical responses take place or have previously taken place and amounts or levels of the NKG2D ligand gene or protein product where a minority of one or more clinical responses take place or previously taken place.

[0218] In certain embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, associated with a particular type of clinical response from amounts or levels associated with one or more other types of clinical response. In particular embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, associated with clinical response (e.g., CR and / or PR) from the amounts or levels that are associated with no clinical response (e.g., PD). In particular embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, associated with CR or PR from the amounts or levels that are associated with other clinical responses, e.g., NR / SD or PD.

[0219] In some embodiments, the reference value is a predetermined value. In particular embodiments, the reference value has been calculated and / or derived from data from a study. In some embodiments, the study is a clinical study. In particular embodiments, the clinical study is a completed clinical study. In certain embodiments, the data from the study included NKG2D ligand expression, e.g., expression of a NKG2D ligand gene or protein product, in samples taken or obtained from subjects in the study. In particular embodiments, the data from the study includes the number and types of clinical responses experienced by subjects during the study. In certain embodiments, the subjects in the clinical study had or have a clinical response, such as CR or PR. In some embodiments, the clinical response is CR. In certain embodiments, the data from the study includes the number and types of diseases or conditions, such as cancer (e.g. AML). In particular embodiments, the data from the study includes the number and types of treatment experienced by subjects during the study. In certain embodiments, the subjects are or were treated with NK cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D.

[0220] In some embodiments, the expression of a NKG2D ligand gene or protein product is compared to a reference value and an elevated, increased and / or high probability and / or likelihood of a clinical response (e.g., CR or PR) is indicated. In particular embodiments, the expression of a NKG2D ligand gene or protein product is compared to a reference value and a reduced, decreased and / or low probability and / or likelihood of a clinical response (e.g., CR or PR) is indicated.

[0221] In some embodiments, the reference value is within 25%, within 20%, within 15%, within 10%, or within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 25% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 20% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 15% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 15% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 10% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical responsefollowing administration of the treatment. In some embodiments, the reference value is the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment.

[0222] In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is above the reference value, the subject is identified as likely to exhibit a clinical response to the treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is above the reference value, the subject is selected for treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is above the reference value, the subject is administered the treatment.

[0223] In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is below the reference value, the subject is identified as not likely to exhibit a clinical response to the treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is below the reference value, the subject is not selected for treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is below the reference value, the subject is not administered the treatment.

[0224] In some embodiments, the level of amount of a NKG2D ligand is assessed in a biological sample from the subject. In some embodiments, the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells. In some embodiments, the biological sample comprises a blood sample. In some embodiments, the biological sample is a peripheral blood sample. In some embodiments, the biological sample comprises a bone marrow sample. In some embodiments, the biological sample is a bone marrow biopsy.

[0225] In some embodiments, the biological sample is obtained from the subject within about 14 days, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 14 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 10 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 7 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 6 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 5 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 4 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 3 days prior to administration of thegenetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 2 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 1 day prior to administration of the genetically engineered NK cells to the subject.

[0226] In some embodiments, the NKG2D ligand comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA. In some embodiments, the NKG2D ligand comprises MICA and MICB. In some embodiments, the NKG2D ligand comprises MICA and MICB. In some embodiments, the NKG2D ligand comprises ULBP1. In some embodiments, the NKG2D ligand comprises ULBP2. In some embodiments, the NKG2D ligand comprises ULBP3. In some embodiments, the NKG2D ligand comprises ULBP4. In some embodiments, the NKG2D ligand comprises ULBP5. In some embodiments, the NKG2D ligand comprises ULBP6. In some embodiments, the NKG2D ligand comprises ULBP1 and ULBP3. In some embodiments, the NKG2D ligand comprises ULBP2, ULBP5, and ULBP6. In some embodiments, the NKG2D ligand comprises MICA, MIB, and ULBP1. In some embodiments, the NKG2D ligand comprises MICA, MIB, and ULBP3. In some embodiments, the NKG2D ligand comprises MICA, MIB, ULBP1, and ULBP3.

[0227] Methods for determining the amount or level of a NKG2D ligand protein product are known in the art. Suitable methods for assessing, measuring, determining, and / or quantifying the level, amount, or concentration of one or more protein products include, but are not limited to detection with immunoassays, nucleic acid-based or protein-based aptamer techniques, high precision liquid chromatography (HPLC), peptide sequencing, and microarray adaptations of any of the foregoing (including nucleic acid, antibody or protein-protein (i.e., non-antibody) arrays). In some embodiments, the immunoassay is or includes methods or assays that detect proteins based on an immunological reaction, e.g., by detecting the binding of an antibody or antigen binding antibody fragment to a gene product. Immunoassays include, but are not limited to, quantitative immunocytochemistry or immunohistochemistry, ELISA (including direct, indirect, sandwich, competitive, multiple and portable ELISAs), Western blotting (including one, two or higher dimensional blotting or other chromatographic means, optionally including peptide sequencing), enzyme immunoassay, radioimmunoassay, and surface plasmon resonance. In some embodiments, the level or amount of a NKG2D ligand protein product is determined by the number or percentage of cells positive for the NKG2D ligand protein product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand protein product is determined by the intensity of expression of the NKG2D ligand protein product among cells positive for the NKG2D ligand protein product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand protein product is determined by the number or percentage of cells positive for the NKG2D ligand protein product in a biological sample and the intensity of expression of the NKG2D ligand protein product among cells positive for the NKG2D ligand protein product in a biological sample.

[0228] Methods for determining the amount or level of a NKG2D ligand gene product are also known in the art. Suitable methods for assessing, measuring, determining, and / or quantifying the level, amount, or concentration of one or more gene products include, but are not limited to polymerase chain reaction (PCR), including reverse transcriptase (rt) PCR, droplet digital PCR, real-time and quantitative PCR (qPCR) methods, Northern blotting; Southern blotting, e.g., of reverse transcription products and derivatives; array based methods, including blotted arrays, microarrays, or in situ-synthesized arrays; and sequencing, e.g., sequencing by synthesis, pyrosequencing, dideoxy sequencing, or sequencing by ligation, or any other methods known in the art. In some embodiments, the level or amount of a NKG2D ligand gene product is determined by the number or percentage of cells positive for the NKG2D ligand gene product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand gene product is determined by the intensity of expression of the NKG2D ligand gene product among cells positive for the NKG2D ligand gene product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand gene product is determined by the number or percentage of cells positive for the NKG2D ligand gene product in a biological sample and the intensity of expression of the NKG2D ligand gene product among cells positive for the NKG2D ligand gene product in a biological sample.Peripheral Blasts

[0229] In some embodiments, the methods comprise selecting a subject having marrow limited disease (also known as marrow localized disease) and / or less than or equal to a particular percentage of peripheral blasts (blasts in peripheral blood) for treatment. For example, in some cases, a subject having less than or equal to 5% peripheral blasts (e.g., without evidence of extramedullary disease) is predicted to exhibit a clinical response to treatment. Conversely, a subject having greater than 5% peripheral blasts would not be predicted to exhibit a clinical response to treatment. In some embodiments, the subject has acute myeloid leukemia (AML), optionally relapsed / refractory AML (r / r AML). In some embodiments, the subject has relapsed following HCT. In some embodiments, the treatment comprises administration of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D.

[0230] Methods for assessing the percentage of blasts in peripheral blood of a subject are known in the art.

[0231] In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining whether a subject has marrow limited disease and / or selecting a subject for treatment that has marrow limited disease. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining what a subject has marrow limited disease. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises selecting a subject for treatment that has marrow limited disease. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining whetherthe subject has marrow limited disease and selecting the subject for treatment if the subject has marrow limited disease.

[0232] In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining the percentage of peripheral blasts in a subject and / or selecting a subject for treatment that has less than or equal to 5% peripheral blasts. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining the percentage of peripheral blasts in a subject. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises selecting a subject for treatment that has less than or equal to 5% peripheral blasts. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining the percentage of peripheral blasts in a subject and selecting the subject for treatment if the subject that has less than or equal to 5% peripheral blasts.

[0233] In some embodiments, the subject has marrow limited disease (also known as marrow localized disease). In some embodiments, marrow limited disease is defined as less than or equal to 5% peripheral blasts with no evidence of extramedullary disease. Thus, in some embodiments, the subject has less than or equal to 5% peripheral blasts. Thus, in some embodiments, the subject has less than or equal to 5% peripheral blasts with no evidence of extramedullary disease. In some embodiments, the subject has less than 5% peripheral blasts. In some embodiments, the subject has less than 5% peripheral blasts with no evidence of extramedullary disease. In some embodiments, at the time of administration of the lymphodepleting therapy, the subject has less than or equal to 5% peripheral blasts. In some embodiments, at the time of administration of the first dose of the dosing cycle, the subject has less than or equal to 5% peripheral blasts. In some embodiments, at the time of administration of the lymphodepleting therapy, the subject has less than 5% peripheral blasts. In some embodiments, at the time of administration of the first dose of the dosing cycle, the subject has less than 5% peripheral blasts.X. Administration and Dosing

[0234] Further provided herein arc methods of treating a subject having cancer, comprising administering to the subject a composition comprising immune cells (such as NK and / or T cells) engineered to express a cytotoxic receptor complex as disclosed herein. In some embodiments, a therapeutic agent is also administered to the subject. For example, some embodiments of the compositions and methods described herein relate to use of (a) a tumor-directed chimeric antigen receptor and / or tumor-directed chimeric receptor, or use of cells expressing a tumor-directed chimeric antigen receptor and / or tumor-directed chimeric receptor; and (b) a therapeutic agent, for treating a cancer patient. Uses of such engineered immune cells and a therapeutic agent for treating cancer are also provided.

[0235] In certain embodiments, treatment of a subject with genetically engineered cell(s) and a therapeutic agent as described herein achieves one, two, three, four, or more of the following effects,including, for example: (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 (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy. Each of these comparisons are versus, for example, a different therapy for a disease, which includes a cell-based immunotherapy for a disease using cells that do not express the constructs disclosed herein. Advantageously, the non-alloreactive engineered T cells disclosed herein further enhance one or more of the above.

[0236] Administration can be by a variety of routes, including, without limitation, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to an affected tissue. The cells (in particular, NK cells and / or T cells) engineered to express a chimeric receptor complex described herein can be formulated for parenteral administration by injection, e.g., by bolus injection or infusion.

[0237] Doses of immune cells such as NK and / or T 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., I (f- 107, 1O7-1O10, 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 and / or T cells is administered, for example between about 1 x 106cells / kg to about 1 x 108cells / kg.

[0238] In several embodiments, 1 x 108NK cells are administered (2 x 106 / kg for subject under 50kg) three times over a 28-day cycle. In several embodiments, 3 x 108NK cells are administered three times over a 28-day cycle. In several embodiments, 1 x 109NK cells are administered three times over a 28-day cycle.

[0239] In several embodiments, 1.5 x 108NK cells are administered (3 x 106 / kg for a subject under 50kg) two times over a 28-day cycle. In several embodiments, 4.5 x 108NK cells are administered two times over a 28-day cycle. In several embodiments, 1.5 x 109NK cells are administered two times over a 28-day cycle.

[0240] In several embodiments, 1.5 x 109NK cells are administered (3 x 107 / kg for a subject under 50kg) three times over a 28-day cycle. In several embodiments, 3 x 109NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 x 1010NK cells are administered three times over a 28-day cycle. In several embodiments, at least 4.5 x 109NK cells are administered over the cycle.

[0241] 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 toxicides. In several embodiments, a determination about receiving / administering an additional dosing cycle is made at an evaluation 30 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 5 additional cycles are given to a subject.

[0242] 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.

[0243] 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.

[0244] In several embodiments, cytosine arabinoside (Ara-C) is used. 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.

[0245] In several embodiments, the additional agent is fludarabine. 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 / nr, 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 at about 3 days. In several embodiments, the dose of fludarabine is given daily for at about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[0246] 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.

[0247] In several embodiments, the additional agent is cyclophosphamide. In several embodiments, a dose of between about 100 mg / m2- about 100 mg / m2fludarabine is administered, 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 at about 3 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[0248] 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.

[0249] 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.

[0250] In several embodiments, a subject is subject to lymphodepletion at least one time prior to administration of genetically engineered cells as disclosed herein. In several embodiments, lymphodepletion is performed before one or more additional doses of engineered cells are administered. In several embodiments, a dosing cycle is used that comprises lymphodepletion followed by at least two doses of engineered cells as disclosed herein, with the two doses separated by a time interval. In several embodiments, the time interval is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more days (including intervals falling between the time marking a price interval since the last administration, e.g., 84 hours, or 3.5 days). In several embodiments, the dosing cycle itself is approximately 14, 21, 28, 35, 42 or more days. In several embodiments, three doses are administered, ~1 week apart from each other. In several embodiments, two doses are administered ~1 week apart from one another. In several embodiments, a subject receives a first dose on day 0 of the cycle, a second dose on day 7 of the cycle and a third dose on day 14 of the cycle. In several such embodiments, a 28- day cycle is used with primary outcome measures evaluated at day 28 (see e.g., Figure 3A). In several embodiments, a subject receives a first dose on day 0 of the cycle and a second dose on day 7 of the cycle. In several such embodiments, a 28-day cycle is used with primary outcome measures evaluated at day 28 (See e.g., Figure 3B).

[0251] 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. Clinical responses may include complete response (CR; e.g., complete remission), complete response with incomplete hematologic recovery (CRi; e.g., CR with residual thrombocytopenia), morphologic leukemia-free state (MLFS), and partial response (PR; e.g., partial remission). Methods for assessing clinical response are known in the art (Dohner et al., Blood (2017) 129(4):424-47).

[0252] 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 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.

[0253] 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. 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.

[0254] 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. 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.

[0255] 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.

[0256] In some embodiments, among subjects treated according to the method, the overall response rate (ORR) is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65%. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of subjects treated according to the method exhibit a CR or a CRi. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or atleast about 65% of subjects treated according to the method exhibit a CR. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of subjects treated according to the method exhibit a CRi. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of subjects treated according to the method exhibit MLFS. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of subjects treated according to the method exhibit a PR.

[0257] 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 a second 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.

[0258] Depending on the embodiment, various types of cancer can be treated. In some embodiments, the cancer is a NKG2D ligand-expressing cancer. 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 myelodysplastic syndrome. In several embodiments, the cancer is a solid cancer. Additional embodiments provided for herein include treatment or prevention of the following nonlimiting examples of cancers including, but not limited to, acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi sarcoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including but not limited to astrocytomas, spinal cord tumors, brain stem glioma, glioblastoma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell leukemia, renal cell cancer, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to, non-small cell lung cancer, (NSCLC) and small cell lung cancer), pancreatic cancer, bowel cancer, melanoma, ocular cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is livercancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is melanoma.

[0259] 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. 1-38 (or combinations of two or more of SEQ ID NOS: 1-38) and that also exhibit one or more of the functions as compared with the respective SEQ ID NOS. 1-38 (or combinations of two or more of SEQ ID NOS: 1-38) 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, but not limited to IFNg, TNFa, IL-22, CCL3, CCL4, and CCL5), (vii) enhanced ability to stimulate further innate and adaptive immune responses, and (viii) combinations thereof. 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. 1-45 (or combinations of two or more of SEQ ID NOS: 1-45) and that also exhibit one or more of the functions as compared with the respective SEQ ID NOS. 1-45 (or combinations of two or more of SEQ ID NOS: 1-45).

[0260] 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.

[0261] In several embodiments, polynucleotides encoding the disclosed cytotoxic receptor complexes 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 cytotoxic receptor complex.

[0262] Additionally provided, according to several embodiments, is a vector comprising the polynucleotide encoding any of the polynucleotides provided for herein, wherein the polynucleotides are optionally operatively linked to at least one regulatory element for expression of a cytotoxic receptor complex. In several embodiments, the vector is a retrovirus.

[0263] Further provided herein are engineered immune cells (such as NK and / or T cells) comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein. Furtherprovided herein are compositions comprising a mixture of engineered immune cells (such as NK cells and / or engineered T cells), each population comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein.XL Cancer Types and Combination Therapies

[0264] Some embodiments of the compositions and methods described herein relate to administering immune cells comprising a tumor-directed chimeric antigen receptor and / or tumor- directed chimeric receptor to a subject with cancer. Several embodiments provided for herein relate to combination therapies wherein immune cells comprising a tumor-directed chimeric antigen receptor and / or tumor-directed chimeric receptor to a subject with cancer in conjunction with a therapeutic agent (an additional anti-cancer agent). Several embodiments provided for herein relate to combination therapies wherein immune cells comprising a tumor-directed chimeric antigen receptor and / or tumor- directed chimeric receptor to a subject with a solid tumor in conjunction with a therapeutic agent (an additional anti-cancer agent).Cancer Types

[0265] Various embodiments provided for herein include treatment or prevention of the following non-limiting examples of cancers. In some embodiments, the cancer is a NKG2D ligandexpressing cancer. In some embodiments, the cancer is a solid tumor. In several embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a leukemia or a lymphoma. Examples of cancer include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi sarcoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including but not limited to astrocytomas, spinal cord tumors, brain stem glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell leukemia, renal cell cancer, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to, non-small cell lung cancer, (NSCLC) and small cell lung cancer), pancreatic cancer, bowel cancer, melanoma, ocular cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the canceris endometrial cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is melanoma.

[0266] In several embodiments, acute myeloid leukemia (AML) is treated using genetically engineered immune cells in combination with a therapeutic agent (as described more fully below). In several embodiments, the cancer is AML. In several embodiments, the cancer is r / r AML. In some embodiments, the cancer is relapsed to HCT.

[0267] In several embodiments, myelodysplastic syndrome (MDS) is treated using genetically engineered immune cells in combination with a therapeutic agent (as described more fully below). In several embodiments, the cancer is MDS. In several embodiments, the cancer is very high-risk MDS.

[0268] In several embodiments, a solid tumor is treated using genetically engineered immune cells in combination with a therapeutic agent (as described more fully below).

[0269] In several embodiments, breast cancer is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0270] In several embodiments, cervical cancer (e.g., cervical squamous cell carcinoma and / or endocervical adenocarcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0271] In several embodiments, uterine corpus endometrial carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0272] In several embodiments, ovarian serous carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0273] In several embodiments, bladder urothelial carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0274] In several embodiments, colorectal cancer (e.g., colorectal carcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0275] In several embodiments, rectal adenocarcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0276] In several embodiments, gastric cancer is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0277] In several embodiments, a head and neck cancer (e.g., head and neck squamous cell carcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0278] In several embodiments, esophageal carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0279] In several embodiments, hepatocellular carcinoma (HCC) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0280] In several embodiments, lung cancer (e.g., lung squamous cell carcinoma and / or lung adenocarcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0281] In several embodiments, melanoma (e.g., skin cutaneous melanoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[0282] In particular embodiments, the cancer to be treated is one that exhibits an upregulation of NKG2D ligands. In several embodiments, the cancer is associated with a current or previous viral infection. For example, in several embodiments, the cancer to be treated is selected from one or more of head and neck squamous cell carcinoma (HNSCC), cervical carcinoma, esophageal carcinoma, and lung squamous cell carcinoma.

[0283] In some embodiments, the additional anti -cancer agent is a therapeutic agent that upregulates a NKG2D ligand in the subject; or is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, or any combination thereof, or both. Examples of additional anti-cancer agents are described in further detail in the section below.Therapeutic Agents

[0284] In several embodiments, subjects who are administered the genetically engineered immune cells are also administered a therapeutic agent (an additional anti-cancer agent). In several embodiments, the therapeutic agent comprises a chemotherapeutic agent. In several embodiments, the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly (ADP-ribose) polymerase (PARP), or a combination thereof.

[0285] In some embodiments, the chemotherapeutic agent comprises an antimetabolite. In some embodiments, the antimetabolite comprises methotrexate, pemetrexed, cytarabine, 5-fluorouracil (5-FU), capecitabine, gemcitabine, 6-mercaptopurine (6-MP), azathioprine, fludarabine, cladribine, hydroxyurea, or any combination thereof. In some embodiments, the antimetabolite comprises methotrexate. In some embodiments, the antimetabolite comprises pemetrexed. In some embodiments, the antimetabolite comprises cytarabine. In some embodiments, the antimetabolite comprises 5-FU. In some embodiments, the antimetabolite comprises capecitabine. In some embodiments, the therapeutic agent comprises gemcitabine. In some embodiments, the antimetabolite comprises 6-MP. In some embodiments, the antimetabolite comprises azathioprine. In some embodiments, the antimetabolite comprises fludarabine. In some embodiments, the antimetabolite comprises cladribine. In some embodiments, the antimetabolite comprises hydroxyurea. In some embodiments, the therapeutic agent comprises fluorouracil and leucovorin (FOL). In some embodiments, the therapeutic agent comprises capecitabine and ovaliplatin (CAPOX).

[0286] In some embodiments, the chemotherapeutic agent comprises an alkylating agent. In some embodiments, the alkylating agent comprises cyclophosphamide, ifosfamide, chlorambucil, melphalan, temozolomide, carmustine, lomustine, streptozocin, busulfan, procarbazine, cisplatin, carboplatin, oxaliplatin, or any combination thereof. In some embodiments, the therapeutic agent comprises cisplatin. In some embodiments, the therapeutic agent comprises bendamustine. In some embodiments, the therapeutic agent comprises cyclophosphamide. In some embodiments, the therapeutic agent comprises dacarbazine. In some embodiments, the therapeutic agent comprises procarbazine. In some embodiments, the alkylating agent comprises ifosfamide. In some embodiments, the alkylating agent comprises chlorambucil. In some embodiments, the alkylating agent comprises melphalan. In some embodiments, the alkylating agent comprises temozolomide. In some embodiments, the alkylating agent comprises carmustine. In some embodiments, the alkylating agent comprises lomustine. In some embodiments, the alkylating agent comprises streptozocin. In some embodiments, the alkylating agent comprises busulfan. In some embodiments, the alkylating agent comprises carboplatin. In some embodiments, the alkylating agent comprises oxaliplatin. In some embodiments, the therapeutic agent comprises capecitabine and oxaliplatin (CAPOX).

[0287] In some embodiments, the therapeutic agent comprises a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor comprises irinotecan, topotecan, etoposide, or any combination thereof. In some embodiments, the topoisomerase inhibitor comprises irinotecan. In some embodiments, the topoisomerase inhibitor comprises topotecan. In some embodiments, the therapeutic agent comprises etoposide.

[0288] In some embodiments, the therapeutic agent comprises a mitotic inhibitor. In some embodiments, the mitotic inhibitor comprises vincristine, vinblastine, vinorelbine, docetaxel, paclitaxel, eribulin, izabepilone, epothilone, or any combination thereof. In some embodiments, the therapeutic agent comprises vincristine. In some embodiments, the mitotic inhibitor comprises vinorelbine. In some embodiments, the therapeutic agent comprises vinblastine. In some embodiments, the therapeutic agent comprises paclitaxel. In some embodiments, the therapeutic agent comprises docetaxel. In some embodiments, the mitotic inhibitor comprises eribulin. In some embodiments, the mitotic inhibitor comprises izabepilone. In some embodiments, the mitotic inhibitor comprises epothilone.

[0289] In some embodiments, the therapeutic agent comprises an antibiotic. In some embodiments, the antibiotic comprises bleomycin; actinomycin D; an anthracycline, optionally doxorubicin, daunorubicin, or idarubicin, mitomycin, or any combination thereof. In some embodiments, the therapeutic agent comprises bleomycin. In some embodiments, the therapeutic agent comprises actinomycin D. In some embodiments, the therapeutic agent comprises an anthracycline. In some embodiments, the therapeutic agent comprises doxorubicin. In some embodiments, the therapeutic agent comprises daunorubicin. In some embodiments, the therapeutic agent comprises idarubicin. In some embodiments, the therapeutic agent comprises mytomycin.

[0290] In some embodiments, the therapeutic agent comprises a protein kinase inhibitor. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BCR-ABL, c-KIT, EGFR, VEGF, ALK, BRAF, MEK, BTK, JAK, CDK, or any combination thereof. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BCR-ABL. In some embodiments, the protein kinase inhibitor comprises an inhibitor of c-Kit. In some embodiments, the protein kinase inhibitor comprises an inhibitor of EGFR. In some embodiments, the protein kinase inhibitor comprises an inhibitor of VEGF. In some embodiments, the protein kinase inhibitor comprises an inhibitor of ALK. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BRAF. In some embodiments, the protein kinase inhibitor comprises an inhibitor of MEK. In some embodiments, the protein kinase inhibitor comprises an inhibitor of BTK (e.g., ibrutinib). In some embodiments, the protein kinase inhibitor comprises an inhibitor of JAK. In some embodiments, the protein kinase inhibitor comprises an inhibitor of CDK. In some embodiments, the therapeutic agent comprises afatinib. In some embodiments, the therapeutic agent comprises dabrafenib. In some embodiments, the therapeutic agent comprises crizotinib. In some embodiments, the therapeutic agent comprises vemurafenib.

[0291] In some embodiments, the therapeutic agent comprises an inhibitor of BCL2 (e.g., venetoclax). In some embodiments, the therapeutic agent comprises venetoclax. In some embodiments, the therapeutic agent comprises azacitadine. In some embodiments, the therapeutic agent comprises venetoclax and azacitadine. In some embodiments, the therapeutic agent comprises a glucocorticoid (e.g., prednisone). In some embodiments, the therapeutic agent comprises prednisone.

[0292] In some embodiments, the therapeutic agent comprises a proteasome inhibitor. In some embodiments, the proteasome inhibitor comprises bortezomib, carfilzomib, ixazomib, or any combination thereof. In some embodiments, the proteasome inhibitor comprises bortezomib. In some embodiments, the proteasome inhibitor comprises carfilzomib. In some embodiments, the proteasome inhibitor comprises ixazomib.

[0293] In some embodiments, the therapeutic agent comprises a PARP inhibitor. In some embodiments, the PARP inhibitor comprises olaparib, niraparib, rucaparib, or any combination thereof. In some embodiments, the PARP inhibitor comprises olaparib. In some embodiments, the therapeutic agent comprises niraparib. In some embodiments, the PARP inhibitor comprises rucaparib.

[0294] In several embodiments, the therapeutic agent comprises a HD AC inhibitor. In some embodiments, the HDAC inhibitor comprises tricostatin A (TSA), valproic acid, sodium butyrate, sodium valproate (VPA), FR901228, MS-275, phenylbutyrate, PDX101, suberoylanilide hydroxamic acid, or any combination thereof. In some embodiments, the HDAC inhibitor comprises TSA. In some embodiments, the HDAC inhibitor comprises valproic acid. In some embodiments, the HDAC inhibitor comprises sodium buryrate. In some embodiments, the HDAC inhibitor comprises VPA. In some embodiments, the HDAC inhibitor comprises FR901228. In some embodiments, the HDAC inhibitor comprises MS-275. In some embodiments, the HDAC inhibitor comprises phenylbutyrate. In someembodiments, the HD AC inhibitor comprises PDX101. In some embodiments, the HD AC inhibitor comprises suberoylanilide hydroxamic acid.

[0295] In several embodiments, the therapeutic agent comprises a small molecule.

[0296] In several embodiments, the therapeutic agent comprises a monoclonal antibody. In several embodiments, the monoclonal antibody comprises an anti-CD20 antibody, an anti-CTLA4 antibody, an anti-EGFR antibody, an anti-HER2 / neu antibody, an anti-PDl antibody, an anti-PD-Ll antibody, an anti-VEGF antibody, or any combination thereof. In several embodiments, the monoclonal antibody comprises an anti-CD20 antibody (e.g., rituximab). In several embodiments, the monoclonal antibody comprises an anti-CTLA4 antibody. In several embodiments, the monoclonal antibody comprises an anti-EGFR antibody (e.g., cetuximab). In several embodiments, the monoclonal antibody comprises an anti-HER2 / neu antibody. In several embodiments, the monoclonal antibody comprises an anti-PDl antibody. In several embodiments, the monoclonal antibody comprises an anti-PD-Ll antibody. In several embodiments, the monoclonal antibody comprises an anti-VEGF antibody.

[0297] In several embodiments, the therapeutic agent is a hormone therapy or a therapeutic agent that disrupts or modifies a hormone pathway. In some embodiments, the therapeutic agent comprises tamoxifen. In several embodiments, the therapeutic agent comprises hyperthermia. In several embodiments, the therapeutic agent is an additional form of immunotherapy. In several embodiments, the therapeutic agent comprises photodynamic therapy (PDT). In several embodiments, the therapeutic agent comprises radiation therapy. In several embodiments, the therapeutic agent comprises transplanted stem cells.

[0298] In several embodiments, the therapeutic agent increases a ligand of an NKG2D receptor. In some embodiments, the ligand comprises MICA, MICB, ULBP1, 2, 3, 4, 5 and / or 6.

[0299] In some embodiments, the therapeutic agent comprises decitabine. In some embodiments, a dose of decitabine is administered to the subject daily for between about 1 day and about 10 days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of decitabine is administered to the subject daily for five days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of decitabine is between about 10 mg / m2and about 30 mg / m2. In some embodiments, a dose of decitabine is about 20 mg / m2. In some embodiments, decitabine is administered intravenously (IV).

[0300] In some embodiments, the therapeutic agent comprises metformin. In some embodiments, the therapeutic agent comprises gefitinib. In some embodiments, the therapeutic agent comprises erlotinib. In some embodiments, the therapeutic agent comprises sunitinib. In some embodiments, the therapeutic agent comprises trabectedin. In some embodiments, the therapeutic agent comprises sulforaphane.

[0301] In several embodiments, the therapeutic agent is an NK cell engager (e.g., a molecule that binds both an antigen expressed by cells of the cancer and an antigen expressed by NK cells). In several embodiments, the NK cell engager binds to an activating receptor on an NK cell and an antigenexpressed by cells of the cancer. In some embodiments, the activating receptor on the NK cell is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof. In several embodiments, the NK cell engager binds to CD 16. In several embodiments, the NK cell engager binds to NKp30. In several embodiments, the NK cell engager binds to NKp46. In several embodiments, theNK cell engager binds to NKG2D.

[0302] In several embodiments, the therapeutic agent administered is cisplatin. In several embodiments, cisplatin is administered as a IV bolus, or as a series of infusions. In several embodiments, the dose of cisplatin ranges from about 20 mg / m2to about 150 mg / m2. In several embodiments, the dose of cisplatin ranges from about 20 mg / m2to about 30 mg / m2. In several embodiments, the dose of cisplatin ranges from about 30 mg / m2to about 40 mg / m2. In several embodiments, the dose of cisplatin ranges from about 40 mg / m2to about 50 mg / m2. In several embodiments, the dose of cisplatin ranges from about 50 mg / m2to about 60 mg / m2. In several embodiments, the dose of cisplatin ranges from about 60 mg / m2to about 70 mg / m2. In several embodiments, the dose of cisplatin ranges from about 70 mg / m2to about 80 mg / m2. In several embodiments, the dose of cisplatin ranges from about 80 mg / m2to about 90 mg / m2. In several embodiments, the dose of cisplatin ranges from about 90 mg / m2to about 100 mg / m2. In several embodiments, the dose of cisplatin ranges from about 100 mg / m2to about 1 10 mg / m2. In several embodiments, the dose of cisplatin ranges from about 110 mg / m2to about 120 mg / m2. In several embodiments, the dose of cisplatin ranges from about 120 mg / m2to about 130 mg / m2. In several embodiments, the dose of cisplatin ranges from about 130 mg / m2to about 140 mg / m2. In several embodiments, the dose of cisplatin ranges from about 140 mg / m2to about 150 mg / m2.

[0303] Depending on the embodiment, cisplatin is administered IV on a weekly basis for 3, 4, 5, or 6 total administrations. In several embodiments, cisplatin is administered every 3 to weeks for a total of 3 doses. In several embodiments, cisplatin is administered daily for 5 days. In several embodiments, a series of 3 to 4 doses comprises a cycle, and optionally more than one cycle is administered. In several embodiments, more than one cycle is administered.

[0304] In several embodiments, cisplatin is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, cisplatin is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, cisplatin is administered concurrently with engineered immune cells. In several embodiments, cisplatin is administered after administration of engineered immune cells.

[0305] In several embodiments, the therapeutic agent administered is sorafenib. In several embodiments, sorafenib is administered orally (e.g., in a tableted form). In several embodiments, the dose of sorafenib ranges from about 200 mg to about 800 mg per day. In several embodiments, the dose of sorafenib ranges from about 200 mg to about 250 mg per day. In several embodiments, the dose of sorafenib ranges from about 250 mg to about 300 mg per day. In several embodiments, thedose of sorafenib ranges from about 300 mg to about 350 mg per day. In several embodiments, the dose of sorafenib ranges from about 350 mg to about 400 mg per day. In several embodiments, the dose of sorafenib ranges from about 400 mg to about 450 mg per day. In several embodiments, the dose of sorafenib ranges from about 450 mg to about 500 mg per day. In several embodiments, the dose of sorafenib ranges from about 500 mg to about 550 mg per day. In several embodiments, the dose of sorafenib ranges from about 550 mg to about 600 mg per day. In several embodiments, the dose of sorafenib ranges from about 600 mg to about 650 mg per day. In several embodiments, the dose of sorafenib ranges from about 650 mg to about 700 mg per day. In several embodiments, the dose of sorafenib ranges from about 700 mg to about 750 mg per day. In several embodiments, the dose of sorafenib ranges from about 750 mg to about 800 mg per day. In several embodiments, the total dose is broken into two administrations per day, for example separated by 8 to 12 hours. For example, for a total daily dose of 400 mg, a 200 mg administration (e.g., tablet) is ingested by a subject in the morning and a second 200 mg administration is ingested in the evening.

[0306] In several embodiments, sorafenib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, sorafenib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, sorafenib is administered concurrently with engineered immune cells. In several embodiments, sorafenib is administered after administration of engineered immune cells.

[0307] In several embodiments, the therapeutic agent administered is regorafenib. In several embodiments, regorafenib is administered orally (e.g., in a tableted form). In several embodiments, the dose of regorafenib ranges from about 100 mg to about 200 mg per day. In several embodiments, the dose of regorafenib ranges from about 100 mg to about 110 mg per day. In several embodiments, the dose of regorafenib ranges from about 110 mg to about 120 mg per day. In several embodiments, the dose of regorafenib ranges from about 120 mg to about 130 mg per day. In several embodiments, the dose of regorafenib ranges from about 130 mg to about 140 mg per day. In several embodiments, the dose of regorafenib ranges from about 150 mg to about 160 mg per day. In several embodiments, the dose of regorafenib ranges from about 160 mg to about 170 mg per day. In several embodiments, the dose of regorafenib ranges from about 170 mg to about 180 mg per day. In several embodiments, the dose of regorafenib ranges from about 180 mg to about 190 mg per day. In several embodiments, the dose of regorafenib ranges from about 190 mg to about 200 mg per day. In several embodiments, the total dose is broken into two administrations per day, for example separated by 8 to 12 hours. For example, for a total daily dose of 160 mg, an 80 mg administration (e.g., two 40 mg tablets) is ingested by a subject in the morning and a second 80 mg administration is ingested in the evening (e.g., two additional 40 mg tablets).

[0308] In several embodiments, regorafenib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In severalembodiments, regorafenib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, regorafenib is administered concurrently with engineered immune cells. In several embodiments, regorafenib is administered after administration of engineered immune cells.

[0309] In several embodiments, the therapeutic agent administered is atezolizumab. In several embodiments, the atezolizumab is administered as an IV infusion for a duration of 60 minutes. If 60 minutes is tolerated by a subject a shorter, 30-minute, infusion is optionally used. Depending on the embodiment, the dose of atezolizumab varies with the amount of elapsed time between administrations. For example, in several embodiments two weeks are allowed to elapse between administrations. A lower dose is used in such embodiments based on the higher frequency of administration. In several embodiments, three weeks are allowed to elapse between administrations. An intermediate dose is used in such embodiments based on the intermediate frequency of administration. In several embodiments, four weeks are allowed to lapse between administrations. A higher dose is used in such embodiments based on the lower frequency of administration.

[0310] In several embodiments employing administration every two weeks, the atezolizumab is administered in a dose ranging from about 800 to about 900 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 800 to about 810 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 810 to about 820 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 820 to about 830 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 830 to about 840 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 840 to about 850 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 850 to about 860 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 860 to about 870 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 870 to about 880 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 880 to about 890 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 890 to about 900 mg.

[0311] In several embodiments employing administration every three weeks, the atezolizumab is administered in a dose ranging from about 1000 to about 1400 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1000 to about 1050 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1050 to about 1100 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1100 to about 1150 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1150 to about 1200 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1200 to about 1250 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1250 to about 1300 mg. In several embodiments, the atezolizumab is administeredin a dose ranging from about 1300 to about 1350 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1350 to about 1400 mg.

[0312] In several embodiments employing administration every four weeks, the atezolizumab is administered in a dose ranging from about 1500 to about 1800 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1500 to about 1550 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1550 to about 1600 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1600 to about 1640 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1640 to about 1680 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1680 to about 1700 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1700 to about 1750 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1750 to about 1800 mg.

[0313] In several embodiments, atezolizumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, atezolizumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, atezolizumab is administered concurrently with engineered immune cells. In several embodiments, atezolizumab is administered after administration of engineered immune cells.

[0314] In several embodiments, the therapeutic agent administered is bevacizumab. In several embodiments, the bevacizumab is administered as an IV infusion. Depending on the embodiment, bevacizumab is infused every two weeks or every three weeks. In several embodiments, the bevacizumab is administered in a dose ranging from about 2.5 mg / kg to about 20 mg / kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 2.5 mg / kg to about 5 mg / kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 5 mg / kg to about 7.5 mg / kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 7.5 mg / kg to about 10 mg / kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 10 mg / kg to about 12.5 mg / kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 12.5 mg / kg to about 15 mg / kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 15 mg / kg to about 17.5 mg / kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 17.5 mg / kg to about 20 mg / kg.

[0315] According to several embodiments, the bevacizumab is optionally combined with one or more of cisplatin, paclitaxel, topotecan, interferon (e.g., interferon alpha), paclitaxel and carboplatin, atezolizumab, pegylated liposomal doxorubicin, carboplatin, gemcitabine, fluoropyrimidine-based chemotherapy, 5 -fluorouracil (5-FU) / leucovorin (LV)Zirinotecan (IFL), and 5-FU / LV / oxaliplatin (FOLFOX4).

[0316] In several embodiments, bevacizumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, bevacizumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, bevacizumab is administered concurrently with engineered immune cells. In several embodiments, bevacizumab is administered after administration of engineered immune cells.

[0317] In several embodiments, the therapeutic agent administered is levantinib. In several embodiments, levantinib is administered orally (e.g., in a tableted form). In several embodiments, the dose of levantinib ranges from about 1 mg to about 2 mg per day. In several embodiments, the dose of levantinib ranges from about 1 mg to about 2 m per day. In several embodiments, the dose of levantinib ranges from about 2 mg to about 3 mg per day. In several embodiments, the dose of levantinib ranges from about 3 mg to about 4 mg per day. In several embodiments, the dose of levantinib ranges from about 4 mg to about 5 mg per day. In several embodiments, the dose of levantinib ranges from about 5 mg to about 6 mg per day. In several embodiments, the dose of levantinib ranges from about 6 mg to about 7 mg per day. In several embodiments, the dose of levantinib ranges from about 7 mg to about 8 mg per day. In several embodiments, the dose of levantinib ranges from about 8 mg to about 9 mg per day. In several embodiments, the dose of levantinib ranges from about 9 mg to about 10 r per day. In several embodiments, the dose of levantinib ranges from about 10 mg to about 11 m ’ per day. In several embodiments, the dose of levantinib ranges from about 11 mg to about 12 mg per day. In several embodiments, the total dose is broken into two administrations per day, for example separated by 8 to 12 hours. For example, for a total daily dose of 10 mg, a 5mg administration (e.g., a 5 mg tablet) is ingested by a subject in the morning and a second 5 mg administration is ingested in the evening (e.g., an additional 5 mg tablet).

[0318] In several embodiments, the levantinib is optionally administered in conjunction with a dose of between about 150 mg and about 300 mg of pembrolizumab (on a 3 week cycle) or a dose of between about 350 mg and about 500 mg of pembrolizumab (on a 6 week cycle). In several embodiments, the dose of pembrolizumab ranges from about 150 mg to about 200 mg. In several embodiments, the dose of pembrolizumab ranges from about 200 mg to about 250 mg. In several embodiments, the dose of pembrolizumab ranges from about 250 mg to about 300 mg. In several embodiments, the dose of pembrolizumab ranges from about 350 mg to about 400 mg. In several embodiments, the dose of pembrolizumab ranges from about 400 mg to about 450 mg. In several embodiments, the dose of pembrolizumab ranges from about 450 mg to about 500 mg.

[0319] In several embodiments, levantinib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, levantinib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, levantinib is administered concurrently with engineeredimmune cells. In several embodiments, levantinib is administered after administration of engineered immune cells.

[0320] In several embodiments, the therapeutic agent administered is nivolumab. In several embodiments, the nivolumab is administered as an IV infusion. Depending on the embodiment, nivolumab is infused every two weeks or every four weeks. With the higher frequency dosing schedule, nivolumab is administered in an amount ranging from about 200 to about 300 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 200 mg to about 210 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 210 mg to about 220 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 220 mg to about 230 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 230 mg to about 240 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 240 mg to about 250 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 250 mg to about 260 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 260 mg to about 270 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 270 mg to about 280 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 280 mg to about 290 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 290 mg to about 300 mg. With the lower frequency dosing schedule, the nivolumab is administered in an amount ranging from about 400 mg to about 500 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 400 mg to about 410 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 410 mg to about 420 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 420 mg to about 430 mg. In several embodiments, the nivolumab is administered in an amount between about 430 mg to about 440 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 440 mg to about 450 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 450 mg to about 460 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 460 mg to about 470 mg. In several embodiments, the nivolumab is administered in an amount between about 470 mg to about 480 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 480 mg to about 490 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 490 mg to about 500 mg.

[0321] In several embodiments, an intermediate 3-week administration cycle is used, with the nivolumab administered in an amount ranging from about 300 mg and about 400 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 300 mg to about 310 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 310 mg to about 320 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 320 mg to about 330 mg. In several embodiments, the nivolumab is administered in an amountranging from about 330 mg to about 340 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 340 mg to about 350 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 350 mg to about 360 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 360 mg to about 370 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 370 mg to about 380 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 380 mg to about 390 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 390 mg to about 400 mg.

[0322] In several embodiments, the nivolumab is administered in conjunction with another agent, such as ipilimumab. In several such embodiments, the nivolumab is administered in an amount ranging from about 1 mg / kg to about 5 mg / kg. In several embodiments, the nivolumab is administered in an amount ranging from about 1 mg / kg to about 2 mg / kg. In several embodiments, the nivolumab is administered in an amount ranging from about 2 mg / kg to about 3 mg / kg. In several embodiments, the nivolumab is administered in an amount ranging from about 3 mg / kg to about 4 mg / kg. In several embodiments, the nivolumab is administered in an amount ranging from about 4 mg / kg to about 5 mg / kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 0.5 mg / kg to about 1.5 mg / kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 0.5 mg / kg to about .75 mg / kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 0.75 mg / kg to about 1.0 mg / kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 1.0 mg / kg to about 1.5 mg / kg. In several embodiments, the ipilimumab is administered on the same day as a nivolumab infusion for 2, 3, 4, or 5 doses, and then nivolumab is administered alone on an ongoing basis at a dose as described above.

[0323] In several embodiments, nivolumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, nivolumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, nivolumab is administered concurrently with engineered immune cells. In several embodiments, nivolumab is administered after administration of engineered immune cells.

[0324] In several embodiments, the therapeutic agent administered is pembrolizumab. In several embodiments, the pembrolizumab is administered as an IV infusion. Depending on the embodiment, nivolumab is infused every two weeks or every four weeks. With the higher frequency dosing schedule, pembrolizumab is administered in an amount ranging from about 150 to about 250 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 150 mg to about 160 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 160 mg to about 170 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 170 mg to about 180 mg. In several embodiments, the pembrolizumabis administered in an amount ranging from about 180 mg to about 190 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 190 mg to about 200 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 200 mg to about 210 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 210 mg to about 220 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 220 mg to about 230 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 230 mg to about 240 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 240 mg to about 250 mg. With the lower frequency dosing schedule, the pembrolizumab is administered in an amount ranging from about 350 mg to about 400 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 350 mg to about 360 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 360 mg to about 370 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 370 mg to about 380 mg. In several embodiments, the pembrolizumab is administered in an amount between about 380 mg to about 390 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 390 mg to about 400 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 400 mg to about 410 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 410 mg to about 420 mg. In several embodiments, the pembrolizumab is administered in an amount between about 420 mg to about 430 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 430 mg to about 440 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 440 mg to about 450 mg.

[0325] In several embodiments, pembrolizumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, pembrolizumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, pembrolizumab is administered concurrently with engineered immune cells. In several embodiments, pembrolizumab is administered after administration of engineered immune cells.

[0326] In several embodiments, the therapeutic agent administered is ipilimumab. In several embodiments, the ipilimumab is administered as an IV infusion. In several embodiments, the ipilimumab is infused every three weeks. In some embodiments, ipilimumab is administered on a ten- week cycle (for example after several three-week cycles). In several embodiments, ipilimumab is administered in an amount ranging from about 1 mg / kg to about 10 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 1 mg / kg to about 2 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 2 mg / kg to about 3 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 3 mg / kg to about 4 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 4 mg / kgto about 5 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 5 mg / kg to about 6 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 6 mg / kg to about 7 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 7 mg / kg to about 8 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 8 mg / kg to about 9 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 9 mg / kg to about 10 mg / kg.

[0327] In several embodiments, ipilimumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, ipilimumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, ipilimumab is administered concurrently with engineered immune cells. In several embodiments, ipilimumab is administered after administration of engineered immune cells.

[0328] In several embodiments, ipilimumab is optionally administered in conjunction with nivolumab, as discussed above.

[0329] In several embodiments, the therapeutic agent administered is ramucirumab. In several embodiments, the ramucirumab is administered as an IV infusion. In several embodiments, the ramucirumab is infused every two weeks. In some embodiments, ipilimumab is administered on a 21 - day cycle (e.g., on day 1). In several embodiments, ramucirumab is administered on a 28-day cycle (e.g., on days 1 and 15). In several embodiments, ramucirumab is administered in an amount ranging from about 5 mg / kg to about 15 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 5 mg / kg to about 6 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 6 mg / kg to about 7 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 7 mg / kg to about 8 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 8 mg / kg to about 9 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 9 mg / kg to about 10 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 10 mg / kg to about 11 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 11 mg / kg to about 12 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 12 mg / kg to about 13 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 13 mg / kg to about 14 mg / kg. In several embodiments, ramucirumab is administered in an amount ranging from about 14 mg / kg to about 15 mg / kg.

[0330] In several embodiments, ramucirumab is optionally administered in conjunction with additional agents. For example, in several embodiments, ramucirumab is administered in conjunction with docetaxel (e.g., at a dose of about 50, about 75, or about 100 mg / m2on day 1 of a 28-day cycle). For example, in several embodiments, ramucirumab is administered in conjunction with eroltinib (e.g., at a dose of about 100, about 150, or about 150 mg daily). For example, in several embodiments,ramucirumab is administered in conjunction with paclitaxel (e.g., at a dose of about 60, about 80, or about 100 mg / m2on days 1, 8, and 15 of a 28-day cycle).

[0331] In several embodiments, ramucirumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, ramucirumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, ramucirumab is administered concurrently with engineered immune cells. In several embodiments, ramucirumab is administered after administration of engineered immune cells.

[0332] In several embodiments, the therapeutic agent administered is cabozantinib. In several embodiments, cabozantinib is administered orally (e.g., in a tableted form). In several embodiments, the dose of cabozantinib ranges from about 10 mg to about 100 mg per day. In several embodiments, the dose of cabozantinib ranges from about 10 mg to about 20 mg per day. In several embodiments, the dose of cabozantinib ranges from about 20 mg to about 30 mg per day. In several embodiments, the dose of cabozantinib ranges from about 30 mg to about 40 mg per day. In several embodiments, the dose of cabozantinib ranges from about 40 mg to about 50 mg per day. In several embodiments, the dose of cabozantinib ranges from about 50 mg to about 60 mg per day. In several embodiments, the dose of cabozantinib ranges from about 60 mg to about 70 mg per day. In several embodiments, the dose of cabozantinib ranges from about 70 mg to about 80 mg per day. In several embodiments, the dose of cabozantinib ranges from about 80 mg to about 90 mg per day. In several embodiments, the dose of cabozantinib ranges from about 90 mg to about 100 mg per day.

[0333] In several embodiments, cabozantinib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, cabozantinib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, cabozantinib is administered concurrently with engineered immune cells. In several embodiments, cabozantinib is administered after administration of engineered immune cells.

[0334] In several embodiments, the therapeutic agent administered is doxorubicin. In several embodiments, doxorubicin is administered intravenously (e.g., by bolus injection). In several embodiments, doxorubicin is administered on day 1 of a 21 -day dosing cycle. In several embodiments, the dose of doxorubicin ranges from about 20 mg / m2to about 100 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 20 mg / m2to about 30 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 30 mg / m2to about 40 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 40 mg / m2to about 50 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 50 mg / m2to about 60 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 60 mg / m2to about 65 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 65 mg / m2to about 70 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 70 mg / m2to about 75 mg / m2. In several embodiments, the dose ofdoxorubicin ranges from about 75 mg / m2to about 80 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 80 mg / m2to about 90 mg / m2. In several embodiments, the dose of doxorubicin ranges from about 90 mg / m2to about 100 mg / m2.

[0335] In several embodiments, doxorubicin is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, doxorubicin is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, doxorubicin is administered concurrently with engineered immune cells. In several embodiments, doxorubicin is administered after administration of engineered immune cells.

[0336] In several embodiments, the therapeutic agent administered is gemcitabine. In several embodiments, gemcitabine is administered intravenously (e.g., by infusion). In several embodiments, the dose of gemcitabine ranges from about 850 mg / m2to about 1500 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 850 mg / m2to about 900 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 900 mg / m2to about 950 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 950 mg / m2to about 1000 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1000 mg / m2to about 1050 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1050 mg / m2to about 1 100 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1100 mg / m2to about 1150 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1150 mg / m2to about 1200 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1200 mg / m2to about 1250 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1250 mg / m2to about 1300 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1300 mg / m2to about 1400 mg / m2. In several embodiments, the dose of gemcitabine ranges from about 1400 mg / m2to about 1500 mg / m2.

[0337] In several embodiments, gemcitabine is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, gemcitabine is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, gemcitabine is administered concurrently with engineered immune cells. In several embodiments, gemcitabine is administered after administration of engineered immune cells.

[0338] In several embodiments, the therapeutic agent administered is cetuximab. In several embodiments, cetuximab is administered intravenously (e.g., by infusion). In several embodiments, cetuximab is given in a weekly dose format. In several embodiments, cetuximab is given in a bi-weekly format. For the weekly format, in several embodiments, an initial dose is infused at an elevated concentration (e.g., as a loading dose) followed by a lower concentration for each subsequent dose. In several embodiments, the first dose of cetuximab ranges from about 300 mg / m2to about 500 mg / m2. In several embodiments, the first dose of cetuximab ranges from about 300 mg / m2to about 325 mg / m2. In several embodiments, the first dose of cetuximab ranges from about 325 mg / m2to about 350 mg / m2.In several embodiments, the first dose of cetuximab ranges from about 350 mg / m2to about 375 mg / m2. In several embodiments, the first dose of cetuximab ranges from about 375 mg / m2to about 400 mg / m2. In several embodiments, the first dose of cetuximab ranges from about 400 mg / m2to about 425 mg / m2. In several embodiments, the first dose of cetuximab ranges from about 425 mg / m2to about 450 mg / m2. In several embodiments, the first dose of cetuximab ranges from about 450 mg / m2to about 475 mg / m2. In several embodiments, the first dose of cetuximab ranges from about 475 mg / m2to about 500 mg / m2. In several embodiments, the subsequent doses of cetuximab range from about 200 mg / m2to about 300 mg / m2. In several embodiments, the subsequent doses of cetuximab range from about 200 mg / m2to about 225 mg / m2. In several embodiments, the subsequent doses of cetuximab range from about 225 mg / m2to about 250 mg / m2. In several embodiments, the subsequent doses of cetuximab range from about 250 mg / m2to about 275 mg / m2. In several embodiments, the subsequent doses of cetuximab range from about 275 mg / m2to about 300 mg / m2. For the bi-weekly format, in several embodiments, each dose of cetuximab ranges from about 400 mg / m2to about 600 mg / m2. In several embodiments, each dose of cetuximab ranges from about 400 mg / m2to about 425 mg / m2. In several embodiments, each dose of cetuximab ranges from about 425 mg / m2to about 450 mg / m2. In several embodiments, each dose of cetuximab ranges from about 450 mg / m2to about 475 mg / m2. In several embodiments, each dose of cetuximab ranges from about 475 mg / m2to about 500 mg / m2. In several embodiments, each dose of cetuximab ranges from about 500 mg / m2to about 525 mg / m2. In several embodiments, each dose of cetuximab ranges from about 525 mg / m2to about 550 mg / m2. In several embodiments, each dose of cetuximab ranges from about 550 mg / m2to about 575 mg / m2. In several embodiments, each dose of cetuximab ranges from about 575 mg / m2to about 600 mg / m2.

[0339] In several embodiments, cetuximab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, cetuximab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, cetuximab is administered concurrently with engineered immune cells. In several embodiments, cetuximab is administered after administration of engineered immune cells.

[0340] In several embodiments, the therapeutic agent administered is irinotecan (e.g., irinotecan hydrochloride). In several embodiments, irinotecan is administered intravenously (e.g., by infusion). In several embodiments, the dose of irinotecan ranges from about 10 mg / m2to about 1000 mg / m2. In several embodiments, the dose of irinotecan ranges from about 20 mg / m2to about 500 mg / m2. In several embodiments, the dose of irinotecan ranges from about 50 mg / m2to about 125 mg / m2. In several embodiments, the dose of irinotecan is about 20 mg / m2. In several embodiments, the dose of irinotecan is about 20 mg / m2. In several embodiments, the dose of irinotecan is about 30 mg / m2. In several embodiments, the dose of irinotecan is about 40 mg / m2. In several embodiments, the dose of irinotecan is about 50 mg / m2. In several embodiments, the dose of irinotecan is about 60 mg / m2. In several embodiments, the dose of irinotecan is about 70 mg / m2. In several embodiments, the dose ofirinotecan is about 75 mg / m2. In several embodiments, the dose of irinotecan is about 80 mg / m2. In several embodiments, the dose of irinotecan is about 90 mg / m2. In several embodiments, the dose of irinotecan is about 100 mg / m2. In several embodiments, the dose of irinotecan is about 120 mg / m2. In several embodiments, the dose of irinotecan is about 125 mg / m2. In several embodiments, the dose of irinotecan is about 150 mg / m2. In several embodiments, the dose of irinotecan is about 180 mg / m2. In several embodiments, the dose of irinotecan is about 200 mg / m2. In several embodiments, the dose of irinotecan is about 240 mg / m2. In several embodiments, the dose of irinotecan is about 250 mg / m2. In several embodiments, the dose of irinotecan is about 300 mg / m2. In several embodiments, the dose of irinotecan is about 320 mg / m2. In several embodiments, the dose of irinotecan is about 350 mg / m2. In several embodiments, the dose of irinotecan is about 360 mg / m2. In several embodiments, the dose of irinotecan is about 400 mg / m2. In several embodiments, the dose of irinotecan is about 450 mg / m2. In several embodiments, the dose of irinotecan is about 480 mg / m2. In several embodiments, the dose of irinotecan is about 500 mg / m2. In several embodiments, the dose of irinotecan is about 600 mg / m2. In several embodiments, the dose of irinotecan is about 700 mg / m2. In several embodiments, the dose of irinotecan is about 750 mg / m2. In several embodiments, the dose of irinotecan is about 800 mg / m2. In several embodiments, the dose of irinotecan is about 900 mg / m2. In several embodiments, the dose of irinotecan is about 1000 mg / m2.

[0341] In several embodiments, one dose of irinotecan is administered each week (e.g., every 7 days). In several embodiments, one dose of irinotecan is administered each two-week period (e.g., every 14 days). In several embodiments, one dose of irinotecan is administered each three-week period (e.g., every 21 days). In several embodiments, two doses of irinotecan are administered each week. In several embodiments, two doses of irinotecan are administered each two-week period (e.g., each 14 days). In several embodiments, two doses of irinotecan are administered each three-week period (e.g., each 21 days).

[0342] In several embodiments, irinotecan is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, irinotecan is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, irinotecan is administered concurrently with engineered immune cells. In several embodiments, irinotecan is administered after administration of engineered immune cells.

[0343] In several embodiments, the therapeutic agent administered is capecitabine. In several embodiments, capecitabine is administered orally. In several embodiments, the dose of capecitabine ranges from about 1000 mg / m2to about 5000 mg / m2. In several embodiments, the dose of capecitabine ranges from about 1250 mg / m2to about 2500 mg / m2. In several embodiments, the dose of capecitabine is about 1250 mg / m2. In several embodiments, the dose of capecitabine is about 2500 mg / m2. In several embodiments, the dose of capecitabine is about 3750 mg / m2. In several embodiments, the dose of capecitabine is about 5000 mg / m2.

[0344] In some embodiments, the dose of capecitabine is administered daily. In some embodiments, the dose of capecitabine is administered daily for two weeks. In some embodiments, the dose of capecitabine is administered daily for two weeks, followed by a one-week rest period, given as a three-week cycle. In some embodiments, the dose is divided between two administrations. For example, in some embodiments, a daily dose of 2500 mg / m2is divided between two administrations of 1250 mg / nr. In some embodiments, 1250 mg / m2capecitabine is administered orally two times a day (e.g., morning and evening) for a 2500 mg / m2total daily dose, for two weeks followed by a one-week rest period, given as a three-week cycle. In some embodiments, a subject is administered a total of 4 cycles. In some embodiments, a subject is administered a total of 8 cycles.

[0345] In several embodiments, capecitabine is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, capecitabine is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, capecitabine is administered concurrently with engineered immune cells. In several embodiments, capecitabine is administered after administration of engineered immune cells.

[0346] In several embodiments, the therapeutic agent administered is vinorelbine. In several embodiments, vinorelbine is administered intravenously (e.g., by infusion). In several embodiments, the dose of vinorelbine ranges from about 5 mg / m2to about 50 mg / m2. In several embodiments, the dose of vinorelbine ranges from about 10 mg / m2to about 45 mg / m2. In several embodiments, the dose of vinorelbine ranges from about 15 mg / m2to about 40 mg / m2. In several embodiments, the dose of vinorelbine ranges from about 20 mg / m2to about 35 mg / m2. In several embodiments, the dose of vinorelbine ranges from about 25 mg / m2to about 30 mg / m2. In several embodiments, the dose of vinorelbine is about 6.25 mg / m2. In several embodiments, the dose of vinorelbine is about 7.5 mg / m2. In several embodiments, the dose of vinorelbine is about 10 mg / m2. In several embodiments, the dose of vinorelbine is about 12.5 mg / m2. In several embodiments, the dose of vinorelbine is about 15 mg / m2. In several embodiments, the dose of vinorelbine is about 17.5 mg / m2. In several embodiments, the dose of vinorelbine is about 18.75 mg / m2. In several embodiments, the dose of vinorelbine is about 20 mg / m2. In several embodiments, the dose of vinorelbine is about 22.5 mg / m2. In several embodiments, the dose of vinorelbine is about 25 mg / m2. In several embodiments, the dose of vinorelbine is about 27.5 mg / m2. In several embodiments, the dose of vinorelbine is about 30 mg / m2.

[0347] In some embodiments, a dose of vinorelbine is administered once each week (e.g., every 7 days). In some embodiments, vinorelbine is administered in a 28-day cycle. In some embodiments, a dose of vinorelbine is administered once each week for four weeks (e.g., in a 28-day cycle).

[0348] In several embodiments, vinorelbine is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, vinorelbine is administered between lymphodepletion and administration of engineeredimmune cells. In several embodiments, vinorelbine is administered concurrently with engineered immune cells. In several embodiments, vinorelbine is administered after administration of engineered immune cells.

[0349] It shall be appreciated that various combinations of the additional anti-cancer agents may also be used. For example, cetuximab and doxorubicin may be used together with engineered immune cells as provided for herein.XII. Additional Cancer Targets

[0350] Some embodiments of the compositions and methods described herein relate to immune cells comprising a chimeric receptor that targets a cancer antigen, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. Additional non-limiting examples of target antigens include: CD70, CD5, CD19; CD123; CD22; CD30; CD171 ; CS1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); TNF receptor family member B cell maturation (BCMA) ; CD38; DLL3; G protein coupled receptor class C group 5, member D (GPRC5D); epidermal growth factor receptor (EGFR) CD138; prostate-specific membrane antigen (PSMA); Fms Like Tyrosine Kinase 3 (FLT3); KREMEN2 (Kringle Containing Transmembrane Protein 2), ALPPL2, Claudin 4, Claudin 6, C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2- 8)aNeu5Ac(2-3)bDGalp(l-4 )bDGlcp(l-l)Cer);); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostatespecific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21 ); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha (FRa or FR1); Folate receptor beta (FRb); Receptor tyrosine-protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weightmelanoma associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ES0-1); Cancer / testis antigen 2 (LAGE-la); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-l or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase; reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase -related protein 2 (TRP-2); Cytochrome P450 IB 1 (CYPIB 1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator oflmprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Gly cation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, Biotin, c-MYC epitope Tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GMl, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL1 IRa, IL13Ra2, CD179b-IGLll, TCRgamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, TimL / HVCRl,CSF2RA (GM-CSFR-alpha), TGFbetaR2, Lews Ag, TCR-betal chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, Leutenizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), Gonadotropin Hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLVl-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC), auto antibody to desmoglein 3 (Dsg3), auto antibody to desmoglein 1 (Dsgl), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1), AFP, GPRC5D, Claudinl 8.2 (CLD18A2 or CLDN18A.2)), P-glycoprotein, STEAP1, Livl, Nectin-4, Cripto, gpA33, BST1 / CD157, low conductance chloride channel, and the antigen recognized by TNT antibody.DEFINITIONS

[0351] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0352] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0353] 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.

[0354] “Preventing” (and grammatical variations thereof such as “prevent” or “prevention”) as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a diseasein a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.

[0355] A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation or cells, 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 cells and / or compositions at effective amounts, e.g., therapeutically effective amounts.

[0356] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0357] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

[0358] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. 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, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0359] 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.

[0360] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0361] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood towhich the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood.

[0362] 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, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0363] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.NON-LIMITING EMBODIMENTS

[0364] Among the embodiments provided herein are:1. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells; and(b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.2. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.3. The method of embodiment 1 or embodiment 2, wherein the therapeutic agent increases expression of a NKG2D ligand in the subject.4. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject.5. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject.6. A method of treating a cancer in a subject, comprising: administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells, and wherein the subject has less than or equal to 5% peripheral blasts.7. The method of embodiment 6, wherein the method 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.8. The method of any one of embodiments 1-5, wherein the subject has less than or equal to 5% peripheral blasts.9. The method of any one of embodiments 1-8, wherein the subject has less than 5% peripheral blasts.10. The method of any one of embodiments 6-9, wherein, prior to administering the genetically engineered NK cells to the subject, the percentage of peripheral blasts is determined in the subject, and if the subject has less than or equal to 5% peripheral blasts, the subject is selected for treatment.11. The method of any one of embodiments 6-10, wherein, prior to administering the genetically engineered NK cells to the subject, the method comprises:(a) determining the percentage of peripheral blasts in the subject; and(b) if the subject has less than or equal to 5% peripheral blasts, selecting the subject for treatment.12. The method of any one of embodiments 1-11, wherein the subject does not have evidence of extramedullary disease.13. The method of any one of embodiments 4-11, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof.14. The method of any one of embodiments 4-1 , wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.15. The method of any one of embodiments 1-14, wherein each of the first, second, and third doses of the genetically engineered NK cells comprises about 1 x 109genetically engineered NK cells or about 1.5 x 109genetically engineered NK cells.16. The method of any one of embodiments 1-15, wherein the dosing cycle is between about 14 days and about 35 days.17. The method of any one of embodiments 1-16, wherein the dosing cycle is about 21 days.18. The method of any one of embodiments 1-16, wherein the dosing cycle is about 28 days.19. The method of any one of embodiments 1-18, wherein the method comprises administering an additional dosing cycle.20. The method of any one of embodiments 1-19, wherein, if the subject exhibits a clinical response, optionally a complete response (CR), following the dosing cycle, the method comprises administering an additional dosing cycle.21. The method of any one of Embodiments 1 to 20, 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.22. The method of any one of embodiments 1-21, wherein the method comprises administration of between one dosing cycle and five dosing cycles.23. The method of any one of Embodiments 19 to 22, wherein the subject is administered a lymphodepleting therapy prior to each cycle.24. The method of any one of embodiments 1-18, wherein the second dose of the genetically engineered cells is administered to the subject about 7 days after administration of the first dose of the genetically engineered cells.25. The method of any one of embodiments 1-24, wherein the third dose of the genetically engineered cells is administered to the subject about 7 days after administration of the second dose of the genetically engineered cells.26. A method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 about 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 about 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; and wherein the subject has less than or equal to 5% peripheral blasts and does not have evidence of extramedullary disease.27. The method of any one of embodiments 1-25, wherein administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells.28. The method of any one of embodiments 1-26, wherein administration of the therapeutic agent is concurrent with administration of a dose of the genetically engineered NK cells, optionally concurrent with administration of the first dose of the genetically engineered NK cells.29. The method of any one of embodiments 1-26, wherein administration of the therapeutic agent is after administration of a dose of the genetically engineered NK cells, optionally after administration of the third dose of the genetically engineered NK cells.30. The method of any one of embodiments 1-3 and 12-29, wherein the lymphodepleting therapy comprises administration of fludarabine (Flu).31. The method of any one of embodiments 1-3 and 12-30, wherein the lymphodepleting therapy comprises administration of three doses of Flu.32. The method of embodiment 31, wherein each dose of Flu comprises between about 10 mg / m2and about 60 mg / m2.33. The method of any one of embodiments 1-3 and 12-32, wherein the lymphodepleting therapy comprises administration of Flu and cyclophosphamide (Cy).34. The method of any one of embodiments 1-3 and 12-33, wherein the lymphodepleting therapy comprises administration of three doses of Cy.35. The method of embodiment 34, wherein the first doses of Flu and Cy are each given 5 days prior to initiation of the dosing cycle; the second doses of Flu and Cy are each given 4 days prior to initiation of the dosing cycle; and the third doses of Flu and Cy are each given 3 days prior to initiation of the dosing cycle.36. The method of embodiment 34 or embodiment 35, wherein each dose of Cy comprises between about 200 mg / m2and about 600 mg / m2.37. The method of any one of embodiments 34-36, wherein each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 300 mg / m2.38. The method of any one of embodiments 34-36, wherein each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 500 mg / m2.39. The method of any one of embodiments 1-3 and 12-32, wherein the lymphodepleting therapy comprises administration of five doses of Flu.40. The method of any one of embodiments 1 -3, 12-32, and 39, wherein the lymphodepleting therapy comprises administration of Flu and cytosine arabinoside (Ara-C).41. The method of any one of embodiments 1-3, 12-32, 39, and 40, wherein the lymphodepleting therapy comprises administration of five doses of Ara-C.42. The method of embodiment 41, 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 3 days prior to initiation of the dosing cycle; and the fifth doses of Flu and Ara-C are each given 4 days prior to initiation of the dosing cycle.43. The method of embodiment 41 or embodiment 42, wherein each dose of Ara-C comprises between about 1 g / m2and about 4 g / m2.44. The method of any one of embodiments 41-43, wherein each dose of Flu comprises about 30 mg / m2and each dose of Ara-C comprises about 2 g / m2.45. The method of any one of embodiments 1-44, wherein the therapeutic agent is a chemotherapeutic agent.46. The method of any one of embodiments 1-45, wherein the therapeutic agent is decitabine.47. The method of embodiment 46, wherein a dose of decitabine is administered to the subject daily for five days prior to initiation of the dosing cycle.48. The method of embodiment 47, wherein a dose of decitabine comprises about 20 mg / m2.49. A method of treating a cancer in a subject, comprising administering to ...

Claims

WHAT IS CLAIMED IS:

1. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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; fii) a second dose of the genetically engineered NK cells administered to the subject between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

2. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof.

3. The method of claim 1, wherein the therapeutic agent is a chemotherapeutic agent.

4. The method of claim 1, wherein the subject has less than or equal to 5% peripheral blasts.

5. The method of claim 1, wherein the subject has less than 5% peripheral blasts.

6. The method of claim 1, wherein, prior to administering the genetically engineeredNK cells to the subject, the percentage of peripheral blasts is determined in the subject, and if the subject has less than or equal to 5% peripheral blasts, the subject is selected for treatment.

7. The method of claim 1, wherein, prior to administering the genetically engineered NK cells to the subject, the method further comprises:(a) determining the percentage of peripheral blasts in the subject; and(b) if the subject has less than or equal to 5% peripheral blasts, selecting the subject for treatment.

8. The method of claim 1, wherein the subject does not have evidence of extramedullary disease.

9. The method of any one of claims 1-9, wherein administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells.

10. The method of any one of claims 1-9, wherein administration of the therapeutic agent is concurrent with administration of a dose of the genetically engineered NK cells, optionally concurrent with administration of the first dose of the genetically engineered NK cells.

11. The method of any one of claims 1-9, wherein administration of the therapeutic agent is after administration of a dose of the genetically engineered NK cells, optionally after administration of the third dose of the genetically engineered NK cells12. The method of claim 1, wherein the lymphodepleting therapy comprises administration of fludarabine (Flu).

13. The method of claim 1, wherein the lymphodepleting therapy comprises administration of three doses of Flu.

14. The method of claim 13, wherein each dose of Flu comprises between about 10 mg / m2and about 60 mg / m2.

15. The method of claim 1 , wherein the lymphodepleting therapy comprises administration of Flu and cyclophosphamide (Cy).

16. The method of claim 15, wherein the lymphodepleting therapy comprises administration of three doses of Cy.

17. The method of claim 16, wherein the first doses of Flu and Cy are each given 5 days prior to initiation of the dosing cycle; the second doses of Flu and Cy are each given 4 days prior to initiation of the dosing cycle; and the third doses of Flu and Cy are each given 3 days prior to initiation of the dosing cycle.

18. The method of claim 16 or claim 17, wherein each dose of Cy comprises between about 200 mg / m2and about 600 mg / m2.

19. The method of claim 16 or claim 17, wherein each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 300 mg / m2.

20. The method of claim 16 or 17, wherein each dose of Flu comprises about 30 mg / m2and each dose of Cy comprises about 500 mg / m2.

21. The method of claim 1, wherein the lymphodepleting therapy comprises administration of five doses of Flu.

22. The method of claim 1 or claim 21, wherein the lymphodepleting therapy comprises administration of Flu and cytosine arabinoside (Ara-C).

23. The method of claim 1 or claim 22, wherein the lymphodepleting therapy comprises administration of five doses of Ara-C.

24. The method of claim 23, 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 3 days prior to initiation of the dosing cycle; and the fifth doses of Flu and Ara-C are each given 4 days prior to initiation of the dosing cycle.

25. The method of claim 23 or claim 24, wherein each dose of Ara-C comprises between about 1 g / m2and about 4 g / m2.

26. The method of claim 23 or claim 24, wherein each dose of Flu comprises about 30 mg / m2and each dose of Ara-C comprises about 2 g / m2.

27. A method of selecting a subject having a cancer for treatment with a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds ligands of natural killer cell group 2D (NKG2D), the method comprising:(a) assessing the level or amount of a NKG2D ligand in a biological sample from a subject having a cancer, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or a polynucleotide encoded by the NKG2D ligand gene;(b) selecting the subject for treatment with a population of NK cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D if the level or amount of the NKG2D ligand is above a reference value; and(c) administering the genetically engineered NK cells to the subject, wherein the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells.

28. The method of claim 27, wherein the genetically engineered NK cells are administered to the 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells.

29. The method of claim 27 or claim 28, wherein the biological sample is obtained from the subject within about 14 days, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administration of the genetically engineered NK cells to the subject.

30. The method of claim 27 or claim 28, wherein the percentage of blasts in the subject’s bone marrow is between about 0% and about 60%, between about 0% and about 50%, between about0% and about 40%, between about 0% and about 30%, or between about 0% and about 20%.

31. The method of claim 27 or claim 28, wherein the NKG2D ligand comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof.

32. The method of claim 27 or claim 28, wherein the NKG2D ligand comprises MICA and MICB.

33. The method of claim 27 or claim 28, wherein the NKG2D ligand comprises ULBP1 and ULBP3.

34. The method of any one of claims 1-26 or claims 28-33, wherein each of the first, second, and third doses of the genetically engineered NK cells comprises about 1 x 109genetically engineered NK cells or about 1.5 x 109genetically engineered NK cells.

35. The method of any one of claims 1-26 or claims 28-34, wherein the dosing cycle is between about 14 days and about 35 days.

36. The method of any one of claims 1-26 or claims 28-34, wherein the dosing cycle is about 21 days.

37. The method of any one of claims 1 -26 or claims 28-34, wherein the dosing cycle is about 28 days.

38. The method of any one of claims 1-26 or claims 28-37, wherein the method comprises administering an additional dosing cycle.

39. The method of any one of claims 1-26 or claims 28-38, 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.

40. The method of any one of claims 1-26 or claims 28-38, 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.

41. The method of any one of claims 1-26 or claims 28-40, wherein the method comprises administration of between one dosing cycle and five dosing cycles.

42. The method of Claim 41, wherein the subject is administered a lymphodepleting therapy prior to each cycle.

43. The method of any one of claims 1-26 or claims 28-42, wherein the second dose of the genetically engineered cells is administered to the subject about 7 days after administration of the first dose of the genetically engineered cells.

44. The method of any one of claims 1-26 or claims 28-43, wherein the third dose of the genetically engineered cells is administered to the subject about 7 days after administration of the second dose of the genetically engineered cells.

45. The method of claim any one of claims 27 to 44, wherein the cancer is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), optionally wherein the cancer is relapsed / refractory (r / r) AML.

46. The method of any one of claims 1 to 26, wherein the therapeutic agent comprises a chemotherapeutic agent and wherein the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly (ADP-ribose) polymerase (PARP), or any combination thereof.

47. The method of any one of claims 1 to 26, wherein the therapeutic agent comprises a monoclonal antibody and wherein the monoclonal antibody comprises an anti-CTLA4 antibody, an anti-EGFR antibody, an anti-HER2 / neu antibody, an anti-PDl antibody, an anti-PD-Ll antibody, an anti-VEGF antibody, or any combination thereof.

48. The method of any one of claims 1 to 26, wherein the therapeutic agent comprises an NK cell engager and wherein the NK cell engager binds to an activating receptor on an NK cell and an antigen expressed by cells of the cancer, optionally wherein the activating receptor is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof.

49. The method of any one of claims 1 -44 or 46-48, wherein the cancer is a carcinoma, a sarcoma, or a melanoma.

50. The method of any one of claims 1-44 or 46-48, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

51. The method of any one of claims 1-44 or 46-48, wherein the cancer comprises liver cancer or colorectal cancer.

52. The method of any one of claims 1-51, wherein the therapeutic agent comprises capecitabine, cisplatin, doxorubicin, regorafenib, or sorafenib.

53. The method of any one of claims 1-44 and 46-48, wherein the cancer comprises bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, or uterine cancer.

54. The method of any one of claims 1-50 or 53, wherein the therapeutic agent comprises doxorubicin or gemcitabine.

55. The method of any one of claims 1-44 and 46-48, wherein the cancer comprises esophageal cancer, head and neck cancer, or lung cancer.

56. The method of any one of claims 1-50 or 55, wherein the therapeutic agent comprises gemcitabine, irinotecan, or vinorelbine.

57. The method of any one of claims 1-44 and 46-48, wherein the cancer comprises melanoma.

58. The method of any one of claims 1-50 or 57, wherein the therapeutic agent comprises a MEK inhibitor, a BRAF inhibitor, an anti-CTLA4 antibody, an anti-LAG3 antibody, an anti-PDl antibody, or an anti-PD-Ll antibody.

59. The method of any one of claims 1-58, wherein the cancer is a relapsed / refractory (R / R) cancer.

60. The method of any one of claims 1-59, wherein the subject has been treated with one prior line of therapy.

61. The method of any one of claims 1-60, wherein the subject has been treated with two prior lines of therapy.

62. The method of any one of claims 1-61, wherein the subject has been treated with three prior lines of therapy.

63. The method of any one of claims 1-62, wherein the subject has an ECOG of 0-2, optionally 0 or 1.

64. The method of any one of claims 1-63, wherein the subject is 18 years of age or older.

65. The method of any one of claims 1-64, wherein the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling region.

66. The method of claim 65, wherein the extracellular binding domain has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42.

67. The method of claim 65 or claim 66, wherein the transmembrane domain comprises a CD8 transmembrane domain.

68. The method of any one of claims 65-67, wherein the intracellular signaling region comprises a co-stimulatory domain and a CD3zeta.

69. The method of claim 68, wherein the co-stimulatory domain comprises an 0X40 domain.

70. The method of any one of claims 1-69, wherein the chimeric receptor has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39.

71. The method of any one of claims 1-70, wherein the genetically engineered NK cells express a membrane-bound interleukin 15 (mbIL15).

72. The method of claim 71, wherein the mbIL15 has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40.

73. The method of any one of claims 1-72, wherein the population of engineered NK cells are allogeneic to the subject.

74. The method of any one of claims 1-73, wherein the population of engineered NK cells are derived from a subject without cancer.

75. The method of any one of claims 1-74, 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.

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 cancer in a subject, wherein:(a) the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) the subject is administered a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

77. 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 cancer in a subject, wherein:(a) the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells; and(b) the subject is administered a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof,wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

78. The use of claim 76 or 77, wherein the therapeutic agent increases expression of a NKG2D ligand in the subject.

79. 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 cancer in a subject, wherein:(a) the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) the subject is administered a therapeutic agent that increases expression of a NKG2D ligand in the subject.

80. 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 cancer in a subject, wherein:(a) the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells; and(b) the subject is administered a therapeutic agent that increases expression of aNKG2D ligand in the subject.

81. 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 cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells, and wherein the subject has less than or equal to 5% peripheral blasts.

82. The use of claim 81 , wherein the population of genetically engineered NK cells is for use with 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.

83. The use of any one of claims 76-82, wherein the subject has less than or equal to 5% peripheral blasts.

84. The use of any one of claims 76-83, wherein the subject has less than 5% peripheral blasts.

85. The use of any one of claims 76-84, wherein the subject does not have evidence of extramedullary disease.

86. The use of any one of claims 79-85, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof.

87. The use of any one of claims 79-86, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

88. 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 cancer in a subject, 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 about 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 about 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; and wherein the subject has less than or equal to 5% peripheral blasts and does not have evidence of extramedullary disease.

89. 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 cancer in a subject, 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 about 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 about 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; and wherein the subject has been administered a dose of about 20 mg / m2decitabine daily for five days prior to administration of the first dose of the genetically engineered NK cells.

90. The use of claim 89, wherein the subject has less than or equal to 5% peripheral blasts, optionally wherein the subject does not have evidence of extramedullary disease.

91. The use of any one of claims 76-90, wherein the subject is administered an additional dosing cycle.

92. The use of any one of claims 76-91, 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, an additional dosing cycle is administered to the subject as a consolidation treatment.

93. The use of any one of claims 76-91, wherein, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the subject is administered an additional dosing cycle as retreatment.

94. The use of any one of claims 76-93, wherein the subject is administered between one dosing cycle and five dosing cycles.

95. The use of Claim 94, wherein the subject is administered a lymphodepleting therapy prior to each cycle.

96. The use of claim any one of claims 77-78 or 80-95, wherein the cancer is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), optionally wherein the cancer is relapsed / refractory (r / r) AML or very high-risk MDS.

97. The use of any one of claims 76-96, 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.

98. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

99. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells; and(b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, and any combination thereof, wherein, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

100. The method of claim 98 or 99, wherein the therapeutic agent increases expression of a NKG2D ligand in the subject.

101. A method of treating a cancer in a subject, comprising:(a) administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 10 days after administration of the second dose of the genetically engineered NK cells, wherein the cancer is a solid tumor and each dose of the genetically engineered NK cells comprises between about 1 x 108genetically engineered NK cells and about 1 x 1010genetically engineered NK cells; and(b) administering to the subject a therapeutic agent that increases expression of a NKG2D ligand in the subject.

102. A method of treating a cancer in a subject, comprising: administering to a subject having a cancer 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 between about 5 days and about 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 between about 5 days and about 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 108genetically engineered NK cells and about 1 x IO10genetically engineered NK cells, and wherein the subject has less than or equal to 5% peripheral blasts.

103. A method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 about 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 about7 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; and wherein the subject has less than or equal to 5% peripheral blasts and does not have evidence of extramedullary disease.

104. A method of treating a cancer in a subject, comprising administering to a subject having a cancer 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 about 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 about 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 has less than or equal to 5% peripheral blasts; and wherein the subject has been administered a dose of about 20 mg / m2decitabine daily for five days prior to administration of the first dose of the genetically engineered NK cells.

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