Dosing regimen for combination therapy

Abstract

Several embodiments of the methods and compositions disclosed herein relate to immune cells engineered to express cytotoxic chimeric receptors and various dosing regimens for administering such cells. In several embodiments, the immune cell expresses a chimeric receptor that targets a ligand of NKG2D on a tumor cell. In several embodiments, the cancer is a leukemia, e.g., acute myelogenous leukemia (e.g., recurrent / refractory acute myelogenous leukemia) or myelodysplastic syndrome. In several embodiments, the tumor is a solid tumor, such as breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, hepatocellular cancer, lung cancer, melanoma, intrahepatic cholangiocarcinoma, or other liver tumor, such as a secondary metastatic tumor from colorectal cancer. In several embodiments, the immune cells are administered in combination with a therapeutic agent, such as an additional anti-cancer agent.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 384,073, filed on November 16, 2022; 63 / 489,946, filed on March 13, 2023; 63 / 499,470, filed on May 1, 2023; and 63 / 523,333, filed on June 26, 2023, each of which is incorporated herein by reference in its entirety. Technical Field

[0002] Several embodiments disclosed herein relate to methods and compositions comprising genetically engineered cells for use in cancer immunotherapy. In several embodiments, the disclosure relates to cells engineered to express a cytotoxic receptor complex, and the administration of such cells according to certain dosing regimens to achieve successful cancer immunotherapy. Background Art

[0003] As our understanding of various cancers increases and the characteristics of cancer cells that can be used to clearly distinguish them from healthy cells, therapeutic agents are being developed that exploit the unique characteristics of cancer cells. Immunotherapy using engineered immune cells is one approach to treating cancer. The material in the sequence listing document is incorporated by reference

[0004] This application incorporates by reference the material in the sequence listing contained in the following XML file filed herewith: File name: NKT096WO_ST26.xml, created on November 15, 2023, and 46,520 bytes in size. Summary of the Invention

[0005] Immunotherapy represents a new technological advancement in the treatment of disease, in which immune cells are engineered to express specific targeting and / or effector molecules that specifically recognize and react with diseased or damaged cells. This represents a promising advance, at least in part due to the potential to specifically target diseased or damaged cells, in contrast to more traditional approaches such as chemotherapy, in which all cells are affected and the desired outcome is that enough healthy cells survive to allow the patient to survive. One immunotherapy approach is to recombinantly express chimeric receptors in immune cells to achieve targeted recognition and destruction of abnormal cells of interest.

[0006] In several embodiments, provided herein is a method of treating cancer in a subject, the method comprising (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to 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 the administration of the first dose of the genetically engineered cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10 In several embodiments, the method comprises administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

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

[0008] In several embodiments, provided herein is a method of treating cancer in a subject, the method comprising (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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 the administration of the first dose of the genetically engineered cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10The method further comprises administering to the subject between one and two genetically engineered NK cells, wherein the cancer is a solid tumor, and wherein lymphodepletion therapy is administered to the subject prior to administering the first dose of the genetically engineered NK cells to the subject. In several embodiments, the method comprises administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0009] In several embodiments, the therapeutic agent when administered is selected from a chemotherapeutic agent, a monoclonal antibody, an 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 in the subject is determined, 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, an additional evaluation of the subject is performed and includes (a) determining the percentage of peripheral primitive cells in the subject, and (b) selecting the subject for treatment if the subject has less than or equal to 5% peripheral primitive cells.

[0012] In several embodiments, the subject has no evidence of extramedullary disease.

[0013] In several embodiments, the administration of the therapeutic agent is performed before the administration of the first dose of the genetically engineered NK cells. In several embodiments, the administration of the therapeutic agent is performed in parallel with the administration of a certain dose of the genetically engineered NK cells, optionally in parallel with the administration of the first dose of the genetically engineered NK cells. In several embodiments, the administration of the therapeutic agent is performed after the administration of a certain dose of the genetically engineered NK cells, optionally after the administration of the third dose of the genetically engineered NK cells. In several embodiments, the therapeutic agent is administered before, in parallel with, and / or after the administration of a certain dose of the genetically engineered NK cells.

[0014] In several embodiments, the lymphodepleting therapy comprises administering fludarabine (Flu). In several embodiments, the lymphodepleting therapy comprises administering three doses of Flu. In several embodiments, each dose of Flu comprises about 10 mg / m 2 and about 60 mg / m2 In several embodiments, the lymphodepleting therapy comprises administering Flu and cyclophosphamide (Cy). In several embodiments, the lymphodepleting therapy comprises administering three doses of Cy. In several embodiments, the first dose of Flu and Cy are each administered 5 days before the start of the dosing cycle, the second dose of Flu and Cy are each administered 4 days before the start of the dosing cycle, and the third dose of Flu and Cy are each administered 3 days before the start of the dosing cycle. In several embodiments, each Cy dose comprises about 200 mg / m 2 and about 600 mg / m 2 In several embodiments, each Flu dose comprises about 30 mg / m 2 Each Cy dose contains approximately 300 mg / m 2 In other embodiments, each Flu dose contains about 30 mg / m 2 Each Cy dose contains approximately 500 mg / m 2 .

[0015] In several embodiments, the lymphodepleting therapy comprises administering five doses of Flu. In several embodiments, the lymphodepleting therapy comprises administering Flu and cytarabine (Ara-C). In several embodiments, the lymphodepleting therapy comprises administering five doses of Ara-C. In several embodiments, a first dose of Flu and Ara-C are each administered 7 days before the start of the dosing cycle, a second dose of Flu and Ara-C are each administered 6 days before the start of the dosing cycle, a third dose of Flu and Ara-C are each administered 5 days before the start of the dosing cycle, a fourth dose of Flu and Ara-C are each administered 3 days before the start of the dosing cycle, and a fifth dose of Flu and Ara-C are each administered 4 days before the start of the dosing cycle. In several embodiments, each Ara-C dose comprises about 1 g / m 2 With about 4 g / m 2 In several embodiments, each Flu dose comprises about 30 mg / m 2 Each Ara-C dose contains approximately 2 g / m 2 .

[0016] In several embodiments, a method is provided for selecting a subject having cancer for treatment with a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D), the method comprising (a) assessing the level or amount of an NKG2D ligand in a biological sample from a subject having cancer, wherein the level or amount of an NKG2D ligand is the level or amount of a protein or polynucleotide encoded by an NKG2D ligand gene, (b) if the level or amount of the NKG2D ligand is above a reference value, selecting the subject for treatment with a population of NK cells genetically engineered to express a chimeric receptor that binds to a ligand of NKG2D, and (c) administering the genetically engineered NK cells to the subject. In several embodiments, the biological sample is obtained from the subject prior to administering 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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10 Genetically engineered NK cells.

[0018] In several embodiments, the biological sample is obtained from the subject within about 14 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day prior to administering 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 dose, the second dose, and the third dose of the genetically engineered NK cells comprises approximately 1 x 10 9 Genetically engineered NK cells or approximately 1.5 x 10 9 Genetically 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 additional dosing cycles.

[0023] In several embodiments, if the subject exhibits a clinical response, optionally a partial remission (PR), complete remission with incomplete hematologic recovery (CRi), or complete remission (CR) after the dosing cycle, the method comprises administering an additional dosing cycle as consolidation therapy.

[0024] In several embodiments, if the subject exhibits a clinical response after 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 administering between one and five dosing cycles.In several embodiments, the subject is administered lymphodepleting therapy prior to each cycle.

[0026] In several embodiments, the subject is administered the second dose of the genetically engineered cells about 7 days after administering the first dose of the genetically engineered cells. In several embodiments, the subject is administered the third dose of the genetically engineered cells about 7 days after administering 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, a poly (ADP-ribose) polymerase (PARP) inhibitor, 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-PD1 antibody, an anti-PD-L1 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 NK cells and an antigen expressed by cells of the cancer, optionally wherein the activating receptor is selected from 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 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-PD1 antibody, or an anti-PD-L1 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 subject has been treated with three (or more) prior lines of therapy. In several embodiments, the subject has an ECOG score 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, the 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 costimulatory domain and a CD3ζ domain. In several embodiments, the costimulatory domain comprises an OX40 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 membrane-bound interleukin 15 (mbIL15). In several embodiments, the mbIL15 has at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or higher sequence identity to the amino acid sequence shown in SEQ ID NO: 40.

[0039] In several embodiments, the engineered NK cell population is allogeneic to the subject. In several embodiments, the engineered NK cell population is 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 herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having 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 the administration of the first dose of the genetically engineered cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the 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 approximately 1 x 10 8 and about 1 x 10 10 In some embodiments, the subject is administered a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof. In some embodiments, the subject is administered lymphodepletion therapy prior to administering the first dose of the genetically engineered NK cells to the subject.

[0042] Also provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein (a) the genetically engineered NK cells are administered to a subject having cancer in an administration 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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x10 10 and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, wherein the subject is administered lymphodepletion therapy prior to administering the first dose of the genetically engineered NK cells to the subject.

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

[0044] Provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein (a) the genetically engineered NK cells are administered to a subject having 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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the 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 approximately 1 x 10 8 and about 1 x 10 10 and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0045] Also provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having 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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells. In several embodiments, each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10 In several embodiments, the subject is administered a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0046] Also provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having cancer in an administration 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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10 The invention relates to a method for producing genetically engineered NK cells of at least 100,000 cells / day, and wherein the subject has less than or equal to 5% of peripheral blasts.

[0047] In several embodiments, the genetically engineered NK cell population is for use with a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, a therapeutic agent that increases expression of an 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 has no evidence of extramedullary disease.

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

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

[0051] Also provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating 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 approximately 7 days after the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject approximately 7 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 The invention relates to a method for treating a subject with a plurality of genetically engineered NK cells, wherein the subject has less than or equal to 5% peripheral blasts and / or has no evidence of extramedullary disease.

[0052] Also provided is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating 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 approximately 7 days after the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject approximately 7 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 The invention provides a method for treating a subject with a first dose of genetically engineered NK cells, wherein the subject has been administered decitabine at a dose of about 20 mg / m2 daily for five days prior to administering 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 has no 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 after the dosing cycle, optionally a partial remission (PR), complete remission with incomplete hematologic recovery (CRi), or complete remission (CR), the subject is administered an additional dosing cycle as consolidation therapy. In several embodiments of the uses provided, if the subject exhibits a clinical response after 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 and five dosing cycles. In several embodiments of the uses provided, the subject is administered lymphodepletion 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 herein is a method of treating cancer in a subject, the method comprising (a) administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the 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 approximately 1 x 10 8 and about 1 x 10 10 and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, wherein the subject is administered lymphodepletion therapy prior to administering the first dose of the genetically engineered NK cells to the subject.

[0056] In several embodiments, a method of treating cancer in a subject is also provided, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10 (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, wherein the subject is administered lymphodepletion therapy prior to administering the first dose of the genetically engineered NK cells to the subject. In several embodiments, the therapeutic agent increases expression of an NKG2D ligand in the subject.

[0057] In several embodiments, the method of treating cancer in a subject comprises (a) administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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 the administration of the first dose of the genetically engineered cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the 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 approximately 1 x 10 8 and about 1 x 10 10 and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0058] Additionally, provided herein is a method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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 the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10 The invention relates to a method for producing genetically engineered NK cells of at least 100,000 cells / day, and wherein the subject has less than or equal to 5% of peripheral blasts.

[0059] Additionally, provided herein is a method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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 approximately 7 days after administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject approximately 7 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 The invention relates to a method for treating a subject with a plurality of genetically engineered NK cells, wherein the subject has less than or equal to 5% peripheral blasts and no evidence of extramedullary disease.

[0060] Also provided herein is a method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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 approximately 7 days after the administration of the first dose of the genetically engineered NK cells, and (iii) a third dose of the genetically engineered NK cells administered to the subject approximately 7 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 genetically engineered NK cells, wherein the subject has less than or equal to 5% peripheral blasts, and wherein prior to administering the first dose of the genetically engineered NK cells, the subject has been administered decitabine at a dose of about 20 mg / m2 daily for five days.

[0061] Further provided herein is a method of treating cancer in a subject, the method comprising: (a) administering to the subject a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (NKG2D), wherein the genetically engineered 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, an NK cell engager, and any combination thereof; wherein, prior to administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy.

[0062] Also provided herein is a method of treating cancer in a subject, the method comprising administering to the subject a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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, an NK cell engager, and any combination thereof; and wherein prior to administering the first dose of the genetically engineered NK cells to the subject, lymphodepletion therapy is administered to the subject.

[0063] Also provided herein is a method of treating cancer in a subject, the method comprising administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an 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 to a ligand for natural killer cell group 2 D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle; and wherein prior to administering the first dose of the genetically engineered NK cells to the subject, lymphodepletion therapy is administered to the subject.

[0064] In some embodiments, the administration 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 the administration of the first dose of the genetically engineered NK cells; and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 1 x 10 8 and about 1 x 10 10 In some embodiments, each of the first dose, the second dose, and the third dose of the genetically engineered NK cells comprises about 1 x 10 9 NK cells or approximately 1.5 x 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of the genetically engineered NK cells comprises approximately 1 x 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 NK cells.

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

[0066] Also provided herein is a method of treating cancer in a subject, the method comprising: (a) administering to the subject a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D), wherein the genetically engineered NK cells are administered during a dosing cycle; and (b) administering to the subject a therapeutic agent that increases expression of NKG2D ligand in the subject.

[0067] Also provided herein is a method of treating cancer in a subject, the method comprising administering to the subject a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (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 an NKG2D ligand in the subject.

[0068] Also provided herein is a method of treating cancer in a subject, the method comprising administering to the subject a therapeutic agent that increases expression of an 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 to a ligand of natural killer cell group 2 D (NKG2D); and wherein the genetically engineered NK cells are administered in a dosing cycle comprising.

[0069] Also provided herein is a method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (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 the administration of the first dose of the genetically engineered NK cells; and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1x 10 10 The invention relates to a method for producing genetically engineered NK cells of at least 100,000 cells / day, and wherein the subject has less than or equal to 5% of 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, an 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% of peripheral blasts. In some embodiments, the subject has less than 5% of peripheral blasts. In some embodiments, the subject has no evidence of extramedullary disease. In some embodiments, before administering the NK cell population to the subject, the percentage of peripheral blasts in the subject is determined. In some embodiments, before administering the NK cell population to the subject, if it is determined that the subject has less than or equal to 5% of peripheral blasts, the subject is selected for treatment. In some embodiments, before administering the NK cell population to the subject, the percentage of peripheral blasts in the subject is determined, and if it is determined that the subject has less than or equal to 5% of peripheral blasts, the subject is selected for treatment. In some embodiments, before administering the NK cell population to the subject, the method includes determining the percentage of peripheral blasts in the subject. In some embodiments, before administering the NK cell population to the subject, if it is determined that the subject has less than or equal to 5% of peripheral blasts, the method includes selecting the subject for treatment. In some embodiments, prior to administering the NK cell population to the subject, the method comprises: (a) determining the percentage of peripheral blasts in the subject; and (b) selecting the subject for treatment if the subject is determined to have less than or equal to 5% peripheral blasts.

[0072] 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 NK cells; and (iii) a third dose of the 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 for selecting a subject having cancer for treatment with a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D), the method comprising: (a) assessing the level or amount of NKG2D ligand in a biological sample from a subject having cancer, wherein the level or amount of NKG2D ligand is the level or amount of a protein or polynucleotide encoded by an NKG2D ligand gene; (b) if the level or amount of NKG2D ligand is higher than a reference value, selecting the subject for treatment with a population of NK cells genetically engineered to express a chimeric receptor that binds to a ligand of NKG2D; and (c) administering the genetically engineered NK cells to the subject, wherein the biological sample is obtained from the subject prior to administering the genetically engineered NK cells.

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

[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 is acute 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 the administration of the first dose of the genetically engineered NK cells; and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1 x 10 8 and about 1 x 10 10 Genetically 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 NKG2D ligands in a population of subjects who have cancer and have been administered the genetically engineered NK cells and have not demonstrated a clinical response after administration of the genetically engineered NK cells. In some embodiments, the reference value is the average level or amount of NKG2D ligands in a population of subjects who have cancer and have been administered the genetically engineered NK cells and have not demonstrated a clinical response after administration of the genetically engineered NK cells.

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

[0081] In some embodiments, each dose of the genetically engineered NK cells comprises about 1 x 10 8 and about 1 x10 10 In some embodiments, each of the first dose, the second dose, and the third dose of the genetically engineered NK cells comprises about 1 x 10 9 NK cells or approximately 1.5 x 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of the genetically engineered NK cells comprises approximately 1 x 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of genetically engineered NK cells comprises 2 × 10 9 NK cells, 3 × 10 9 NK cells, 4 × 10 9 NK cells or 5 × 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of genetically engineered NK cells comprises 2 × 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of genetically engineered NK cells comprises 3 × 10 9In some embodiments, each of the first dose, the second dose, and the third dose of genetically engineered NK cells comprises 4 × 10 9 In some embodiments, each of the first dose, the second dose, and the third dose of genetically engineered NK cells comprises 5 × 10 9 In some embodiments, the NK cells are genetically engineered NK cells.

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

[0083] In some embodiments, lymphodepletion therapy is administered to the subject prior to administering the first dose of the genetically engineered NK cells to the subject.

[0084] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological 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 the 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 the administration of the second dose of the genetically engineered cells.

[0086] In some embodiments, the administration cycle is between about 14 days and about 35 days. In some embodiments, the administration cycle is about 21 days. In some embodiments, the administration 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, the first, 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 cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (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 approximately 7 days after the administration of the first dose of the genetically engineered NK cells; and (iii) a third dose of the genetically engineered NK cells administered to the subject approximately 7 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 genetically engineered NK cells; and wherein the subject has less than or equal to 5% peripheral blasts and no 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 after the dosing cycle, the method comprises administering an additional dosing cycle. In some embodiments, if the subject exhibits a clinical response after the dosing cycle, the method comprises administering an additional dosing cycle as consolidation therapy. In some embodiments, if the subject exhibits a complete remission (CR) after the dosing cycle, the method comprises administering an additional dosing cycle. In some embodiments, if the subject exhibits a complete remission (CR) after the dosing cycle, the method comprises administering an additional dosing cycle as consolidation therapy. In some embodiments, if the subject exhibits a complete remission with incomplete hematological recovery (CRi) after the dosing cycle, the method comprises administering an additional dosing cycle. In some embodiments, if the subject exhibits CRi after the dosing cycle, the method comprises administering an additional dosing cycle as consolidation therapy.

[0089] In some embodiments, if the subject exhibits a clinical response after 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 after the dosing cycle and subsequently exhibits disease progression, the method comprises administering an additional dosing cycle as retreatment. In some embodiments, the method comprises administering between one and five dosing cycles. In some embodiments, the method consists of administering one dosing cycle. In some embodiments, the method consists of administering two dosing cycles. In some embodiments, the method consists of administering three dosing cycles. In some embodiments, the method consists of administering four dosing cycles. In some embodiments, the method consists of administering five dosing cycles. In some embodiments, the method consists of administering no more than five dosing cycles. In some embodiments, lymphodepletion therapy is administered to the subject prior to each cycle.

[0090] In some embodiments, the therapeutic agent is administered before administering the first dose of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered before administering the lymphodepletion therapy. In some embodiments, the therapeutic agent is administered after administering the lymphodepletion therapy, and until administering a certain dose of genetically engineered NK cells. In some embodiments, the administration of the therapeutic agent is carried out in parallel with the administration of a certain dose of the genetically engineered NK cells. In some embodiments, the administration of the therapeutic agent is carried out in parallel with the administration of the first dose of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered after administering a certain dose of the genetically engineered NK cells. In some embodiments, the therapeutic agent is administered after administering the third dose of the genetically engineered NK cells.

[0091] In some embodiments, the first dosing cycle is initiated after the subject has been administered 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, one or more additional cycles are administered depending on the status of the subject's cancer (e.g., in the event of progression or development of another cancer). In some embodiments, one or more additional cycles are administered as retreatment in the event of disease progression. In some embodiments, if the subject demonstrates a clinical response to a dosing cycle and subsequently demonstrates disease progression, one or more additional cycles are administered as retreatment. In some embodiments, one or more additional cycles are administered as consolidation therapy after the subject demonstrated a clinical response to a previous cycle. In several embodiments, no additional cycles are required when the subject demonstrates a response (e.g., complete remission).

[0092] In some embodiments, the lymphodepleting therapy comprises administering fludarabine (Flu). In some embodiments, the lymphodepleting therapy comprises administering three doses of Flu. In some embodiments, each dose of Flu comprises about 10 mg / m 2 and about 60 mg / m 2 In some embodiments, each Flu dose contains about 30 mg / m 2 . In some embodiments, the lymphodepleting therapy comprises administering Flu and cyclophosphamide (Cy). In some embodiments, the lymphodepleting therapy comprises administering three doses of Cy. In some embodiments, the first dose of Flu and Cy are each administered 5 days before the start of the dosing cycle; the second dose of Flu and Cy are each administered 4 days before the start of the dosing cycle; and the third dose of Flu and Cy are each administered 3 days before the start of the dosing cycle. In some embodiments, each Cy dose contains about 200 mg / m 2 and about 600 mg / m 2 In some embodiments, each Cy dose comprises about 300 mg / m 2 In some embodiments, each Cy dose comprises about 500 mg / m 2 In some embodiments, each Flu dose contains about 30 mg / m 2 Each Cy dose contains approximately 300 mg / m 2 In some embodiments, each Flu dose contains about 30 mg / m 2 Each Cy dose contains approximately 500 mg / m 2In some embodiments, 30 mg / m 2 Flu and 500 mg / m 2 The Cy of each is administered five, four, and three days prior to the start of the dosing cycle. In several embodiments, about two days are allowed to pass between the third dose of cyclophosphamide and fludarabine and the start of the dosing cycle.

[0093] In some embodiments, the lymphodepleting therapy comprises administering fludarabine (Flu). In some embodiments, the lymphodepleting therapy comprises administering three to five doses of Flu. In some embodiments, each Flu dose comprises about 10 mg / m 2 and about 60 mg / m 2 In some embodiments, each Flu dose contains about 30 mg / m 2 In some embodiments, the lymphodepleting therapy comprises administering Flu and cytarabine (Ara-C). In some embodiments, the lymphodepleting therapy comprises five doses of Ara-C. In some embodiments, the lymphodepleting therapy comprises five daily doses of Ara-C and five daily doses of fludarabine, wherein the first dose of Ara-C and fludarabine is administered seven days before the start of the dosing cycle. In some embodiments, the first dose of Flu and Ara-C is each administered seven days before the start of the dosing cycle; the second dose of Flu and Ara-C is each administered six days before the start of the dosing cycle; the third dose of Flu and Ara-C is each administered five days before the start of the dosing cycle; the fourth dose of Flu and Ara-C is each administered four days before the start of the dosing cycle; and the fifth dose of Flu and Ara-C is each administered three days before the start of the dosing cycle. In some embodiments, about two days are allowed to pass between the fifth dose of Ara-C and fludarabine and the start of the dosing cycle. In some embodiments, each Ara-C dose contains about 1 g / m 2 With about 4 g / m 2 In some embodiments, each Ara-C dose comprises about 2 g / m 2 In some embodiments, each Flu dose contains about 30 mg / m 2 Each Ara-C dose contains approximately 2 g / m 2 In some embodiments, about 30 mg / m 2 Flu and about 2 g / m 2 The Ara-C was administered seven, six, five, four and three days prior to the start of the dosing cycle, respectively.

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

[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, a poly (ADP-ribose) polymerase (PARP) inhibitor, 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, streptozotocin, 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 streptozotocin. In some embodiments, the alkylating agent comprises busulfan. In some embodiments, the alkylating agent comprises carboplatin. In some embodiments, the alkylating agent comprises oxaliplatin.

[0098] 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, an 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; anthracyclines, optionally doxorubicin, daunorubicin or idarubicin, mitomycin, or any combination thereof. In some embodiments, the antibiotic comprises bleomycin; actinomycin D; anthracyclines. 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 some embodiments, 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 (eg, venetoclax). In some embodiments, the therapeutic agent comprises a glucocorticoid (eg, 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-PD1 antibody, an anti-PD-L1 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-PD1 antibody. In some embodiments, the monoclonal antibody comprises an anti-PD-L1 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 CD16, NKp30, NKp46, NKG2D, and any combination thereof.

[0106] In some embodiments, the therapeutic agent is decitabine. In some embodiments, prior to starting the dosing cycle, the subject is administered a dose of decitabine daily for five days. In some embodiments, the dose of decitabine comprises about 20 mg / m 2 .

[0107] Also provided herein is a method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (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 approximately 7 days after the administration of the first dose of the genetically engineered NK cells; and (iii) a third dose of the genetically engineered NK cells administered to the subject approximately 7 days after the administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises approximately 1.5 x 10 9 genetically engineered NK cells; and wherein prior to administering the first dose of the genetically engineered NK cells, the subject has been administered approximately 20 mg / m2 The dose of decitabine lasts for five days.

[0108] In some embodiments, the subject has less than or equal to 5% peripheral blasts. In some embodiments, the subject has no evidence of extramedullary disease.

[0109] In some embodiments, before administering the NK cell population to the subject, the percentage of peripheral primitive cells in the subject is determined. In some embodiments, before administering the NK cell population to the subject, if it is determined that the subject has less than or equal to 5% peripheral primitive cells, the subject is selected for treatment. In some embodiments, before administering the NK cell population to the subject, the percentage of peripheral primitive cells in the subject is determined, and if it is determined that the subject has less than or equal to 5% peripheral primitive cells, the subject is selected for treatment. In some embodiments, before administering the NK cell population to the subject, the method includes determining the percentage of peripheral primitive cells in the subject. In some embodiments, before administering the NK cell population to the subject, if it is determined that the subject has less than or equal to 5% peripheral primitive cells, the method includes selecting the subject for treatment. In some embodiments, before administering the NK cell population to the subject, the method includes: (a) determining the percentage of peripheral primitive cells in the subject; and (b) if it is determined that the subject has less than or equal to 5% peripheral primitive cells, the subject is selected 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 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 bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, stomach 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 bladder cancer. In some embodiments, the cancer is bone cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is 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-PD1 antibody, or an anti-PD-L1 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 relapsed after hematopoietic cell transplantation (HCT). In some embodiments, the cancer cells have an fms-like tyrosine kinase 3 (FLT3) mutation or an isocitrate dehydrogenase (IDH) 1 / 2 mutation.

[0119] In some embodiments, the subject has been treated with one, two, three, or four prior lines of treatment. In some embodiments, the subject has been treated with one prior line of treatment. In some embodiments, the subject has been treated with two prior lines of treatment. In some embodiments, the subject has been treated with three prior lines of treatment. In some embodiments, the subject has been treated with four prior lines of treatment. In some embodiments, if the subject has a targetable FLT3 mutant cancer or a targetable IHD 1 / 2 mutant cancer, the subject has been treated with four prior lines of treatment. In some embodiments, the subject has a targetable FLT3 mutant cancer. In some embodiments, the subject has a targetable IHD 1 / 2 mutant cancer.

[0120] In some embodiments, the subject has an ECOG of 0 to 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 CD3ζ domain. In some embodiments, the costimulatory domain comprises an OX40 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 shown in SEQ ID NO:39.

[0122] In some embodiments, the genetically engineered NK cells express 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 of 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 of SEQ ID NO: 38. In several embodiments, the mbIL15 has at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 40. In several embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the population of genetically engineered NK cells is allogeneic to the subject. In some embodiments, the population of genetically engineered cells is 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, individual doses of the engineered NK cells are administered to the subject on an outpatient basis.

[0124] Also provided herein is a use of a combination for treating cancer in a subject, the combination comprising: (a) a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (NKG2D), and (b) a therapeutic agent selected from a chemotherapeutic agent, a monoclonal antibody, an 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 administering the first dose of the genetically engineered NK cells to the subject, lymphodepletion therapy is administered to the subject.

[0125] Also provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein the genetically engineered NK cells are administered to the subject 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, an NK cell engager, and any combination thereof; and wherein, prior to administering the first dose of the genetically engineered NK cells to the subject, lymphodepletion therapy is administered to the subject.

[0126] Also provided herein is a use of a therapeutic agent selected from a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, for treating 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 to a ligand for natural killer cell group 2 D (NKG2D); wherein the genetically engineered NK cells are administered to the subject in a dosing cycle; and wherein, prior to administering the first dose of the genetically engineered NK cells to the subject, lymphodepletion therapy is administered to the subject.

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

[0128] Also provided herein is a combination for treating cancer in a subject, the combination comprising: (a) a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D), and (b) a therapeutic agent that increases expression of NKG2D ligand in the subject.

[0129] Also provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating 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 NKG2D ligand in the subject.

[0130] Also provided herein is a use of a therapeutic agent that increases expression of an NKG2D ligand in a subject for treating cancer in the subject; wherein the subject has been treated with or is a candidate for treatment with a natural killer (NK) cell population genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D).

[0131] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody, an NK cell adapter, 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 an NK cell adapter. In some embodiments, lymphocyte depletion therapy is administered to the subject prior to administering the first dose of the genetically engineered NK cells to the subject.

[0132] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological 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 administration 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 the administration of the first dose of the genetically engineered NK cells; and (iii) a third dose of the genetically engineered NK cells administered to the subject between about 5 days and about 10 days after the administration of the second dose of the genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 1 x 10 8 and about 1 x 10 10 In some embodiments, each dose of the genetically engineered NK cells comprises about 1 x 10 9 In some embodiments, each dose of the genetically engineered NK cells comprises about 1.5 x 10 9 NK cells between the two.

[0134] In some embodiments, the first dosing cycle is initiated after the subject is administered 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, one or more additional cycles are administered depending on the status of the subject's cancer (e.g., in the event of progression or development of another cancer). In several embodiments, additional cycles are not required if the subject demonstrates a response (e.g., complete remission). BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figures 1A-1B depict non-limiting schematic diagrams of polynucleotides encoding cytotoxic receptor constructs comprising a binding portion for an NKG2D ligand and encoding either mbIL15 (Receptor B) or not encoding mbIL15 (Receptor A).

[0136] 2A-2B depict non-limiting schematic diagrams of polynucleotides encoding cytotoxic receptor constructs comprising an NKG2D receptor domain (eg, fragment) and encoding mbIL15 (Receptor B) or not encoding mbIL15 (Receptor A).

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

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

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

[0140] FIG5A shows the IC values for specific killing of A-431 cells treated with NK cells expressing NKG2D chimeric receptors from donors with the indicated CD16 phenotypes in combination with 10 μg / mL cetuximab or isotype control antibody. 50 curve.

[0141] 5B-5C show the increased percent killing efficacy of cells expressing the NKG2D chimeric receptor in combination with cetuximab when co-cultured with A-431 ( FIG. 5B ) or NCI-H2228 cells ( FIG. 5C ).

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

[0143] FIG7 shows the expression of NucRed™ or NKG2D ligands by NucRed™-labeled UMUC3 cells.

[0144] FIG8A , FIG8B , and FIG8C show the expression of NKG2D ligands by UMUC3 cells after treatment with the indicated concentrations of gemcitabine for 24 hours, 48 hours, or 72 hours, respectively.

[0145] Figure 9 shows the IC of gemcitabine for UMUC3 cells 50 curve.

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

[0147] Some embodiments of the methods and compositions provided herein relate to engineered immune cells, therapeutic agents, and combinations thereof for immunotherapy. In several embodiments, the engineered cells are engineered in various ways, for example, to express a receptor complex that induces cytotoxicity. As used herein, the term "cytotoxic receptor complex" should be given its ordinary meaning and should also refer to (unless otherwise indicated) chimeric antigen receptors (CARs), chimeric receptors (also referred to as activating chimeric receptors in the case of NKG2D chimeric receptors). In various embodiments, the cells are further engineered to achieve modification of the responsiveness of the cells to non-tumor tissues and / or other therapeutic cells. I. Cell Type

[0148] Some embodiments of the methods and compositions provided herein involve cells, such as immune cells. For example, immune cells (such as NK cells or T cells) can be engineered to contain a chimeric receptor (such as an NKG2D ligand-directed chimeric receptor) or to contain a nucleic acid encoding a chimeric receptor as described herein. Additional embodiments involve engineering a second group of cells to express another cytotoxic receptor complex, such as the NKG2D chimeric receptor complex disclosed herein.

[0149] Traditional cancer treatments rely on surgery, radiation therapy, chemotherapy, or a combination of these. As research has yielded a deeper understanding of the mechanisms underlying certain cancers, this knowledge has been used to develop targeted cancer therapies. Targeted therapy is a cancer treatment method that uses drugs that target specific genes or proteins found in cancer cells or cells that support cancer growth (such as blood vessel cells) to reduce or prevent cancer cell growth. More recently, genetic engineering has enabled the development of approaches that harness aspects of the immune system to fight cancer. 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 cells (NK cells), or a combination thereof, as described in more detail below. In some embodiments, the immune cells include T cells. In some embodiments, the immune cells include NK cells. In some embodiments, the immune cells include both T cells and NK cells.

[0150] To promote cancer immunotherapy, polynucleotides, polypeptides, and vectors encoding chimeric receptors comprising a target-binding moiety (e.g., an extracellular binder of a ligand expressed by cancer cells) and a cytotoxic signaling complex are also provided herein. For example, some embodiments include polynucleotides, polypeptides, or vectors encoding, for example, activating chimeric receptors comprising an NKG2D extracellular domain specific for tumor markers (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, etc.) to promote immune cell targeting of cancer and exert cytotoxic effects against cancer cells. Engineered immune cells (e.g., NK cells and / or T cells) expressing such chimeric receptors are also provided herein. In several embodiments, polynucleotides, polypeptides, and vectors encoding constructs comprising an extracellular domain comprising two or more subdomains (e.g., a first ligand-binding receptor and a second ligand-binding receptor) and a cytotoxic signaling complex are also provided herein. Also provided are engineered immune cells (e.g., NK cells and / or T cells) expressing such bispecific constructs (in some embodiments, the first ligand-binding domain and the second ligand-binding domain target the same ligand). Also provided herein are methods of treating cancer and other uses of such cells for cancer immunotherapy. Engineered cells for immunotherapy

[0151] In several embodiments, cells of the immune system are engineered to have enhanced cytotoxic effects on target cells (such as tumor cells). For example, cells of the immune system can be engineered to contain tumor-directed chimeric receptors and / or tumor-directed CARs as described herein. In several embodiments, white blood cells or leukocytes are used because their natural function is to protect the body from the growth of abnormal cells and infectious diseases. There are many types of white blood cells that play specific roles in the human immune system and are therefore preferred starting points for the cell engineering disclosed herein. White blood cells include granulocytes and agranulocytes (having granules present or absent in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells (such as those described below or otherwise herein) can be engineered to contain chimeric antigen receptors, such as NKG2D ligand-directed chimeric receptors, or nucleic acids encoding chimeric receptors. In several embodiments, immune cells engineered to express a chimeric receptor are engineered to also express (e.g., bicistronic expression) a membrane-bound interleukin-15 (mbIL15) domain. As discussed in more detail below, in several embodiments, the therapeutic cells are further genetically modified to enhance the cytotoxicity and / or persistence of the cells. In several embodiments, the genetic modifications enhance the cells' ability to resist signals from the tumor microenvironment that would otherwise reduce the efficacy or lifespan of the therapeutic cells. Monocytes for immunotherapy

[0152] In some embodiments, immune cells include monocytes. Monocytes are a subtype of white blood cells. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are associated with the adaptive immune system and perform primary functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of ingesting cellular material or whole cells, followed by digestion and destruction of the engulfed cellular material. In several embodiments, monocytes are used in combination with one or more additional engineered cells as disclosed herein. Several embodiments of the methods and compositions disclosed herein involve monocytes engineered to express activating chimeric receptors that target ligands on tumor cells (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others). In some embodiments, monocytes engineered to express chimeric receptors are also engineered to express (e.g., bicistronic expression) a membrane-bound interleukin-15 (mbIL15) domain. In several embodiments, the monocytes are autologous. In some embodiments, the monocytes are allogeneic cells. Lymphocytes for immunotherapy

[0153] In some embodiments, the immune cells include lymphocytes. Lymphocytes (the other major subtype of white blood cells) 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 include T cells. Thus, in some embodiments, the immune cells include NK cells. In some embodiments, the immune cells include B cells. While B cells are engineered according to several embodiments disclosed herein, several embodiments also involve engineered T cells or engineered NK cells (in some embodiments, using a mixture of T cells and NK cells from the same donor or different donors). Several embodiments of the methods and compositions disclosed herein involve lymphocytes engineered to express activating chimeric receptors that target ligands on tumor cells (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others). In some embodiments, the lymphocytes engineered to express the chimeric receptor are engineered to also express (e.g., bicistronic expression) 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 can be distinguished from other lymphocyte subtypes (e.g., B cells or NK cells) based on the presence of T cell receptors on their cell surface. T cells can be divided into various subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated constant T cells, and γδ 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, γδ 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 T cell types to be engineered to express the cytotoxic receptor complexes disclosed herein. In several embodiments, specific techniques (e.g., stimulation with cytokines) are used to enhance the expansion / collection of T cells with a specific marker signature. For example, in several embodiments, activation of certain human T cells (e.g., CD4+ T cells, CD8+ T cells) is achieved by using CD3 and / or CD28 as stimulatory molecules. In several embodiments, methods for treating or preventing cancer or infectious diseases are provided, comprising administering a therapeutically effective amount of T cells expressing a cytotoxic receptor complex and / or homing moiety as described herein. In several embodiments, the T cells are autologous. In some embodiments, the T cells are allogeneic. Several embodiments of the methods and compositions disclosed herein involve T cells engineered to express an activating chimeric receptor that targets a ligand on tumor cells (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (etc.)). In some embodiments, the T cells engineered to express the chimeric receptor are also engineered to express (e.g., bicistronic expression) a membrane-bound interleukin 15 (mbIL15) costimulatory domain. NK cells for immunotherapy

[0155] In some embodiments, immune cells include NK cells. Thus, in several embodiments, methods for treating or preventing cancer or infectious diseases are provided, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex and / or homing moiety as described herein. In several embodiments, methods for treating cancer are provided, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex. In several embodiments, the NK cells are autologous cells. In some embodiments, the NK cells are allogeneic cells. In several embodiments, NK cells are preferred because NK cells have a relatively high natural cytotoxic potential. In several embodiments, unexpectedly, the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, resulting in even more effective activity against target cells (e.g., tumors or other diseased cells). Several embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express activating chimeric receptors that target ligands on tumor cells (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (etc.)). In some embodiments, the NK cells engineered to express the chimeric receptors are also engineered to express (e.g., bicistronic expression) a membrane-bound interleukin 15 (mbIL15) domain. Thus, in some embodiments, NK cells are engineered to express (e.g., bicistronic expression) a chimeric receptor (e.g., NKG2D ACR) and mbIL15.

[0156] In several embodiments, immortalized NK cells are used and engineered as disclosed herein. In some embodiments, the NK cells are derived from the NK-92 cell line. NK-92 cells are derived from NK cells but lack the major inhibitory receptors displayed by normal NK cells, while retaining most of the activating receptors. Some embodiments of the NK-92 cells described herein relate to NK-92 cells that have been engineered to silence certain additional inhibitory receptors (e.g., SMAD3), thereby allowing upregulation of interferon-γ (IFNγ), granzyme B, and / or perforin production. Additional information related to the NK-92 cell line is disclosed in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, which are incorporated herein by reference in their entirety. In several embodiments, NK-92 cells are used in combination with one or more other cell types disclosed herein. For example, in one embodiment, NK-92 cells are used in combination with NK cells as disclosed herein. In additional embodiments, 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 immunotherapy methods disclosed herein. In several embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. In several embodiments, HSCs are used to exploit their ability to engraft for long-term production of blood cells, which can result in a continuous source of targeted anti-cancer effector cells, for example, to combat cancer remission. In several embodiments, this ongoing production helps 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 types disclosed herein. Several embodiments of the methods and compositions disclosed herein involve hematopoietic stem cells engineered to express activating chimeric receptors targeting ligands on tumor cells (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others). In some embodiments, the HSC engineered to express the chimeric receptor is engineered to also express (eg, bicistronic expression) 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 immunotherapy methods disclosed herein. In some embodiments, induced pluripotent stem cells (iPSCs) are used in the immunotherapy methods disclosed herein. In several embodiments, iPSCs are used to exploit their ability to differentiate and derive into non-pluripotent cells, including but not limited to CD34 cells, hematopoietic endothelial 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 containing one or more genetic modifications at a selected site by differentiating iPSCs, or less differentiated cells containing the same genetic modifications at the same selected site. In several embodiments, iPSCs are used to generate iPSC-derived NK or T cells. In several embodiments, the cells are engineered to express homing moieties and / or cytotoxic receptor complexes. In several embodiments, iPSCs are used in combination with one or more additional engineered cell types disclosed herein. Several embodiments of the methods and compositions disclosed herein involve induced pluripotent stem cells engineered to express activating chimeric receptors that target ligands on tumor cells (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (etc.)). In some embodiments, the iPSCs engineered to express the chimeric receptors are also engineered to express (e.g., bicistronic expression) a membrane-bound interleukin 15 (mbIL15) costimulatory domain. In several embodiments, the engineered iPSCs are differentiated into NK, T, or other immune cells, such as for use in the compositions or methods provided herein. II. Extracellular Domain (Tumor Binder)

[0159] Some embodiments of the compositions and methods described herein relate to chimeric receptors comprising an extracellular domain comprising 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 chimeric receptors (also referred to as activating chimeric receptors) comprising an extracellular domain comprising a ligand-binding domain that binds a ligand expressed by tumor cells as described herein. In some embodiments, the ligand-binding domain binds to a ligand for NKG2D. Depending on the embodiment, the ligand-binding domain 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 a wild-type or non-wild-type sequence of an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single domain antibody (sdAb), a vH or vL domain, a camelid VHH domain, or a non-immunoglobulin scaffold (e.g., a DARPIN, affibody, affilin, adnectin, affitin, repebody, fynomer, alphabody, avimer, atrimer, centyrin, probentin, anticalin, kunitz domain, armadillo repeat protein, autoantigen, receptor, or ligand). In some embodiments, the tumor binding domain contains more than one antigen binding domain. Antigen binding proteins

[0161] In several embodiments, antigen-binding proteins are provided. As used herein, the term "antigen-binding protein" should be given its ordinary meaning and should 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 the binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds to the antigen or from the light chain of an antibody that binds to the antigen. In still some embodiments, the antigen-binding fragment comprises all six CDRs (three from the heavy chain and three from the light chain) from an antibody that binds to the antigen. 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 chain and / or light chain CDRs. In some embodiments, the antigen-binding fragment is an antibody fragment.

[0162] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody analogs. Other specific examples include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a camelid heavy-chain antibody; VHH fragment), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments. These molecules can be derived from any mammalian source, such as human, mouse, rat, rabbit, or pig, dog, or camelid. Antibody fragments can compete with intact (e.g., natural) antibodies for target antigen binding and can be produced by modification of intact antibodies (e.g., enzymatic or chemical cleavage) or de novo synthesis using recombinant DNA technology or peptide synthesis. Antigen-binding proteins can include, for example, surrogate protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic scaffolds containing, for example, biocompatible polymers. Additionally, 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 multiple polypeptide chains. For example, the antigen-binding protein may include, but is not limited to, diabodies; intrabodies; domain antibodies (a single VL or VH domain or two or more VH domains connected by a peptide linker); macrobodies (two scFvs fused to an Fc region); triabodies; tetrabodies; minibodies (scFvs fused to a CH3 domain); peptibodies (one or more peptides attached to an Fc region); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions); small modular immunopharmaceuticals; and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, 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" should be given its ordinary meaning and should also refer to a tetrameric molecule, wherein each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light" chain (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 carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0165] Within the light and heavy chains, the variable (V) and constant (C) regions are connected by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. The variable regions of each light / heavy chain pair form the antibody combining site, resulting in two binding sites for an intact immunoglobulin.

[0166] Immunoglobulin chains exhibit the same overall 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 contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0167] Human light chains are classified as kappa (κ) and lambda (λ) light chains. An antibody "light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa (κ) light chains and lambda (λ) light chains refer to the two major antibody light chain isotypes. A light chain can comprise 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 (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε) chains, and define the antibody 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 its naturally occurring conformation in an antibody molecule, and generally determines the class to which the antibody belongs. A heavy chain can include a polypeptide comprising, from amino-terminus to carboxyl-terminus, a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4).

[0169] The IgG class is further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4. The IgA class is further divided into subclasses, namely IgA1 and IgA2. IgM has multiple subclasses, including but not limited to IgM1 and IgM2. The heavy chains in IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains in IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via interpolypeptide disulfide bonds between the CL domain and the CH1 domain (e.g., between the light chain and the heavy chain) and between the hinge regions of the antibody heavy chains.

[0170] In some embodiments, the antigen-binding protein is an antibody. As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be monoclonal or polyclonal, multi-chain or single-chain or complete immunoglobulins, and can be from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. Antibodies can be "humanized," "chimeric," or non-human. Antibodies can include complete immunoglobulins of any isotype, and include, for example, chimeric antibodies, humanized antibodies, human antibodies, and bispecific antibodies. Complete antibodies typically comprise at least two full-length heavy chains and two full-length light chains. Antibody sequences can be derived from only a single species, or can be "chimeric," that is, different parts of the antibody can be derived from two different species, as further described below. Unless otherwise indicated, the term "antibody" also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains, as long as the antibody retains the same or similar binding and / or function as an antibody comprising two full-length light chains and a heavy chain. For example, antibodies having 1, 2, 3, 4 or 5 amino acid residues substituted, inserted or deleted at the N-terminus and / or C-terminus of the heavy chain and / or light chain are included in the definition, as long as the antibody retains the same or similar combination and / or function as the antibody comprising two full-length heavy chains and two full-length light chains. The example of an antibody includes monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies and synthetic antibodies. In some embodiments, monoclonal antibodies and polyclonal antibodies are provided. As used herein, the term "polyclonal antibody" should be given its ordinary meaning, and should also refer to antibody colonies that typically vary widely in composition and binding specificity. As used herein, the term "monoclonal antibody" ("mAb") should be given its ordinary meaning, and should also refer to one or more in antibody colonies with the same sequence. Monoclonal antibodies bind to the antigen at a specific epitope on the antigen.

[0171] In some embodiments, the antigen-binding protein is an antibody fragment or antigen-binding fragment. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an antigen epitope (e.g., through binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CHI domains, linear antibodies, single-domain antibodies (e.g., sdAb (VL or VH)), Camelidae VHH domains, multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be introduced into single-domain antibodies, large antibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be transplanted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). Antibody fragments can include Fab, Fab', F(ab')2, and / or Fv fragments that contain at least one CDR of an immunoglobulin sufficient to confer specific antigen binding to a cancer antigen (e.g., CD19). Antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.

[0172] In some embodiments, a Fab fragment is provided. A Fab fragment is a monovalent fragment having VL, VH, CL, and CH1 domains; a F(ab')2 fragment is a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; an Fd fragment has VH and CH1 domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has an antigen-binding fragment of a VH domain, a VL domain, or a VH or VL domain. In some embodiments, these antibody fragments can be introduced into single-domain antibodies, single-chain antibodies, large antibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. In some embodiments, the antibody comprises at least one CDR as described herein.

[0173] In several embodiments, single-chain variable fragments are also provided herein. As used herein, the term "single-chain variable fragment" ("scFv") should be given its ordinary meaning and should also refer to a fusion protein in which the VL and VH regions are connected by 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 clarity, unless otherwise specified, a "single-chain variable fragment" is not an antibody or antibody fragment as defined herein. A diabody is a bivalent antibody comprising two polypeptide chains, each of which comprises a VH and a VL domain connected by a linker that is configured to reduce or disallow pairing between the two domains on the same chain, thereby allowing each domain to pair with a complementary domain on the other polypeptide chain. According to several embodiments, if the two polypeptide chains of a diabody are identical, the diabody resulting from their pairing will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to create diabodies with two different antigen-binding sites. Similarly, triabodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which may be the same or different.

[0174] In several embodiments, an antigen-binding protein comprises one or more CDRs. As used herein, the term "CDR" should be given its ordinary meaning and also refers to the complementarity-determining region (also known as the "minimal recognition unit" or "hypervariable region") within an antibody variable sequence. CDRs allow an 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 are typically aligned by framework regions to form a structure that specifically binds to a specific epitope or domain on the target protein. From N-terminus to C-terminus, naturally occurring light and heavy chain variable regions typically conform to the following order of these elements: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. For the heavy chain variable region, the order from N-terminus to C-terminus is generally: FW-H1, CDR-H1, FW-H2, CDR-H2, FW-H3, CDR-H3, and FW-H4. For the light chain variable region, the order from N-terminus to C-terminus is generally: FW-L1, CDR-L1, FW-L2, CDR-L2, FW-L3, CDR-L3, FW-L4. A numbering system has been designed to assign numbers to the amino acids occupying 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 and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. Using this system, the complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (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 can utilize CDRs defined according to any of these systems. For any given embodiment comprising more than one CDR, the CDRs can be defined according to any of the Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or conformational definitions, or a combination of any of the foregoing.Any CDR may be interpreted individually or in the context of a variable domain as appropriate by one skilled in the art according to any of these numbering systems.One or more CDRs may be covalently or non-covalently incorporated into a molecule to make it an antigen binding protein.

[0175] In some embodiments, the antigen-binding proteins provided herein comprise one or more CDRs as part of a larger polypeptide chain. In some embodiments, the antigen-binding protein covalently links the one or more CDRs to another polypeptide chain. In some embodiments, the antigen-binding protein non-covalently incorporates the one or more CDRs. In some embodiments, the antigen-binding protein may comprise at least one CDR described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support, framework, or scaffold capable of displaying one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that bind to an antigen in a localized surface area. Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs), or may have one or more modifications relative to a naturally occurring polypeptide or fold, such as amino acid additions, deletions, and / or substitutions. Depending on the embodiment, the scaffold may be derived from polypeptides of multiple different species (or more than one species), such as humans, non-human primates or other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0176] Depending on the embodiment, the biocompatible framework structures are based on protein scaffolds or backbones other than immunoglobulin domains. In some such embodiments, those framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and / or tendamistat domain.

[0177] In some embodiments, antigen-binding proteins with more than one binding site are also provided. In several embodiments, the binding sites are identical to each other, while in some embodiments, the binding sites are different from each other. For example, antibodies typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody will bind to two different epitopes, which can be located on the same or different protein targets. In several embodiments, this is particularly advantageous because bispecific chimeric antigen receptors can give engineered cells the ability to target multiple tumor markers. For example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6, etc., as well as other tumor markers such as CD70, CD123, CD19, Her2, mesothelin, claudin 6, BCMA, EGFR, or any other marker disclosed herein or understood in the art as a tumor-specific antigen or tumor-associated antigen can be bound by a bispecific antibody. Natural killer cell family domains that bind tumor ligands

[0178] In several embodiments, the ability of engineered immune cells (such as NK cells) to recognize and destroy tumor cells is exploited. NK cells express both inhibitory and activating receptors on their cell surfaces. Inhibitory receptors bind to self-molecules expressed on the surface of healthy cells (thus preventing immune responses against "self" cells), while activating receptors bind to ligands expressed on abnormal cells (such as tumor cells). When the balance between inhibitory and activating receptor activation favors the 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. Surface expression of various NKG2D ligands is typically low in healthy cells but is upregulated, for example, during 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 functions of NK cells. In several embodiments, T cells are engineered to express an extracellular domain that binds one or more tumor ligands and activates them. For example, in several embodiments, T cells are engineered to express the NKG2D receptor as a binding / activating moiety. In several embodiments, NK cells are engineered to express an extracellular domain that binds one or more tumor ligands and activates them. For example, in several embodiments, NK cells are engineered to express the NKG2D receptor as a binding / activating moiety. In several embodiments, the engineered cells disclosed herein are engineered to express another member of the NKG2 family, such as NKG2A, NKG2C, and / or NKG2E. Combinations of such receptors are engineered in some embodiments. Additionally, in several embodiments, other receptors, such as killer cell immunoglobulin-like receptors (KIRs), are expressed.

[0180] In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a full-length NKG2D as an extracellular component, thereby recognizing a ligand on the surface of a tumor cell (e.g., a hepatocyte). In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a functional fragment of an NKG2D (e.g., a human NKG2D) as an extracellular component, thereby recognizing a ligand on the surface of a tumor cell (e.g., a hepatocyte). In one embodiment, the full-length NKG2D (e.g., a full-length human NKG2D) has the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the full-length NKG2D (e.g., a full-length human NKG2D) has the amino acid sequence of SEQ ID NO: 43. In several embodiments, the full-length NKG2D or a functional fragment thereof is human NKG2D. Additional information regarding chimeric receptors for use with the methods and compositions disclosed herein can be found in PCT Patent Publication No. WO / 2018 / 183385, which is incorporated herein by reference in its entirety.

[0181] In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a functional fragment of NKG2D as an extracellular component, thereby recognizing a ligand on the surface of a tumor cell or other diseased cell. In one embodiment, the functional fragment of NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 25. In several embodiments, the NKG2D fragment has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to full-length wild-type NKG2D. In several embodiments, the nucleic acid sequence encoding the fragment may have one or more additional mutations compared to SEQ ID NO: 25, but retain ligand binding function or, in some embodiments, have 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 concatemer; such embodiments provide enhanced ligand binding activity. In several embodiments, the sequence encoding the NKG2D fragment is optionally fully or partially codon-optimized. In one embodiment, the sequence encoding the codon-optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28. Advantageously, according to several embodiments, the functional fragment lacks its native transmembrane or intracellular domain, but retains its ability to bind to NKG2D ligands and transduce activation signals upon ligand binding. Another advantage of such NKG2D fragments is that expression of DAP10 is not required to localize NKG2D to the cell membrane. 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 the embodiments disclosed herein), are engineered to express one or more chimeric receptors targeting, for example, CD70, CD19, CD123, Her2, mesothelin, claudin-6, BCMA, EGFR, and NKG2D ligands such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. In several embodiments, such cells also express (eg, bicistronic expression) mbIL15.

[0182] In several embodiments, the cytotoxic receptor complex is configured to dimerize. Dimerization can comprise homodimers or heterodimers, depending on the embodiment. In several embodiments, dimerization results in improved ligand recognition by the cytotoxic receptor complex (and, therefore, the NK cells expressing the receptor), leading to a reduction (or absence) of adverse toxic effects. In several embodiments, the cytotoxic receptor complex employs internal dimers or duplication of one or more component subunits. For example, in several embodiments, the cytotoxic receptor complex can optionally comprise a first NKG2D extracellular domain coupled to a second NKG2D extracellular domain and a transmembrane / signaling domain (or a single transmembrane domain and a single signaling domain).

[0183] In several embodiments, the various domains / subdomains are separated by a linker, for example, using a GS3 linker (SEQ ID NOs: 15 and 16, nucleotide and protein, respectively) (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 in accordance with various embodiments disclosed herein include, but are not limited to, those encoded by SEQ ID NOs: 17, 19, 21, or 23. In several embodiments, the other linker comprises a peptide sequence of one of SEQ ID NOs: 18, 20, 22, and 24. This provides the potential to separate the various component parts of the receptor complex along the polynucleotide, which can enhance the expression, stability, and / or function of the receptor complex. III. Cytotoxicity Signaling Complexes

[0184] Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as chimeric receptors for NKG2D ligands (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6), that include a cytotoxic signaling complex. As disclosed herein, according to several embodiments, the chimeric receptors provided comprise one or more transmembrane domains and / or intracellular domains that, when the one or more extracellular domains bind to a ligand on the surface of a target cell, initiate a cytotoxic signaling cascade. Thus, in some embodiments, the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain (e.g., CD3ζ) and a costimulatory signaling domain.

[0185] In several embodiments, the chimeric receptor comprises at least one transmembrane domain, at least one costimulatory domain, and / or at least one signaling domain. In some embodiments, more than one component part constitutes a given domain, for example, a costimulatory domain can comprise two subdomains. Furthermore, in some embodiments, a domain can serve multiple functions, for example, a transmembrane domain can also serve to provide a signaling function. IV. Transmembrane domain

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

[0187] However, in several embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein typically expressed on both T cells and NK cells. In several embodiments, the transmembrane domain comprises CD8α. In several embodiments, the transmembrane domain comprises the CD8 (e.g., CD8α) hinge and the CD8 (e.g., CD8α) transmembrane region.

[0188] In several embodiments, the transmembrane domain comprises a "hinge", such as a CD8α hinge. In several embodiments, the hinge of CD8α has the nucleic acid sequence of SEQ ID NO: 1. In several embodiments, the CD8α 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 CD8α hinge having the sequence of SEQ ID NO: 1. In several embodiments, the hinge of CD8α comprises the amino acid sequence of SEQ ID NO: 2. In several embodiments, the CD8α hinge can be truncated or modified so 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 CD8α transmembrane region. In several embodiments, the CD8α transmembrane region is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 3. In several embodiments, the CD8α transmembrane region is truncated or modified and is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 3. In several embodiments, the CD8α transmembrane region comprises the amino acid sequence of SEQ ID NO: 4. In several embodiments, the CD8α transmembrane region is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CD8α having the sequence of SEQ ID NO: 4.

[0190] In summary, 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 to 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 the chimeric receptor provided herein comprises a stimulatory molecule. Thus, some embodiments of the compositions and methods described herein relate to chimeric receptors comprising stimulatory molecules (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors). In several embodiments, in addition to various transmembrane domains and signaling domains (and combinations of transmembrane / signaling domains), additional stimulatory molecules may also be provided. These may be, for example, certain molecules that further enhance the activity of immune cells. In some embodiments, cytokines may be used. For example, certain interleukins, such as IL-2 and / or IL-15 (as non-limiting examples), are used. In some embodiments, immune cells used for therapy are engineered to express such molecules in a secreted form. In other embodiments, such stimulatory molecules are engineered to be membrane-bound, acting as autocrine stimulatory molecules (or even as paracrine stimulators of neighboring cells).

[0193] In several embodiments, the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15). In some embodiments, IL15 is expressed by a separate cassette on a construct comprising any of the CARs disclosed herein. In some embodiments, IL15 and any of the CARs disclosed herein are expressed by the same cassette. In some embodiments, the chimeric receptor and IL15 are separated by a nucleic acid sequence encoding a cleavage site (e.g., a proteolytic cleavage site or a T2A, P2A, E2A, or F2A self-cleavage peptide cleavage site). In some embodiments, the chimeric receptor and IL15 are separated by a T2A sequence. In some embodiments, the T2A sequence comprises SEQ ID NO: 10. In some embodiments, the T2A sequence comprises SEQ ID NO: 45.

[0194] In some embodiments, IL15 is membrane-bound IL15 (mbIL15). In some embodiments, mbIL15 comprises a native IL15 sequence (e.g., a native human IL15 sequence) and at least one transmembrane domain. In some embodiments, the native IL15 sequence is encoded by a sequence that has at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 11. In some embodiments, the native IL15 sequence comprises a peptide sequence that has at least 85%, at least 90%, or 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 being coupled 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, mbIL15 may comprise additional components, such as a leader sequence and / or 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, mbIL15 comprises an amino acid sequence having at least about 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 40. In some embodiments, mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0196] In some embodiments, the tumor antigen-directed CAR and / or tumor ligand-directed chimeric receptor is encoded by a polynucleotide encoding one or more cytoplasmic protease cleavage sites. Such sites are recognized and cleaved by cytoplasmic proteases, which can result in the separation (and separate expression) of the various component parts of the receptor encoded by the polynucleotide. In some embodiments, the tumor antigen-directed CAR and / or tumor ligand-directed chimeric receptor is encoded by a polynucleotide encoding one or more self-cleaving peptides (e.g., 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 component parts of the engineered cytotoxic receptor complex can be delivered to NK cells or T cells in a single vector or via multiple vectors. Thus, as schematically shown in the accompanying figures, the construct can be encoded by a single polynucleotide, but also includes a cleavage site, allowing the downstream elements of the construct to be expressed by the cell as separate proteins (as in the case of IL-15 in some embodiments). In several embodiments, a T2A cleavage site is used. In several embodiments, the T2A cleavage site is encoded by the nucleic acid sequence of SEQ ID NO: 9. In several embodiments, the 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%, or at least 95% sequence identity to 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 several embodiments, 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%, or at least 95% sequence identity to 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%, or at least 95% sequence identity to 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 NK cells enhances the cytotoxic effect of the engineered NK cells by increasing NK cell proliferation and / or lifespan. In several embodiments, mbIL15 and the CAR are encoded by the same polynucleotide. 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 encoding 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 the amino acid sequence of the 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 may be truncated or modified. In some embodiments, mbIL15 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequence of SEQ ID NO: 35. In several embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 40. In several embodiments, mbIL15 is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to mbIL15 having the sequence of SEQ ID NO: 40. Membrane-bound IL15 sequences are found in PCT Publications WO 2018 / 183385 and WO 2020 / 056045, each of which is hereby expressly incorporated by reference in its entirety and with respect to membrane-bound IL15 sequences. VI. Signaling Domain

[0198] Some embodiments of the compositions and methods described herein relate to chimeric receptors comprising an intracellular signaling domain (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors). For example, immune cells engineered according to several embodiments disclosed herein can comprise at least one subunit of the CD3 T cell receptor complex (or fragment thereof). In several embodiments, the intracellular signaling domain comprises a CD3 zeta subunit. In several embodiments, CD3 zeta is encoded by the nucleic acid sequence of SEQ ID NO: 7. In several embodiments, CD3 zeta can be truncated or modified such that it is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 7. In several embodiments, the CD3 zeta domain comprises the amino acid sequence of SEQ ID NO: 8. In several embodiments, the CD3 zeta domain is truncated or modified. In some embodiments, the CD3 zeta domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the CD3 zeta domain having the sequence of SEQ ID NO: 8.

[0199] In several embodiments, unexpectedly enhanced signaling is achieved by using multiple signaling domains whose activities act synergistically. For example, in several embodiments, the intracellular signaling domain further comprises an OX40 domain. In several embodiments, the OX40 domain is an intracellular signaling domain. In several embodiments, the OX40 intracellular signaling domain is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 5. In several embodiments, the OX40 intracellular signaling domain can be truncated or modified such that it is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 5. In several embodiments, the OX40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 6. In several embodiments, the OX40 intracellular signaling domain is truncated or modified. In some embodiments, the OX40 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, OX40 is used as the sole signaling domain in the chimeric receptor construct, however, in several embodiments, OX40 can be used with one or more other domains. For example, in some embodiments, a combination of OX40 and CD3 zeta is used. For example, in some embodiments, the intracellular signaling domain comprises CD3 zeta and OX40 or a signaling portion thereof. In some embodiments, the intracellular signaling domain comprises CD3 zeta and OX40. As another example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0200] In several embodiments, the signaling domain comprises a 4-1BB 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 4-1BB 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%, or at least 95% sequence identity with SEQ ID NO: 29. In several embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 30. In several embodiments, the 4-1BB intracellular signaling domain is truncated or modified. In several embodiments, the 4-1BB intracellular signaling domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the 4-1BB intracellular signaling domain having the sequence of SEQ ID NO: 30. In several embodiments, 4-1BB is used as the sole signaling domain in the chimeric receptor construct, however, in several embodiments, 4-1BB can be used in conjunction with one or more other domains. For example, in some embodiments, a combination of 4-1BB and CD3ζ is used. For example, in some embodiments, the intracellular signaling domain comprises CD3ζ and 4-1BB or a signaling portion thereof. In some embodiments, the intracellular signaling domain comprises CD3ζ and 4-1BB. As another example, in some embodiments, a combination of CD28, OX40, 4-1BB and / or CD3ζ is used.

[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%, or 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%, or 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 in conjunction with one or more other domains. For example, in some embodiments, a combination of CD28 and CD3 zeta is used. For example, in some embodiments, the intracellular signaling domain comprises CD3 zeta and CD28 or a signaling portion thereof. In some embodiments, the intracellular signaling domain comprises CD3 zeta and CD28. As another example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used. VII. Cytotoxic Receptor Complex Constructs

[0202] Some embodiments of the compositions and methods described herein involve chimeric receptors, such as activating chimeric receptors (ACRs) that target ligands of NKG2D. Expression of these cytotoxic receptor complexes in immune cells (such as genetically modified non-alloreactive T cells and / or NK cells) allows for the targeting and destruction of specific target cells (such as cancer cells). Non-limiting examples of such cytotoxic receptor complexes are discussed in more detail below.

[0203] In several embodiments, polynucleotides encoding a tumor binder / CD8 hinge-CD8TM / OX40 / CD3ζ chimeric receptor complex are provided (see FIG2A , Chimeric Receptor A). The polynucleotide comprises or consists of: an NKG2D ligand binding portion, a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3ζ domain. In several embodiments, the polynucleotide further encodes a 2A cleavage site and an mbIL-15 domain as described herein (see FIG2A , Chimeric Receptor B, representing a polynucleotide structure in which a single polynucleotide encodes both the receptor and mbIL-15). In several embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence derived from a combination of sequences disclosed herein, or the receptor complex comprises an amino acid sequence derived from a combination of sequences disclosed herein. In several embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs as described herein, such as those sequences included herein as examples of components. In several embodiments, the encoding nucleic acid sequence or 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 a combination of one or more SEQ ID NOs as described herein. It should be understood that when sequences are combined (e.g., to create restriction sites), some sequence variability, extensions, and / or truncations of the disclosed sequences may result, for example, due to ease or efficiency of cloning. In several embodiments, the chimeric receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence of one or more of the SEQ ID NOs as provided herein, or a portion thereof (e.g., excluding a portion of the mbIL15 sequence and / or the self-cleaving peptide sequence), or is identical within a range defined by any two of the foregoing percentages.

[0204] In several embodiments, polynucleotides encoding a chimeric NKG2D / CD8α hinge / CD8α transmembrane domain / OX40 / CD3ζ activating receptor complex are provided (see FIG. 2B , NKG2D ACR A). The polynucleotide comprises or consists of a fragment of an NKG2D receptor capable of binding to a ligand of the NKG2D receptor, as described herein, a CD8α hinge, a CD8α transmembrane domain, an OX40 domain, and a CD3ζ domain. In several embodiments, the receptor complex is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 33. In yet another embodiment, the chimeric receptor comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the sequence of the chimeric receptor can be encoded by a nucleic acid sequence different from SEQ ID NO: 33, but retains expression of an amino acid sequence that is 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, depending on the embodiment. In several embodiments, although the chimeric receptor amino acid sequence may differ from SEQ ID NO: 40, the chimeric receptor retains, or in some embodiments, has enhanced, NK cell activation and / or cytotoxicity. Additionally, in several embodiments, the construct can optionally be co-expressed with mbIL15, such as mbIL15 encoded by SEQ ID NO: 35 or 37 ( FIG. 2B , NKG2DACR B, represents a polynucleotide construct in which a single polynucleotide encodes both the receptor and mbIL15). In several embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 36, 38, or 40. In several embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 36. In several embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 38. In several embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the amino acid sequence of mbIL15 may differ from SEQ ID NO: 36, 38, or 40, but, depending on the embodiment, remains 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 mbIL15 may differ from SEQ ID NO: 36, but, depending on the embodiment, remains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 36.In some embodiments, the amino acid sequence of mbIL15 may differ from SEQ ID NO: 38, but, depending on the embodiment, remains 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 mbIL15 may differ from SEQ ID NO: 40, but, depending on the embodiment, remains 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 regarding chimeric receptors for use with the methods and compositions disclosed herein can be found in PCT Patent Publication No. WO 2018 / 183385, filed March 27, 2018, which is incorporated herein by reference in its entirety. VIII. Treatment Methods

[0206] Some embodiments relate to methods for treating, improving, inhibiting or preventing cancer using cells or immune cells comprising chimeric antigen receptors and / or activating chimeric receptors as disclosed herein. Some embodiments relate to methods for treating, improving, inhibiting or preventing cancer using cells or immune cells comprising chimeric receptors provided herein in combination with therapeutic agents. In some embodiments, the methods include treating or preventing cancer. In some embodiments, the methods include administering a therapeutically effective amount of immune cells expressing a tumor-directed chimeric antigen receptor and / or a tumor-directed chimeric receptor as described herein. Examples of cancer types that can be treated in this manner 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 a chemotherapeutic agent, a monoclonal antibody, an 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 an NK cell engager. In some embodiments, the therapeutic agent increases the expression of NKG2D ligands in the subject.

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

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

[0211] In certain embodiments, treatment of a subject with one or more genetically engineered cells and therapeutic agents as described herein achieves one, two, three, four or more of the following effects, including, for example: (i) reducing or ameliorating the severity of a disease or symptom associated therewith; (ii) reducing the duration of symptoms associated with the disease; (iii) preventing the progression of a disease or a symptom associated therewith; (iv) regression of a disease or a symptom associated therewith; (v) preventing the development or onset of symptoms associated with the disease; (vi) preventing the recurrence of symptoms associated with the disease; (vii) reducing the number of hospitalizations for a subject; (viii) reducing the length of hospitalization; (ix) increasing the survival of a subject with the disease; (x) reducing the number of symptoms associated with the disease; and (xi) enhancing, improving, supplementing, complementing or strengthening one or more prophylactic or therapeutic effects of another therapy. Advantageously, the non-alloreactive engineered T cells disclosed herein further enhance one or more of the above.

[0212] Administration of engineered cells can be carried out by a variety of routes, including but not limited to intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to the affected tissue. Administration of therapeutic agents can be carried out by a variety of routes, including but not limited to intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to the affected tissue.

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

[0214] Also disclosed herein is the use of any of the NKG2D ligand binding domains disclosed herein, any of the chimeric receptors disclosed herein, any of the cells and therapeutic agents disclosed herein in the manufacture of a medicament for treating cancer. IX. Selection of Subjects

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

[0216] In some embodiments, the method includes selecting a subject whose level or amount of an NKG2D ligand is above a reference value. For example, in some cases, a subject whose level or amount of an NKG2D ligand is above a reference value is predicted to exhibit a clinical response to treatment. Conversely, a subject whose expression level or amount of an NKG2D ligand is below a reference value is not 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 includes administering a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to an NKG2D ligand.

[0217] In some embodiments, comparison of the level or amount of an NKG2D ligand to a reference value for an 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 and / or associated with treatment. In some embodiments, the level or amount of an NKG2D ligand in a biological sample is compared to a reference value (e.g., an NKG2D ligand reference value). In some embodiments, the reference value is a value for the level, amount, or concentration of an NKG2D ligand. In some embodiments, the reference value is or is derived from the amount or level of an RNA gene product or a protein gene product. In certain embodiments, the reference value is an amount or level of an NKG2D ligand gene or protein product or a converted form thereof that is a boundary or threshold that distinguishes between an amount or level of an NKG2D ligand gene or protein product or a converted form thereof that indicates an increased, elevated, or high likelihood of a clinical response (e.g., CR or PR) and / or a probability of a clinical response (e.g., CR or PR) following administration of the treatment, and an amount or measurement of the NKG2D ligand gene or protein product that indicates an absent or low likelihood and / or a decreased, decreased, or low probability of a clinical response (e.g., CR or PR) following administration of the treatment. In some embodiments, the reference value is a boundary, dividing line, and / or threshold between an amount or level of an NKG2D ligand gene or protein product when a majority of one or more clinical responses occur or have previously occurred and an amount or level of an NKG2D ligand gene or protein product when a minority of one or more clinical responses occur or have previously occurred.

[0218] In certain embodiments, a reference value is an amount or level of an NKG2D ligand gene or protein product, or its converted form, that is a borderline between, or a threshold value that distinguishes between, an amount or level of an NKG2D ligand gene or protein product, or its converted form, associated with a particular type of clinical response and an amount or level associated with one or more other types of clinical responses. In specific embodiments, a reference value is an amount or level of an NKG2D ligand gene or protein product, or its converted form, that is a borderline between, or a threshold value that distinguishes between, an amount or level of an NKG2D ligand gene or protein product, or its converted form, associated with a clinical response (e.g., CR and / or PR) and an amount or level associated with no clinical response (e.g., PD). In specific embodiments, a reference value is an amount or level of an NKG2D ligand gene or protein product, or its converted form, that is a borderline between, or a threshold value that distinguishes between, an amount or level of an NKG2D ligand gene or protein product, or its converted form, associated with CR or PR and an amount or level associated with other clinical responses (e.g., NR / SD or PD).

[0219] In some embodiments, the reference value is a predetermined value. In specific 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 specific embodiments, the clinical study is a completed clinical study. In certain embodiments, the data from the study includes NKG2D ligand expression (e.g., expression of the NKG2D ligand gene or protein product) in samples taken or obtained from subjects in the study. In specific embodiments, the data from the study includes the number and type of clinical responses experienced by subjects during the study. In certain embodiments, subjects in the clinical study had or have had a clinical response, such as a complete remission (CR) or partial remission (PR). In some embodiments, the clinical response was a complete remission (CR). In certain embodiments, the data from the study includes the number and type of diseases or conditions, such as cancer (e.g., AML). In specific embodiments, the data from the study includes the number and type of treatments received by subjects during the study. In certain embodiments, the subjects were or were treated with NK cells genetically engineered to express a chimeric receptor that binds to an NKG2D ligand.

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

[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 NKG2D ligands in a population of subjects suffering from cancer and being administered treatment and not showing a clinical response after administration of the treatment. In some embodiments, the reference value is within 25% of the average level or amount of NKG2D ligands in a population of subjects suffering from cancer and being administered treatment and not showing a clinical response after administration of the treatment. In some embodiments, the reference value is within 20% of the average level or amount of NKG2D ligands in a population of subjects suffering from cancer and being administered treatment and not showing a clinical response after administration of the treatment. In some embodiments, the reference value is within 15% of the average level or amount of NKG2D ligands in a population of subjects suffering from cancer and being administered treatment and not showing a clinical response after administration of the treatment. In some embodiments, the reference value is within 15% of the average level or amount of NKG2D ligands in a population of subjects suffering from cancer and being administered treatment and not showing a clinical response after administration of the treatment. In some embodiments, the reference value is within 10% of the average level or amount of NKG2D ligand in a population of subjects who have cancer and have been administered a treatment and have not demonstrated a clinical response after administration of the treatment. In some embodiments, the reference value is within 5% of the average level or amount of NKG2D ligand in a population of subjects who have cancer and have been administered a treatment and have not demonstrated a clinical response after administration of the treatment. In some embodiments, the reference value is the average level or amount of NKG2D ligand in a population of subjects who have cancer and have been administered a treatment and have not demonstrated a clinical response after administration of the treatment.

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

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

[0224] In some embodiments, the level of the amount of NKG2D ligand in a biological sample from a subject is assessed. In some embodiments, a biological sample is obtained from a subject prior to administering 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, a biological sample is obtained from a subject within about 14 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 14 days before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 10 days before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 7 days before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 6 days before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 5 days before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 4 days before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 3 days before administering genetically engineered NK cells to a subject. In some embodiments, a biological sample is obtained from a subject within about 2 days before administering genetically engineered NK cells to a subject. In some embodiments, the biological sample is obtained from the subject within about 1 day prior to administering 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 ligands include MICA, MIB, and ULBP3. In some embodiments, the NKG2D ligands include MICA, MIB, ULBP1, and ULBP3.

[0227] Methods for determining the amount or level of NKG2D ligand protein products 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, immunoassay detection, nucleic acid-based or protein-based aptamer technology, high-precision liquid chromatography (HPLC), peptide sequencing, and microarray modifications of any of the foregoing (including nucleic acid, antibody, or protein-protein (i.e., non-antibody) arrays). In some embodiments, an immunoassay is or includes a method or assay for detecting a protein based on an immune response, such as by detecting 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, multiplex, and portable ELISAs), Western blotting (including one-, two-, or more-dimensional blotting or other chromatographic approaches, optionally including peptide sequencing), enzyme immunoassay, radioimmunoassay, and surface plasmon resonance. In some embodiments, the level or amount of an NKG2D ligand protein product is determined by the number or percentage of cells in a biological sample that are positive for the NKG2D ligand protein product. In some embodiments, the level or amount of the NKG2D ligand protein product is determined by the intensity of expression of the NKG2D ligand protein product in cells that are positive for the NKG2D ligand protein product in the biological sample. In some embodiments, the level or amount of the NKG2D ligand protein product is determined by the number or percentage of cells that are positive for the NKG2D ligand protein product in the biological sample and the intensity of expression of the NKG2D ligand protein product in cells that are positive for the NKG2D ligand protein product in the biological sample.

[0228] Methods for determining the amount or level of an 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, and real-time and quantitative PCR (qPCR) methods); Northern blotting; Southern blotting, such as Southern blotting of reverse transcription products and derivatives; array-based methods, including blot arrays, microarrays, or in situ synthesis arrays; and sequencing, such as by sequencing by synthesis, pyrosequencing, dideoxy sequencing, or sequencing by ligation, or any other method known in the art. In some embodiments, the level or amount of an NKG2D ligand gene product is determined by the number or percentage of cells in a biological sample that are positive for the NKG2D ligand gene product. In some embodiments, the level or amount of an NKG2D ligand gene product is determined by the intensity of expression of the NKG2D ligand gene product in cells in a biological sample that are positive for the NKG2D ligand gene product. In some embodiments, the level or amount of the NKG2D ligand gene product is determined by the number or percentage of cells in the biological sample that are positive for the NKG2D ligand gene product and the intensity of expression of the NKG2D ligand gene product in the cells in the biological sample that are positive for the NKG2D ligand gene product. peripheral protozoa

[0229] In some embodiments, the method includes selecting for treatment a subject with bone marrow-confined disease (also known as bone marrow-localized disease) and / or with less than or equal to a certain percentage of peripheral blasts (blasts in the peripheral blood). For example, in some cases, a subject with less than or equal to 5% peripheral blasts (e.g., no evidence of extramedullary disease) is predicted to exhibit a clinical response to treatment. Conversely, a subject with 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, treatment includes administering a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for NKG2D.

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

[0231] In some embodiments, before administering the NK cell population to the subject, the method includes determining whether the subject suffers from a bone marrow-restricted disease and / or selecting a subject with a bone marrow-restricted disease for treatment. In some embodiments, before administering the NK cell population to the subject, the method includes determining whether the subject suffers from a bone marrow-restricted disease. In some embodiments, before administering the NK cell population to the subject, the method includes selecting a subject with a bone marrow-restricted disease for treatment. In some embodiments, before administering the NK cell population to the subject, the method includes determining whether the subject suffers from a bone marrow-restricted disease, and if the subject suffers from a bone marrow-restricted disease, the subject is selected for treatment.

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

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

[0234] Also provided herein are methods for treating a subject suffering from 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 (a) the use of a tumor-directed chimeric antigen receptor and / or a tumor-directed chimeric receptor, or a cell expressing a tumor-directed chimeric antigen receptor and / or a tumor-directed chimeric receptor for treating a cancer patient; and (b) the use of a therapeutic agent for treating a cancer patient. Also provided are the uses of such engineered immune cells and therapeutic agents for treating cancer.

[0235] In certain embodiments, treatment of a subject with one or more genetically engineered cells and therapeutic agents as described herein achieves one, two, three, four or more of the following effects, including, for example: (i) reducing or ameliorating the severity of a disease or symptom associated therewith; (ii) reducing the duration of symptoms associated with the disease; (iii) preventing the progression of a disease or a symptom associated therewith; (iv) regression of a disease or a symptom associated therewith; (v) preventing the development or onset of symptoms associated with the disease; (vi) preventing the recurrence of symptoms associated with the disease; (vii) reducing the number of hospitalizations for a subject; (viii) reducing the length of hospitalization; (ix) increasing the survival of a subject with the disease; (x) reducing the number of symptoms associated with the disease; and (xi) enhancing, improving, supplementing, complementing or strengthening one or more preventive or therapeutic effects of another therapy. Each of these comparisons is relative to, for example, a different therapy for a disease, including a cell-based immunotherapy for a disease using cells that do not express a construct disclosed herein. Advantageously, the non-allogeneic reactive engineered T cells disclosed herein further enhance one or more of the above.

[0236] Administration can be carried out by a variety of routes, including but not limited to intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to the affected tissue. Cells (particularly NK cells and / or T cells) engineered to express the chimeric receptor complexes described herein can be formulated for parenteral administration by injection (e.g., by bolus injection or infusion).

[0237] The dosage of immune cells (such as NK and / or T cells) for a given subject can be readily determined based on their weight, disease type and status, and the desired aggressiveness of treatment, but will range from about 10 5 cells / kg to about 10 12 cells / kg (e.g., 10 5 -107 , 10 7 -10 10 , 10 10 -10 12 cells / kg and 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, about 1 x 10 6 cells / kg and approximately 1 x 10 8 cells / kg.

[0238] In several embodiments, 1 × 10 8 NK cells were administered three times (2 × 10 6 / kg). In several embodiments, 3 × 10 8 In some embodiments, 1 × 10 NK cells are administered three times over a 28-day period. 9 NK cells were administered three times.

[0239] In several embodiments, 1.5 × 10 8 NK cells were administered twice (3 × 10 for subjects less than 50 kg) 6 / kg). In several embodiments, 4.5 × 10 8 In some embodiments, 1.5 × 10 NK cells are administered twice over a 28-day cycle. 9 NK cells were administered twice.

[0240] In several embodiments, 1.5 × 10 9 NK cells were administered three times (3 × 10 7 / kg). In several embodiments, 3 × 10 9 In some embodiments, 1.5 × 10 NK cells are administered three times over a 28-day cycle. 10 In some embodiments, at least 4.5 × 10 NK cells are administered during a cycle. 9 NK cells.

[0241] In several embodiments, subjects who exhibit at least partial remission will receive at least one additional dosing cycle. Depending on the embodiment, as long as the subject exhibits an anti-tumor response and tolerates the engineered NK cells, the dosing cycle can continue. In several embodiments, if the subject does not respond (e.g., no tumor response) and / or if the therapy is not tolerated, the subject will not receive additional dosing cycles. However, as discussed herein, in several embodiments, the disclosed dosing regimens have limited or no adverse effects or toxicity. In several embodiments, a decision to accept / administer additional dosing cycles is made at the time of evaluation 30 days after the start of the dosing cycle (whether the first dosing cycle or subsequent cycles). In several embodiments, no more than 5 additional cycles are administered to the subject.

[0242] In some embodiments, a dose of NK cells for a dosing cycle is administered on an outpatient basis. In some embodiments, two doses of NK cells for a dosing cycle are administered on an outpatient basis. In some embodiments, each dose of NK cells for a dosing cycle is administered on an outpatient basis.

[0243] In several embodiments, one or more preparatory treatments are performed before administering engineered NK cells. In several embodiments, lymphocyte clearance therapy (also referred to as "lymphocyte clearance") is performed before administering engineered NK cells. In several embodiments, lymphocyte clearance is performed before each dosing cycle. In several embodiments, a combination of chemotherapeutic agents is used for lymphocyte clearance. In several embodiments, a single chemotherapeutic agent is used for lymphocyte clearance. In several embodiments in which a combination of chemotherapeutic agents is used, agents with different mechanisms of action are optionally used. In several embodiments, agents of different classes are optionally used. In several embodiments, antimetabolites are used. In several embodiments, antimetabolites inhibit and / or prevent cell replication. In several embodiments, antimetabolites are altered nucleotides that destroy DNA replication, making them effective targets for rapidly dividing tumor cells.

[0244] In several embodiments, cytarabine (Ara-C) is used. In several embodiments, about 0.2 to about 10 g / m 2 Doses of Ara-C between, including about 0.2 g / m 2 , about 0.5 g / m 2 , about 1.0 g / m 2 , about 1.5 g / m 2 , about 2.0 g / m 2 , about 2.5 g / m 2 , about 3.0 g / m 2 , about 3.5 g / m2 , about 4.0 g / m 2 , about 5.0 g / m 2 , about 6.0 g / m 2 , about 7.0 g / m 2 , about 8.0 g / m 2 , about 9.0 g / m 2 , about 10.0 about 1.5 g / m 2 In some embodiments, about 2 g / m 2 In some embodiments, the dosage of Ara-C is administered daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, the dosage of Ara-C is administered daily for about 5 days. In some embodiments, the dosage can be divided and administered, for example, twice daily, if desired. In some embodiments, an additional agent is used in combination with Ara-C. In some embodiments, the additional agent is also an antimetabolite. In some embodiments, the additional agent inhibits one or more of DNA polymerase α, ribonucleotide reductase, and / or DNA primase, thereby inhibiting DNA synthesis.

[0245] In some embodiments, the additional agent is fludarabine. In some embodiments, about 5.0 mg / m 2 - Approximately 200 mg / m 2 Fludarabine doses between, including approximately 5.0 mg / m 2 , about 10.0 mg / m 2 , about 15.0 mg / m 2 , about 20.0 mg / m 2 , about 25.0 mg / m 2 , about 30.0 mg / m 2 , about 35.0 mg / m 2 , about 40.0 mg / m 2 , about 45.0 mg / m 2 , about 50.0 mg / m 2 , about 60.0 mg / m 2 , about 70.0 mg / m 2 , about 80.0 mg / m 2 , about 90.0 mg / m 2 , about 100.0 mg / m 2 , about 125.0 mg / m 2 , about 150.0 mg / m 2 , about 175.0 mg / m 2, about 200.0 mg / m 2 In some embodiments, about 30 mg / m 2 In some embodiments, the dosage of fludarabine is administered daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, the dosage of fludarabine is administered daily for about 3 days. In some embodiments, the dosage of fludarabine is administered daily for about 5 days. In some embodiments, if desired, the dosage can be divided and administered, for example, twice a day.

[0246] In several embodiments, a combination of fludarabine and Ara-C is used, wherein the daily dose of fludarabine is about 20 mg / m 2 and 40 mg / m 2 and the daily dose of Ara-C is about 1.5 g / m 2 With 2.5 g / m 2 In several embodiments, a combination of fludarabine and Ara-C is used, wherein the daily dose of fludarabine is about 30 mg / m 2 , and the daily dose of Ara-C is about 2 g / m 2 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 beginning about 7 days prior to the first administration of engineered NK cells (e.g., day -7 to day -3). In several embodiments, lymphodepletion is initiated on day 5 prior to administration of engineered NK cells. In several embodiments, the combination is advantageously used not only as a lymphodepletion regimen, but also as an anti-cancer agent (in addition to the engineered NK cells). In several embodiments, the lymphodepletion regimen acts synergistically with the engineered NK cells to provide effector reduction and / or elimination of cancer cells.

[0247] In some embodiments, the additional agent is cyclophosphamide. In some embodiments, about 100 mg / m 2 - Approximately 100 mg / m 2 Fludarabine doses between, including approximately 100.0 mg / m 2 , about 200 mg / m 2 , about 300 mg / m 2 , about 400 mg / m 2 , about 500 mg / m 2 , about 600 mg / m 2 , about 700 mg / m 2 , about 800 mg / m 2, about 900 mg / m 2 , about 1000 mg / m 2 In some embodiments, about 300 mg / m 2 In several embodiments, about 500 mg / m 2 In some embodiments, the dose of cyclophosphamide is administered daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, the dose of cyclophosphamide is administered daily for about 3 days. In some embodiments, the dose can be divided and administered, for example, twice a day, if desired.

[0248] In several embodiments, a combination of fludarabine and cyclophosphamide is used. In several embodiments, cyclophosphamide (300 mg / m 2 ) and fludarabine (30 mg / m 2 In some embodiments, cyclophosphamide (500 mg / m 2 ) and fludarabine (30 mg / m 2 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 certain dose of genetically engineered cells as described herein or its composition is applied to a subject every day, every other day, every few days, every three days, once a week, twice a week, three times a week or once every two weeks. In other embodiments, two, three or four dosages of one or more genetically engineered cells as described herein or its composition are applied to a subject every day, every few days, every three days, once a week or once every two weeks. In some embodiments, one or more dosages of one or more genetically engineered cells as described herein or its composition are applied for 2 days, 3 days, 5 days, 7 days, 14 days or 21 days. In certain embodiments, a certain dose of one or more genetically engineered cells as described herein or its composition is applied for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or longer.

[0250] In several embodiments, the subject undergoes at least one lymphocyte clearance before administering the genetically engineered cells as disclosed herein. In several embodiments, lymphocyte clearance is performed before administering the engineered cells of one or more additional dosages. In several embodiments, the administration cycle used includes lymphocyte clearance, followed by at least two doses of engineered cells as disclosed herein, the two doses being 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 days or more (including the interval between the time of the marked price interval since the last administration, for example 84 hours or 3.5 days). In several embodiments, the administration cycle itself is about 14, 21, 28, 35, 42 days or more. In several embodiments, three doses are administered, about 1 week apart from each other. In several embodiments, two doses are administered, about 1 week apart from each other. In several embodiments, the subject receives a first dose on cycle day 0, a second dose on cycle day 7, and a third dose on cycle day 14. In several such embodiments, a 28-day cycle is used, and the primary outcome measure is assessed on day 28 (see, e.g., FIG. 3A ). In several embodiments, the subject receives a first dose on cycle day 0, and a second dose on cycle day 7. In several such embodiments, a 28-day cycle is used, and the primary outcome measure is assessed on day 28 (see, e.g., FIG. 3B ).

[0251] In some embodiments, if the subject exhibits a clinical response after the dosing cycle, an additional dosing cycle is administered to the subject as consolidation therapy. Clinical responses can include complete remission (CR; e.g., complete remission), complete remission with incomplete hematologic recovery (CRi; e.g., CR with residual thrombocytopenia), morphological leukemia-free state (MLFS), and partial remission (PR; e.g., partial remission). Methods for assessing clinical response are known in the art (Döhner et al., Blood (2017) 129(4): 424-47).

[0252] For example, in some embodiments, if the subject exhibits a complete remission (CR) after a dosing cycle, the subsequent dosing cycle is administered as a consolidation therapy. In some embodiments, if the subject exhibits a complete remission with incomplete hematological recovery (CRi) after a dosing cycle, the subsequent dosing cycle is administered as a consolidation therapy. In some embodiments, if the subject exhibits a morphologically leukemic-free state (MLFS) after a dosing cycle, the subsequent dosing cycle is administered as a consolidation therapy. In some embodiments, if the subject exhibits a partial remission (PR) after a dosing cycle, the subsequent dosing cycle is administered as a consolidation therapy.

[0253] In some embodiments, if the subject does not show a clinical response from a dosing cycle, an additional dosing cycle is administered to the subject. In some embodiments, if the subject does not show a CR after a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if the subject does not show a CR after a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if the subject does not show MLFS after a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if the subject does not show a PR after a dosing cycle, a subsequent dosing cycle is administered.

[0254] In some embodiments, if the 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 the 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 the 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 the 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 the 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 and five dosing cycles. In some embodiments, the dosing regimen consists of between one and five dosing cycles. In some embodiments, the dosing regimen consists of between one 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, lymphodepletion therapy is administered to the subject prior to each dosing cycle.

[0256] In some embodiments, the overall response rate (ORR) in subjects treated according to the methods 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 the subjects treated according to the methods exhibit a CR or 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 the subjects treated according to the methods 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 the subjects treated according to the methods exhibit 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 the subjects treated according to the methods 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 the subjects treated according to the methods exhibit PR.

[0257] In several embodiments, if subsequent dosing cycles are required (e.g., the subject requires further treatment), lymphocyte clearance is performed before the start of each dosing cycle. For example, in several embodiments, the subject undergoes lymphocyte clearance, receives multiple doses of engineered cells according to the cycle, is evaluated at the end of the cycle time, and, if deemed necessary, undergoes a second lymphocyte clearance followed by a second dosing cycle. In such embodiments where multiple dosing cycles are used, the first and second dosing cycles do not need to be the same (e.g., the first cycle may have 2 doses, while the second cycle 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 cancer that expresses NKG2D ligands. In several embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a leukemia or lymphoma. In several embodiments, the cancer treated is acute myeloid leukemia (AML). In several embodiments, the cancer treated is myelodysplastic syndrome. In several embodiments, the cancer is a solid cancer. Additional embodiments provided herein include treating or preventing the following non-limiting examples of cancer, including but not limited to acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi's 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 gliomas, glioblastomas, craniopharyngiomas, ependymoblastomas, ependymomas, medulloblastomas, medullary epithelioma), breast cancer, bronchial tumors, Burkitt's lymphoma 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 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 uterine 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, the present invention also provides the following nucleic acid and amino acid sequences, which have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges therein) sequence identity and / or homology compared to the corresponding nucleic acid or amino acid sequences of SEQ ID NO. 1-38 (or a combination of two or more of SEQ ID NO: 1-38), and further exhibit one or more of the following functions compared to the corresponding SEQ ID NO. 1-38 (or a combination of two or more of SEQ ID NO: 1-38), including but not limited to: (i) enhanced proliferation; (ii) enhanced activation; (iii) enhanced cytotoxic activity against cells presenting the following ligands, to which NK cells bearing receptors encoded by the nucleic acid and amino acid sequences bind; (iv) enhanced homing to tumors or sites of infection; (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 further stimulate innate and adaptive immune responses; and (viii) combinations of the above. In some embodiments, the present invention also provides the following nucleic acid and amino acid sequences, which have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges therein) sequence identity and / or homology compared to the corresponding nucleic acid or amino acid sequences of SEQ ID NOs: 1-45 (or a combination of two or more of SEQ ID NOs: 1-45), and further exhibit one or more of the functions compared to the corresponding SEQ ID NOs: 1-45 (or a combination of two or more of SEQ ID NOs: 1-45).

[0260] In addition, in several embodiments, amino acid sequences corresponding to any of the nucleic acids disclosed herein are provided, accounting for the degeneracy of the nucleic acid code. Furthermore, sequences (whether nucleic acid or amino acid) that differ from those explicitly disclosed herein but that possess functional similarity or equivalence are contemplated as being within the scope of this disclosure. The foregoing includes mutants, truncations, substitutions, or other types of modifications.

[0261] In several embodiments, the polynucleotide encoding the disclosed cytotoxic receptor complex is mRNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is operably linked to at least one regulatory element for expressing the cytotoxic receptor complex.

[0262] According to several embodiments, a vector is additionally provided, comprising a polynucleotide encoding any of the polynucleotides provided herein, wherein the polynucleotide is optionally operably linked to at least one regulatory element for expressing 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 a polynucleotide, vector, or cytotoxic receptor complex as disclosed herein. Further provided herein are compositions comprising a mixture of engineered immune cells (such as NK cells and / or engineered T cells), each population comprising a polynucleotide, vector, or cytotoxic receptor complex as disclosed herein. XI. Cancer Type and Combination Therapy

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

[0265] Various embodiments provided herein include treating or preventing the following non-limiting examples of cancer. In some embodiments, the cancer is a cancer that expresses NKG2D ligands. In some embodiments, the cancer is a solid tumor. In several embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a leukemia or lymphoma. Examples of cancer include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi's 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 gliomas, craniopharyngiomas, ependymoblastomas, ependymomas, medulloblastomas, medullary epithelioma), breast cancer, bronchial tumors, Burkitt's lymphoma, cervical cancer, colorectal 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 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.

[0266] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat acute myeloid leukemia (AML) (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 relapsed to HCT.

[0267] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat myelodysplastic syndrome (MDS) (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, genetically engineered immune cells are used in combination with therapeutic agents to treat solid tumors (as described more fully below).

[0269] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat breast cancer.

[0270] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat cervical cancer (eg, cervical squamous cell carcinoma and / or endocervical adenocarcinoma).

[0271] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat uterine corpus endometrial cancer.

[0272] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat ovarian serous carcinoma.

[0273] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat bladder urothelial cancer.

[0274] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat colorectal cancer (eg, colorectal carcinoma).

[0275] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat rectal adenocarcinoma.

[0276] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat gastric cancer.

[0277] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat head and neck cancer (eg, head and neck squamous cell carcinoma).

[0278] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat esophageal cancer.

[0279] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat hepatocellular carcinoma (HCC).

[0280] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat lung cancer (eg, squamous cell lung carcinoma and / or adenocarcinoma of the lung).

[0281] In several embodiments, genetically engineered immune cells are used in combination with therapeutic agents to treat melanoma (eg, cutaneous melanoma).

[0282] In certain embodiments, the cancer to be treated is a cancer that exhibits 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 the following: head and neck squamous cell carcinoma (HNSCC), cervical cancer, esophageal cancer, and lung squamous cell carcinoma.

[0283] In some embodiments, the additional anticancer agent is a therapeutic agent that upregulates NKG2D ligands in the subject; or is selected from a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, or any combination thereof; or both. Examples of additional anticancer agents are described in further detail in the following sections. therapeutic agents

[0284] In several embodiments, a therapeutic agent (an additional anti-cancer agent) is also administered to the subject to whom the genetically engineered immune cells are administered. 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, a poly (ADP-ribose) polymerase (PARP) inhibitor, 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 agents include fluorouracil and leucovorin (FOL).In some embodiments, the therapeutic agents include capecitabine and oxaliplatin (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, streptozotocin, 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 streptozotocin. 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, ixabepilone, an 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 ixabepilone. In some embodiments, the mitotic inhibitor comprises an 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 mitomycin.

[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., venetola). In some embodiments, the therapeutic agent comprises venetola. In some embodiments, the therapeutic agent comprises azacitidine. In some embodiments, the therapeutic agent comprises venetola and azacitidine. 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 includes an HDAC inhibitor. In some embodiments, the HDAC inhibitor includes 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 includes TSA. In some embodiments, the HDAC inhibitor includes valproic acid. In some embodiments, the HDAC inhibitor includes sodium butyrate. In some embodiments, the HDAC inhibitor includes VPA. In some embodiments, the HDAC inhibitor includes FR901228. In some embodiments, the HDAC inhibitor includes MS-275. In some embodiments, the HDAC inhibitor includes phenylbutyrate. In some embodiments, the HDAC inhibitor includes PDX101. In some embodiments, the HDAC inhibitor includes 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-PD1 antibody, an anti-PD-L1 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-PD1 antibody. In several embodiments, the monoclonal antibody comprises an anti-PD-L1 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 alters a hormone pathway. In some embodiments, the therapeutic agent comprises tamoxifen. In several embodiments, the therapeutic agent comprises increasing body temperature. In several embodiments, the therapeutic agent is another 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 the ligands of the NKG2D receptor. In some embodiments, the ligands include MICA, MICB, ULBP1, 2, 3, 4, 5, and / or 6.

[0299] In some embodiments, the therapeutic agent comprises decitabine. In some embodiments, prior to administering the first dose of engineered NK cells, the subject is administered a dose of decitabine daily for between about 1 day and about 10 days. In some embodiments, prior to administering the first dose of engineered NK cells, the subject is administered a dose of decitabine daily for five days. In some embodiments, the dose of decitabine is about 10 mg / m 2 and about 30 mg / m 2 In some embodiments, the dose of decitabine is about 20 mg / m 2 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 to 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 the NK cell and an antigen expressed by cells of the cancer. In some embodiments, the activating receptor on the NK cell is selected from CD16, NKp30, NKp46, NKG2D, and any combination thereof. In several embodiments, the NK cell engager binds to CD16. In several embodiments, the NK cell engager binds to NKp30. In several embodiments, the NK cell engager binds to NKp46. In several embodiments, the NK cell engager binds to NKG2D.

[0302] In several embodiments, the therapeutic agent administered is cisplatin. In several embodiments, cisplatin is administered as an IV bolus or as a series of infusions. In several embodiments, the dose range of cisplatin is from about 20 mg / m 2 Up to about 150 mg / m 2 In several embodiments, the dosage range of cisplatin is from about 20 mg / m 2 Up to about 30 mg / m 2 In several embodiments, the dosage range of cisplatin is from about 30 mg / m 2 Up to 40 mg / m 2 In several embodiments, the dose range of cisplatin is from about 40 mg / m 2 Up to 50 mg / m 2 In several embodiments, the dosage range of cisplatin is from about 50 mg / m 2 Up to about 60 mg / m 2 In several embodiments, the dosage range of cisplatin is from about 60 mg / m 2 Up to 70 mg / m 2 In several embodiments, the dosage range of cisplatin is from about 70 mg / m 2 Up to about 80 mg / m 2 In several embodiments, the dosage range of cisplatin is from about 80 mg / m 2 Up to about 90 mg / m 2In several embodiments, the dosage range of cisplatin is from about 90 mg / m 2 to about 100 mg / m 2 In several embodiments, the dose range of cisplatin is from about 100 mg / m 2 to about 110 mg / m 2 In several embodiments, the dose range of cisplatin is from about 110 mg / m 2 to about 120 mg / m 2 In several embodiments, the dose range of cisplatin is from about 120 mg / m 2 to about 130 mg / m 2 In several embodiments, the dose range of cisplatin is from about 130 mg / m 2 to about 140 mg / m 2 In several embodiments, the dose range of cisplatin is from about 140 mg / m 2 Up to about 150 mg / m 2 .

[0303] Depending on the embodiment, cisplatin is administered IV weekly for a total of 3, 4, 5, or 6 doses. In several embodiments, cisplatin is administered every 3 to several 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 constitutes 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 administered therapeutic agent is sorafenib. In several embodiments, sorafenib is administered orally (e.g., in tablet form). In several embodiments, the dosage range of sorafenib is from about 200 mg / day to about 800 mg / day. In several embodiments, the dosage range of sorafenib is from about 200 mg / day to about 250 mg / day. In several embodiments, the dosage range of sorafenib is from about 250 mg / day to about 300 mg / day. In several embodiments, the dosage range of sorafenib is from about 300 mg / day to about 350 mg / day. In several embodiments, the dosage range of sorafenib is from about 350 mg / day to about 400 mg / day. In several embodiments, the dosage range of sorafenib is from about 400 mg / day to about 450 mg / day. In several embodiments, the dosage range of sorafenib is from about 450 mg / day to about 500 mg / day. In several embodiments, the dosage range of sorafenib is from about 500 mg / day to about 550 mg / day. In several embodiments, the dosage range of sorafenib is from about 550 mg / day to about 600 mg / day. In several embodiments, the dosage range of sorafenib is from about 600 mg / day to about 650 mg / day. In several embodiments, the dosage range of sorafenib is from about 650 mg / day to about 700 mg / day. In several embodiments, the dosage range of sorafenib is from about 700 mg / day to about 750 mg / day. In several embodiments, the dosage range of sorafenib is from about 750 mg / day to about 800 mg / day. In several embodiments, the total dose is divided into two daily administrations, for example, 8 to 12 hours apart. For example, for a total daily dose of 400 mg, the subject takes 200 mg (e.g., a tablet) in the morning and a second 200 mg 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 administered therapeutic agent is regorafenib. In several embodiments, regorafenib is administered orally (e.g., in tablet form). In several embodiments, the dosage range of regorafenib is from about 100 mg / day to about 200 mg / day. In several embodiments, the dosage range of regorafenib is from about 100 mg / day to about 110 mg / day. In several embodiments, the dosage range of regorafenib is from about 110 mg / day to about 120 mg / day. In several embodiments, the dosage range of regorafenib is from about 120 mg / day to about 130 mg / day. In several embodiments, the dosage range of regorafenib is from about 130 mg / day to about 140 mg / day. In several embodiments, the dosage range of regorafenib is from about 150 mg / day to about 160 mg / day. In several embodiments, the dosage range of regorafenib is from about 160 mg / day to about 170 mg / day. In several embodiments, the dosage range of regorafenib is from about 170 mg / day to about 180 mg / day. In several embodiments, the dosage range of regorafenib is from about 180 mg / day to about 190 mg / day. In several embodiments, the dosage range of regorafenib is from about 190 mg / day to about 200 mg / day. In several embodiments, the total dose is divided into two daily administrations, for example, 8 to 12 hours apart. For example, for a total daily dose of 160 mg, the subject takes 80 mg (e.g., two 40 mg tablets) in the morning and takes the second 80 mg (e.g., two additional 40 mg tablets) in the evening.

[0308] In several embodiments, regorafenib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, 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, atezolizumab is administered as an IV infusion over a period of 60 minutes. If the subject tolerates 60 minutes, a shorter 30-minute infusion is optionally used. Depending on the embodiment, the dosage of atezolizumab varies with the amount of time that passes between administrations. For example, in several embodiments, two weeks are allowed to pass between administrations. Based on a higher frequency of administration, a lower dose is used in such embodiments. In several embodiments, three weeks are allowed to pass between administrations. Based on an intermediate frequency of administration, an intermediate dose is used in such embodiments. In several embodiments, four weeks are allowed to pass between administrations. Based on a lower frequency of administration, a higher dose is used in such embodiments.

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

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

[0312] In several embodiments employing administration every four weeks, atezolizumab is administered at a dose ranging from about 1500 to about 1800 mg. In several embodiments, atezolizumab is administered at a dose ranging from about 1500 to about 1550 mg. In several embodiments, atezolizumab is administered at a dose ranging from about 1550 to about 1600 mg. In several embodiments, atezolizumab is administered at a dose ranging from about 1600 to about 1640 mg. In several embodiments, atezolizumab is administered at a dose ranging from about 1640 to about 1680 mg. In several embodiments, atezolizumab is administered at a dose ranging from about 1680 to about 1700 mg. In several embodiments, atezolizumab is administered at a dose ranging from about 1700 to about 1750 mg. In several embodiments, atezolizumab is administered at 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, bevacizumab is administered as an IV infusion. Depending on the embodiment, bevacizumab is infused every two weeks or every three weeks. In several embodiments, bevacizumab is administered at a dose ranging from about 2.5 mg / kg to about 20 mg / kg. In several embodiments, bevacizumab is administered at a dose ranging from about 2.5 mg / kg to about 5 mg / kg. In several embodiments, bevacizumab is administered at a dose ranging from about 5 mg / kg to about 7.5 mg / kg. In several embodiments, bevacizumab is administered at a dose ranging from about 7.5 mg / kg to about 10 mg / kg. In several embodiments, bevacizumab is administered at a dose ranging from about 10 mg / kg to about 12.5 mg / kg. In several embodiments, bevacizumab is administered at a dose ranging from about 12.5 mg / kg to about 15 mg / kg. In several embodiments, bevacizumab is administered at a dose ranging from about 15 mg / kg to about 17.5 mg / kg. In several embodiments, bevacizumab is administered at a dose ranging from about 17.5 mg / kg to about 20 mg / kg.

[0315] According to several embodiments, 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) / irinotecan (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 administered therapeutic agent is lenvatinib. In several embodiments, lenvatinib is administered orally (e.g., in tablet form). In several embodiments, the dosage range of lenvatinib is from about 1 mg / day to about 12 mg / day. In several embodiments, the dosage range of lenvatinib is from about 1 mg / day to about 2 mg / day. In several embodiments, the dosage range of lenvatinib is from about 2 mg / day to about 3 mg / day. In several embodiments, the dosage range of lenvatinib is from about 3 mg / day to about 4 mg / day. In several embodiments, the dosage range of lenvatinib is from about 4 mg / day to about 5 mg / day. In several embodiments, the dosage range of lenvatinib is from about 5 mg / day to about 6 mg / day. In several embodiments, the dosage range of lenvatinib is from about 6 mg / day to about 7 mg / day. In several embodiments, the dosage range of lenvatinib is from about 7 mg / day to about 8 mg / day. In several embodiments, the dose range of lenvatinib is from about 8 mg / day to about 9 mg / day. In several embodiments, the dose range of lenvatinib is from about 9 mg / day to about 10 mg / day. In several embodiments, the dose range of lenvatinib is from about 10 mg / day to about 11 mg / day. In several embodiments, the dose range of lenvatinib is from about 11 mg / day to about 12 mg / day. In several embodiments, the total dose is divided into two administrations per day, for example, 8 to 12 hours apart. For example, for a total daily dose of 10 mg, the subject takes 5 mg (e.g., a 5 mg tablet) in the morning and takes a second 5 mg (e.g., an additional 5 mg tablet) in the evening.

[0318] In several embodiments, lenvatinib is optionally administered in combination with pembrolizumab at a dose of between about 150 mg and about 300 mg (3-week cycle) or between about 350 mg and about 500 mg (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, lenvatinib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, lenvatinib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, lenvatinib is administered concurrently with engineered immune cells. In several embodiments, lenvatinib is administered after administration of engineered immune cells.

[0320] In several embodiments, the administered therapeutic agent is nivolumab. In several embodiments, nivolumab is administered as an IV infusion. Depending on the embodiment, nivolumab is infused every two weeks or every four weeks. When using a more frequent dosing regimen, nivolumab is administered in an amount ranging from about 200 to about 300 mg. In several embodiments, nivolumab is administered in an amount ranging from about 200 mg to about 210 mg. In several embodiments, nivolumab is administered in an amount ranging from about 210 mg to about 220 mg. In several embodiments, nivolumab is administered in an amount ranging from about 220 mg to about 230 mg. In several embodiments, nivolumab is administered in an amount ranging from about 230 mg to about 240 mg. In several embodiments, nivolumab is administered in an amount ranging from about 240 mg to about 250 mg. In several embodiments, nivolumab is administered in an amount ranging from about 250 mg to about 260 mg. In several embodiments, nivolumab is administered in an amount ranging from about 260 mg to about 270 mg. In several embodiments, nivolumab is administered in an amount ranging from about 270 mg to about 280 mg. In several embodiments, nivolumab is administered in an amount ranging from about 280 mg to about 290 mg. In several embodiments, nivolumab is administered in an amount ranging from about 290 mg to about 300 mg. When a less frequent dosing regimen is used, nivolumab is administered in an amount ranging from about 400 mg to about 500 mg. In several embodiments, nivolumab is administered in an amount ranging from about 400 mg to about 410 mg. In several embodiments, nivolumab is administered in an amount ranging from about 410 mg to about 420 mg. In several embodiments, nivolumab is administered in an amount ranging from about 420 mg to about 430 mg. In several embodiments, nivolumab is administered in an amount between about 430 mg and about 440 mg. In several embodiments, nivolumab is administered in an amount ranging from about 440 mg to about 450 mg. In several embodiments, nivolumab is administered in an amount ranging from about 450 mg to about 460 mg. In several embodiments, nivolumab is administered in an amount ranging from about 460 mg to about 470 mg. In several embodiments, nivolumab is administered in an amount between about 470 mg and about 480 mg. In several embodiments, nivolumab is administered in an amount ranging from about 480 mg to about 490 mg. In several embodiments, 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, wherein nivolumab is administered in an amount ranging from about 300 mg to about 400 mg. In several embodiments, nivolumab is administered in an amount ranging from about 300 mg to about 310 mg. In several embodiments, nivolumab is administered in an amount ranging from about 310 mg to about 320 mg. In several embodiments, nivolumab is administered in an amount ranging from about 320 mg to about 330 mg. In several embodiments, nivolumab is administered in an amount ranging from about 330 mg to about 340 mg. In several embodiments, nivolumab is administered in an amount ranging from about 340 mg to about 350 mg. In several embodiments, nivolumab is administered in an amount ranging from about 350 mg to about 360 mg. In several embodiments, nivolumab is administered in an amount ranging from about 360 mg to about 370 mg. In several embodiments, nivolumab is administered in an amount ranging from about 370 mg to about 380 mg. In several embodiments, nivolumab is administered in an amount ranging from about 380 mg to about 390 mg. In several embodiments, nivolumab is administered in an amount ranging from about 390 mg to about 400 mg.

[0322] In several embodiments, nivolumab is administered in combination with another agent, such as ipilimumab. In several such embodiments, nivolumab is administered in an amount ranging from about 1 mg / kg to about 5 mg / kg. In several embodiments, nivolumab is administered in an amount ranging from about 1 mg / kg to about 2 mg / kg. In several embodiments, nivolumab is administered in an amount ranging from about 2 mg / kg to about 3 mg / kg. In several embodiments, nivolumab is administered in an amount ranging from about 3 mg / kg to about 4 mg / kg. In several embodiments, nivolumab is administered in an amount ranging from about 4 mg / kg to about 5 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 0.5 mg / kg to about 1.5 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 0.5 mg / kg to about 0.75 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 0.75 mg / kg to about 1.0 mg / kg. In several embodiments, ipilimumab is administered in an amount ranging from about 1.0 mg / kg to about 1.5 mg / kg. In several embodiments, ipilimumab is administered for 2, 3, 4, or 5 doses on the same day as the nivolumab infusion, and then nivolumab is continued alone at the doses 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, pembrolizumab is administered as an IV infusion. Depending on the embodiment, nivolumab is infused every two weeks or every four weeks. When using a more frequent dosing regimen, pembrolizumab is administered in an amount ranging from about 150 to about 250 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 150 mg to about 160 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 160 mg to about 170 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 170 mg to about 180 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 180 mg to about 190 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 190 mg to about 200 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 200 mg to about 210 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 210 mg to about 220 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 220 mg to about 230 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 230 mg to about 240 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 240 mg to about 250 mg. When a less frequent dosing regimen is used, pembrolizumab is administered in an amount ranging from about 350 mg to about 400 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 350 mg to about 360 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 360 mg to about 370 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 370 mg to about 380 mg. In several embodiments, pembrolizumab is administered in an amount between about 380 mg and about 390 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 390 mg to about 400 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 400 mg to about 410 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 410 mg to about 420 mg. In several embodiments, pembrolizumab is administered in an amount between about 420 mg to about 430 mg. In several embodiments, pembrolizumab is administered in an amount ranging from about 430 mg to about 440 mg. In several embodiments, 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 administered therapeutic agent is ipilimumab. In several embodiments, ipilimumab is administered as an IV infusion. In several embodiments, ipilimumab is infused every three weeks. In some embodiments, ipilimumab is administered in ten-week cycles (e.g., 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 / kg to 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 combination with nivolumab, as discussed above.

[0329] In several embodiments, the administered therapeutic agent is ramucirumab. In several embodiments, ramucirumab is administered as an IV infusion. In several embodiments, ramucirumab is infused every two weeks. In some embodiments, ipilimumab is administered in a 21-day cycle (e.g., on day 1). In several embodiments, ramucirumab is administered in 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 combination with an additional agent. For example, in several embodiments, ramucirumab is administered with docetaxel (e.g., at about 50, about 75, or about 100 mg / m2 on day 1 of a 28-day cycle). 2 For example, in several embodiments, ramucirumab is administered in combination with eroltinib (e.g., at a dose of about 100, about 150, or about 150 mg per day). For example, in several embodiments, ramucirumab is administered in combination with paclitaxel (e.g., at a dose of about 60, about 80, or about 100 mg / m2 on days 1, 8, and 15 of a 28-day cycle). 2 doses) in combination.

[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 administered therapeutic agent is cabozantinib. In several embodiments, cabozantinib is administered orally (e.g., in tablet form). In several embodiments, the dose of cabozantinib ranges from about 10 mg / day to about 100 mg / day. In several embodiments, the dose of cabozantinib ranges from about 10 mg / day to about 20 mg / day. In several embodiments, the dose of cabozantinib ranges from about 20 mg / day to about 30 mg / day. In several embodiments, the dose of cabozantinib ranges from about 30 mg / day to about 40 mg / day. In several embodiments, the dose of cabozantinib ranges from about 40 mg / day to about 50 mg / day. In several embodiments, the dose of cabozantinib ranges from about 50 mg / day to about 60 mg / day. In several embodiments, the dose of cabozantinib ranges from about 60 mg / day to about 70 mg / day. In several embodiments, the dose of cabozantinib ranges from about 70 mg / day to about 80 mg / day. In several embodiments, the dosage range of cabozantinib is from about 80 mg / day to about 90 mg / day. In several embodiments, the dosage range of cabozantinib is from about 90 mg / day to about 100 mg / 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 and engineered immune cells are administered concurrently. 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 range of doxorubicin is from about 20 mg / m 2 to about 100 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 20 mg / m 2Up to about 30 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 30 mg / m 2 Up to 40 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 40 mg / m 2 Up to 50 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 50 mg / m 2 Up to about 60 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 60 mg / m 2 Up to about 65 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 65 mg / m 2 Up to 70 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 70 mg / m 2 Up to 75 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 75 mg / m 2 Up to about 80 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 80 mg / m 2 Up to about 90 mg / m 2 In several embodiments, the dosage range of doxorubicin is from about 90 mg / m 2 to about 100 mg / m 2 .

[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 dosage range of gemcitabine is from about 850 mg / m 2 to about 1500 mg / m 2 In several embodiments, the dosage range of gemcitabine is from about 850 mg / m 2 to about 900 mg / m 2In several embodiments, the dosage range of gemcitabine is from about 900 mg / m 2 to about 950 mg / m 2 In several embodiments, the dosage range of gemcitabine is from about 950 mg / m 2 to about 1000 mg / m 2 In several embodiments, the dosage range of gemcitabine is from about 1000 mg / m 2 to about 1050 mg / m 2 In several embodiments, the dose range of gemcitabine is from about 1050 mg / m 2 to about 1100 mg / m 2 In several embodiments, the dose range of gemcitabine is from about 1100 mg / m 2 to about 1150 mg / m 2 In several embodiments, the dose range of gemcitabine is from about 1150 mg / m 2 to about 1200 mg / m 2 In several embodiments, the dose range of gemcitabine is from about 1200 mg / m 2 to about 1250 mg / m 2 In several embodiments, the dosage range of gemcitabine is from about 1250 mg / m 2 to about 1300 mg / m 2 In several embodiments, the dose range of gemcitabine is from about 1300 mg / m 2 to about 1400 mg / m 2 In several embodiments, the dose range of gemcitabine is from about 1400 mg / m 2 to about 1500 mg / m 2 .

[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 and engineered immune cells are administered concurrently. In several embodiments, gemcitabine is administered after administration of engineered immune cells.

[0338] In several embodiments, the administered therapeutic agent is cetuximab. In several embodiments, cetuximab is administered intravenously (e.g., by infusion). In several embodiments, cetuximab is administered in a weekly dosage form. In several embodiments, cetuximab is administered in a biweekly dosage form. For the weekly dosage form, in several embodiments, an initial dose is infused at an increasing concentration (e.g., as a loading dose), followed by each subsequent dose at a lower concentration. In several embodiments, the first dose of cetuximab ranges from about 300 mg / m 2 to about 500 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 300 mg / m 2 to about 325 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 325 mg / m 2 Up to about 350 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 350 mg / m 2 Up to 375 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 375 mg / m 2 to about 400 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 400 mg / m 2 Up to 425 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 425 mg / m 2 Up to about 450 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 450 mg / m 2 to about 475 mg / m 2 In several embodiments, the first dose of cetuximab ranges from about 475 mg / m 2 to about 500 mg / m 2 In several embodiments, subsequent doses of cetuximab range from about 200 mg / m 2 to about 300 mg / m 2 In several embodiments, subsequent doses of cetuximab range from about 200 mg / m 2 Up to 225 mg / m 2 In several embodiments, subsequent doses of cetuximab range from about 225 mg / m 2Up to about 250 mg / m 2 In several embodiments, subsequent doses of cetuximab range from about 250 mg / m 2 Up to 275 mg / m 2 In several embodiments, subsequent doses of cetuximab range from about 275 mg / m 2 to about 300 mg / m 2 For the biweekly regimen, in several embodiments, each dose of cetuximab ranges from about 400 mg / m 2 to about 600 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 400 mg / m 2 Up to 425 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 425 mg / m 2 Up to about 450 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 450 mg / m 2 to about 475 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 475 mg / m 2 to about 500 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 500 mg / m 2 to about 525 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 525 mg / m 2 Up to about 550 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 550 mg / m 2 to about 575 mg / m 2 In several embodiments, each dose of cetuximab ranges from about 575 mg / m 2 to about 600 mg / m 2 .

[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 dosage range of irinotecan is from about 10 mg / m 2 to about 1000 mg / m 2 In several embodiments, the dose range of irinotecan is from about 20 mg / m 2 to about 500 mg / m 2 In several embodiments, the dose range of irinotecan is from about 50 mg / m 2 Up to 125 mg / m 2 In several embodiments, the dose of irinotecan is about 20 mg / m 2 In several embodiments, the dose of irinotecan is about 20 mg / m 2 In several embodiments, the dose of irinotecan is about 30 mg / m 2 In several embodiments, the dose of irinotecan is about 40 mg / m 2 In several embodiments, the dose of irinotecan is about 50 mg / m 2 In several embodiments, the dose of irinotecan is about 60 mg / m 2 In several embodiments, the dose of irinotecan is about 70 mg / m 2 In several embodiments, the dose of irinotecan is about 75 mg / m 2 In several embodiments, the dose of irinotecan is about 80 mg / m 2 In several embodiments, the dose of irinotecan is about 90 mg / m 2 In several embodiments, the dose of irinotecan is about 100 mg / m 2 In several embodiments, the dose of irinotecan is about 120 mg / m 2 In several embodiments, the dose of irinotecan is about 125 mg / m 2 In several embodiments, the dose of irinotecan is about 150 mg / m 2 In several embodiments, the dose of irinotecan is about 180 mg / m 2 In several embodiments, the dose of irinotecan is about 200 mg / m 2 In several embodiments, the dose of irinotecan is about 240 mg / m 2 In several embodiments, the dose of irinotecan is about 250 mg / m 2In several embodiments, the dose of irinotecan is about 300 mg / m 2 In several embodiments, the dose of irinotecan is about 320 mg / m 2 In several embodiments, the dose of irinotecan is about 350 mg / m 2 In several embodiments, the dose of irinotecan is about 360 mg / m 2 In several embodiments, the dose of irinotecan is about 400 mg / m 2 In several embodiments, the dose of irinotecan is about 450 mg / m 2 In several embodiments, the dose of irinotecan is about 480 mg / m 2 In several embodiments, the dose of irinotecan is about 500 mg / m 2 In several embodiments, the dose of irinotecan is about 600 mg / m 2 In several embodiments, the dose of irinotecan is about 700 mg / m 2 In several embodiments, the dose of irinotecan is about 750 mg / m 2 In several embodiments, the dose of irinotecan is about 800 mg / m 2 In several embodiments, the dose of irinotecan is about 900 mg / m 2 In several embodiments, the dose of irinotecan is about 1000 mg / m 2 .

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

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

[0343] In several embodiments, the therapeutic agent administered is capecitabine. In several embodiments, capecitabine is administered orally. In several embodiments, the dosage range of capecitabine is from about 1000 mg / m 2 to about 5000 mg / m 2 In several embodiments, the dose range of capecitabine is from about 1250 mg / m 2 to about 2500 mg / m 2 In several embodiments, the dose of capecitabine is about 1250 mg / m 2 In several embodiments, the dose of capecitabine is about 2500 mg / m 2 In several embodiments, the dose of capecitabine is about 3750 mg / m 2 In several embodiments, the dose of capecitabine is about 5000 mg / m 2 .

[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, in three-week cycles. In some embodiments, the dose is divided into two administrations. For example, in some embodiments, a daily dose of 2500 mg / m 2 1250 mg / m divided into two doses 2 In some embodiments, for 2500 mg / m 2 Total daily dose, taken orally twice a day (e.g., morning and evening), is 1250 mg / m 2 Capecitabine is administered for two weeks, followed by a one-week rest period, in three-week cycles. In some embodiments, the subject is administered for a total of four cycles. In some embodiments, the subject is administered for a total of eight 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 dosage range of vinorelbine is from about 5 mg / m 2 Up to 50 mg / m2 In several embodiments, the dosage range of vinorelbine is from about 10 mg / m 2 Up to 45 mg / m 2 In several embodiments, the dosage range of vinorelbine is from about 15 mg / m 2 Up to 40 mg / m 2 In several embodiments, the dosage range of vinorelbine is from about 20 mg / m 2 Up to about 35 mg / m 2 In several embodiments, the dosage range of vinorelbine is from about 25 mg / m 2 Up to about 30 mg / m 2 In several embodiments, the dose of vinorelbine is about 6.25 mg / m 2 In several embodiments, the dose of vinorelbine is about 7.5 mg / m 2 In several embodiments, the dose of vinorelbine is about 10 mg / m 2 In several embodiments, the dose of vinorelbine is about 12.5 mg / m 2 In several embodiments, the dose of vinorelbine is about 15 mg / m 2 In several embodiments, the dose of vinorelbine is about 17.5 mg / m 2 In several embodiments, the dose of vinorelbine is about 18.75 mg / m 2 In several embodiments, the dose of vinorelbine is about 20 mg / m 2 In several embodiments, the dose of vinorelbine is about 22.5 mg / m 2 In several embodiments, the dose of vinorelbine is about 25 mg / m 2 In several embodiments, the dose of vinorelbine is about 27.5 mg / m 2 In several embodiments, the dose of vinorelbine is about 30 mg / m 2 .

[0347] In some embodiments, a dose of vinorelbine is administered once per 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 per 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 engineered immune 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 will be appreciated that various combinations of additional anticancer agents may also be used. For example, cetuximab and doxorubicin may be used with engineered immune cells as provided herein. XII. Additional Cancer Targets

[0350] Some embodiments of the compositions and methods described herein relate to immune cells comprising chimeric receptors that target cancer antigens, 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 known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); TNF receptor family member B cell maturation antigen (BCMA); CD38; DLL3; G protein-coupled receptor class C 5 member D (GPRC5D); epidermal growth factor receptor (EGFR) CD138; prostate-specific membrane antigen (PSMA); Fms-like tyrosine kinase inhibitor (FMS) inhibitor. 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(ll)Cer); Th1 antigen (Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitopes expressed on acute leukemias or lymphomas but not on hematopoietic progenitor cells, glycosylated CD43 epitopes 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-11Ra); Prostate stem cell antigen (PSCA); serine protease 21 (testesin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha (FRa or FR1); folate receptor beta (FRb); receptor tyrosine kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); mutated elongation factor 2 (ELF2M); ephrin B2;Fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (promegaly factor) subunit beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein composed of the breakpoint cluster region (BCR) and Abelson murine leukemia virus 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(ll)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related protein (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 protein 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); urokinase 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pan-nexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative 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 cancer tumor antigen-1 (PCT Al 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 protein (ML-IAP); ERG (transmembrane serine protease 2 (TMPRSS2) ETS fusion gene);N-acetylglucosamine transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; 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 protein (BORIS or brother of regulator of imprinting sites factor), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosomal protein binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumin; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the 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); mucin-like hormone containing an EGF-like module receptor-like protein 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like protein 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1TROP2, GFRα4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialylated Lewis antigen);Fucosyl-GMl, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, ILl lRa, IL13Ra2, CD179b-IGLll, TCRγ-δ, NKG2D, CD32 (FCGR2A), Tn ag, Timl- / HVCR1, CSF2RA (GM-CSFR-α), TGFβR2, Lews Ag, TCR-βl chain, TCR-β2 chain, TCR-γ chain, TCR-δ chain, FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, guanylate cyclase C (GCC), autoantibodies to desmosomal mucin 3 (Dsg3), autoantibodies to desmosomal mucin 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, claudin 1 8.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Livl, connexin-4, Cripto, gpA33, BST1 / CD157, low-conductance chloride channels, and antigens recognized by TNT antibodies. definition

[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)) can include amino acid residues containing natural and / or non-natural amino acid residues. These terms also include post-expression modifications of the polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, a polypeptide can contain modifications relative to the native or natural sequence, as long as the protein retains the desired activity. These modifications may be intentional (e.g., through site-directed mutagenesis) or may be accidental (e.g., through mutations in the host producing the protein or errors due to PCR amplification).

[0352] As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject (e.g., patient) to whom one or more 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 ape. The subject can be male or female and can be of any suitable age, including infants, young children, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0353] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to the complete or partial improvement or alleviation of a disease, condition, or disorder, or a symptom, adverse effect, outcome, or phenotype associated therewith. Desirable therapeutic effects include, but are not limited to, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, alleviation or slowing of the disease state, and remission or improvement of prognosis. The terms do not imply complete cure of the disease or complete elimination of any symptom or effect on all symptoms or outcomes.

[0354] As used herein, "prevention" (and grammatical variations thereof, such as "prevent" or "prevention") includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.

[0355] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation or cell) is an amount effective to achieve a desired therapeutic outcome (e.g., for treating a disease, condition, or disorder) and / or therapeutic pharmacokinetic or pharmacodynamic effect at the necessary dosage and for the necessary period of time. The therapeutically effective amount can vary depending on factors such as the disease state, the age, sex, and weight of the subject, and the cell population being administered. In some embodiments, provided methods comprise administering cells and / or compositions in an effective amount (e.g., a therapeutically effective amount).

[0356] As used herein, the term "about" refers to the typical error range of the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments involving the value or parameter itself.

[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 range format. It should be understood that the description in range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Therefore, it should be considered that the description of a range specifically discloses all possible sub-ranges and each numerical value within the range. Language such as "up to," "at least," "greater than," "less than," "between," etc. includes the enumerated numbers. Terms such as "about" or "approximately" preceding a number include the enumerated numbers. For example, where a range of values is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other stated values or intermediate values within the stated range are encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range and are also encompassed within the claimed subject matter, subject to any explicitly excluded limits within the stated range. When a stated range includes one or two limitations, ranges excluding either or both of those included limitations are also encompassed within the claimed subject matter. This applies regardless of the width of the range.

[0359] In addition, when a sequence is disclosed as "comprising" a nucleotide or amino acid sequence, such reference should also include sequences "comprising," "consisting of," or "consisting essentially of" the recited sequence, unless otherwise indicated.

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

[0361] Unless otherwise defined, all art terms, symbols, and other technical and scientific terms or nomenclature used herein are intended to have the same meaning as commonly understood meanings applicable to the claimed subject matter. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from commonly understood meanings.

[0362] All publications (including patent documents, scientific articles, and databases) mentioned 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. To the extent that definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over those 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] The embodiments provided herein include: 1. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering 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 approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, Wherein, before administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphocyte depletion therapy. 2. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, Wherein, before administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphocyte depletion therapy. 3. The method of embodiment 1 or embodiment 2, wherein the therapeutic agent increases the expression of NKG2D ligands in the subject. 4. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering 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 approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject. 5. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject. 6. A method of treating cancer in a subject, the method comprising: A population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (NKG2D) is administered to a subject having cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 genetically 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, an NK cell engager, a therapeutic agent that increases the expression of an 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 primitive cells in the subject is determined, and the subject is selected for treatment if the subject has less than or equal to 5% peripheral primitive cells. 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) selecting the subject for treatment if the subject has less than or equal to 5% peripheral blasts. 12. The method of any one of embodiments 1-11, wherein the subject has no evidence of extramedullary disease. 13. The method of any one of embodiments 4-11, wherein the therapeutic agent is selected from a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof. 14. The method of any one of embodiments 4-13, wherein lymphodepletion therapy is administered to the subject prior to administering the first dose of the genetically engineered NK cells to the subject. 15. The method of any one of embodiments 1-14, wherein each of the first dose, the second dose, and the third dose of the genetically engineered NK cells comprises approximately 1 x 10 9 Genetically engineered NK cells or approximately 1.5 x 10 9 Genetically engineered NK cells. 16. The method of any one of embodiments 1-15, wherein the dosing period is between about 14 days and about 35 days. 17. The method of any one of embodiments 1-16, wherein the dosing period is about 21 days. 18. The method of any one of embodiments 1-16, wherein the dosing period is about 28 days. 19. The method of any one of embodiments 1-18, wherein the method comprises administering additional dosing cycles. 20. The method of any one of embodiments 1-19, wherein if the subject exhibits a clinical response, optionally a complete remission (CR), after 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 after 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 administering between one and five dosing cycles. 23. The method of any one of embodiments 19 to 22, wherein the subject is administered lymphodepletion 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 approximately 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 approximately 7 days after administration of the second dose of the genetically engineered cells. 26. A method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells about 7 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells about 7 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1.5 x 10 9 genetically engineered NK cells; and wherein the subject has less than or equal to 5% peripheral blasts and no evidence of extramedullary disease. 27. A method according to any one of embodiments 1-25, wherein the therapeutic agent is administered prior to administering the first dose of the genetically engineered NK cells. 28. A method according to any one of embodiments 1-26, wherein the administration of the therapeutic agent is performed in parallel with the administration of a certain dose of the genetically engineered NK cells, optionally in parallel with the administration of the first dose of the genetically engineered NK cells. 29. A method according to any one of embodiments 1-26, wherein the administration of the therapeutic agent is subsequent to the administration of a dose of the genetically engineered NK cells, optionally subsequent to the 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 administering fludarabine (Flu). 31. The method of any one of embodiments 1-3 and 12-30, wherein the lymphodepletion therapy comprises administering three doses of Flu. 32. The method of embodiment 31, wherein each Flu dose comprises about 10 mg / m 2 and about 60 mg / m 2 between. 33. The method of any one of embodiments 1-3 and 12-32, wherein the lymphodepleting therapy comprises administering Flu and cyclophosphamide (Cy). 34. The method of any one of embodiments 1-3 and 12-33, wherein the lymphodepletion therapy comprises administering three doses of Cy. 35. The method of embodiment 34, wherein a first dose of Flu and Cy are each administered 5 days before the start of the dosing cycle; a second dose of Flu and Cy are each administered 4 days before the start of the dosing cycle; and a third dose of Flu and Cy are each administered 3 days before the start of the dosing cycle. 36. The method of embodiment 34 or embodiment 35, wherein each Cy dose comprises about 200 mg / m 2 and about 600 mg / m 2 between. 37. The method of any one of embodiments 34-36, wherein each Flu dose comprises about 30 mg / m 2 Each Cy dose contains approximately 300 mg / m 2 . 38. The method of any one of embodiments 34-36, wherein each Flu dose comprises about 30 mg / m 2 Each Cy dose contains approximately 500 mg / m 2 . 39. The method of any one of embodiments 1-3 and 12-32, wherein the lymphodepletion therapy comprises administering five doses of Flu. 40. The method of any one of embodiments 1-3, 12-32, and 39, wherein the lymphodepleting therapy comprises administering Flu and cytarabine (Ara-C). 41. The method of any one of embodiments 1-3, 12-32, 39, and 40, wherein the lymphodepleting therapy comprises administering five doses of Ara-C. 42. The method of embodiment 41, wherein a first dose of Flu and Ara-C are each administered 7 days before the start of the dosing cycle; a second dose of Flu and Ara-C are each administered 6 days before the start of the dosing cycle; a third dose of Flu and Ara-C are each administered 5 days before the start of the dosing cycle; a fourth dose of Flu and Ara-C are each administered 3 days before the start of the dosing cycle; and a fifth dose of Flu and Ara-C are each administered 4 days before the start of the dosing cycle. 43. The method of embodiment 41 or embodiment 42, wherein each Ara-C dose comprises about 1 g / m 2 With about 4 g / m 2 between. 44. The method of any one of embodiments 41-43, wherein each Flu dose comprises about 30 mg / m 2 Each Ara-C dose contains approximately 2 g / m 2 . 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 the subject is administered a dose of decitabine daily for five days prior to initiating the dosing cycle. 48. The method of embodiment 47, wherein the dose of decitabine comprises about 20 mg / m 2 . 49. A method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells about 7 days after admini...

Claims

1. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering 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 approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject, Wherein, before administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphocyte depletion therapy.

2. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an 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 according to claim 1, wherein Prior to administering the genetically engineered NK cells to the subject, the percentage of peripheral blasts in the subject is determined, and if the subject has less than or equal to 5% peripheral blasts, the subject is selected for treatment.

7. The method according to 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) selecting the subject for treatment if the subject has less than or equal to 5% peripheral blasts.

8. The method of claim 1, wherein the subject has no evidence of extramedullary disease.

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

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

12. The method of claim 1, wherein the lymphodepleting therapy comprises administering fludarabine (Flu).

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

14. The method of claim 13, wherein each Flu dose comprises about 10 mg / m 2 and about 60 mg / m 2 between.

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

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

17. The method of claim 16, wherein a first dose of Flu and Cy are each administered 5 days before the start of the dosing cycle; a second dose of Flu and Cy are each administered 4 days before the start of the dosing cycle; and a third dose of Flu and Cy are each administered 3 days before the start of the dosing cycle.

18. The method of claim 16 or claim 17, wherein each Cy dose comprises about 200 mg / m 2 and about 600 mg / m 2 between.

19. The method of claim 16 or claim 17, wherein each Flu dose comprises about 30 mg / m 2 Each Cy dose contains approximately 300 mg / m 2 .

20. The method of claim 16 or 17, wherein each Flu dose comprises about 30 mg / m 2 Each Cy dose contains approximately 500 mg / m 2 .

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

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

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

24. The method of claim 23, wherein a first dose of Flu and Ara-C are each administered 7 days before the start of the dosing cycle; a second dose of Flu and Ara-C are each administered 6 days before the start of the dosing cycle; a third dose of Flu and Ara-C are each administered 5 days before the start of the dosing cycle; a fourth dose of Flu and Ara-C are each administered 3 days before the start of the dosing cycle; and a fifth dose of Flu and Ara-C are each administered 4 days before the start of the dosing cycle.

25. The method of claim 23 or claim 24, wherein each Ara-C dose comprises about 1 g / m 2 About 4g / m 2 between.

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

27. A method of selecting a subject having cancer for treatment with a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (NKG2D), the method comprising: (a) assessing the level or amount of an NKG2D ligand in a biological sample from a subject having cancer, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or polynucleotide encoded by an NKG2D ligand gene; (b) if the level or amount of the NKG2D ligand is above the reference value, selecting the subject for treatment with a population of NK cells genetically engineered to express a chimeric receptor that binds to the ligand of NKG2D; and (c) administering the genetically engineered NK cells to the subject, wherein the biological sample is obtained from the subject prior to administering 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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 Genetically 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, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day prior to administering 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 about 0% 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 ligands include MICA and MICB.

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

34. The method of any one of claims 1-26 or 28-33, wherein each of the first, second, and third doses of the genetically engineered NK cells comprises approximately 1 x 10 9 Genetically engineered NK cells or approximately 1.5 x 10 9 Genetically engineered NK cells.

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

37. The method of any one of claims 1-26 or claims 28-34, wherein the dosing period 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 to 26 or claims 28 to 38, wherein If the subject exhibits a clinical response after the dosing cycle, optionally a partial remission (PR), complete remission with incomplete hematologic recovery (CRi), or complete remission (CR), the method comprises administering an additional dosing cycle as consolidation therapy.

40. The method of any one of claims 1-26 or claims 28-38, wherein If the subject exhibits a clinical response after the dosing cycle and subsequently exhibits disease progression, the method includes 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 administering between one and five dosing cycles.

42. The method of claim 41, wherein the subject is administered 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 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, a poly (ADP-ribose) polymerase (PARP) inhibitor, 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-PD1 antibody, an anti-PD-L1 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 a NK cell engager, and wherein the NK cell engager binds to an activating receptor on a NK cell and an antigen expressed by cells of the cancer, optionally wherein the activating receptor is selected from 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 carcinoma, sarcoma, or melanoma.

50. The method of any one of claims 1-44 or 46-48, wherein the cancer is selected from 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-PD1 antibody, or an anti-PD-L1 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 over 18 years of age.

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 shown 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 costimulatory domain and CD3ζ.

69. The method of claim 68, wherein the costimulatory domain comprises an OX40 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 shown in SEQ ID NO:

39.

71. The method of any one of claims 1-70, wherein the genetically engineered NK cells express 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 shown in SEQ ID NO:

40.

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

74. The method of any one of claims 1-73, wherein the engineered NK cell population is 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 to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein: (a) administering the genetically engineered NK cells to a subject having cancer in an administration cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering 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 approximately 1 x 10 8 and about 1 x 10 10 between 100 and 200 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, Wherein, before administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphocyte depletion therapy.

77. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein: (a) administering the genetically engineered NK cells to a subject having cancer in an administration cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, Wherein, before administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphocyte depletion therapy.

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

79. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein: (a) administering the genetically engineered NK cells to a subject having cancer in an administration cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering 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 approximately 1 x 10 8 and about 1 x 10 10 between 100 and 200 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

80. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein: (a) administering the genetically engineered NK cells to a subject having cancer in an administration cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

81. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating cancer in a subject, wherein the genetically engineered NK cells are administered to a subject having cancer in an administration cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells, and wherein the subject has less than or equal to 5% peripheral blasts.

82. The use according to claim 81, wherein the genetically engineered NK cell population is for use with a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, a therapeutic agent that increases the expression of NKG2D ligands 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 has no evidence of extramedullary disease.

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

87. The use according to any one of claims 79 to 86, wherein Prior to administering the first dose of the genetically engineered NK cells to the subject, lymphodepletion therapy is administered to the subject.

88. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating 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) administering to the subject a second dose of the genetically engineered NK cells about 7 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells about 7 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1.5 x 10 9 genetically engineered NK cells; and wherein the subject has less than or equal to 5% peripheral blasts and no evidence of extramedullary disease.

89. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer cell group 2 D (NKG2D) for treating 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) administering to the subject a second dose of the genetically engineered NK cells about 7 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells about 7 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1.5 x 10 9 genetically engineered NK cells; and Before administering the first dose of the genetically engineered NK cells, the subject has been administered approximately 20 mg / m 2 The dose of decitabine was continued for five days.

90. The use of claim 89, wherein the subject has less than or equal to 5% peripheral blasts, optionally wherein the subject has no evidence of extramedullary disease.

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

92. The use according to any one of claims 76 to 91, wherein If the subject exhibits a clinical response after the dosing cycle, optionally a partial remission (PR), complete remission with incomplete hematologic recovery (CRi), or complete remission (CR), the subject is administered an additional dosing cycle as consolidation therapy.

93. The use according to any one of claims 76 to 91, wherein If the subject exhibits a clinical response after 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 and five dosing cycles.

95. The use of claim 94, wherein the subject is administered lymphodepletion therapy prior to each cycle.

96. The use of 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 cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering 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 approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, Wherein, before administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphocyte depletion therapy.

99. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof, Wherein, before administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphocyte depletion therapy.

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

101. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that are genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering 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 approximately 1 x 10 8 and about 1 x 10 10 genetically engineered NK cells; and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

102. A method of treating cancer in a subject, the method comprising: A population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (NKG2D) is administered to a subject having cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of the genetically engineered NK cells; (ii) administering to the subject a second dose of the genetically engineered NK cells between about 5 days and about 10 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells between about 5 days and about 10 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1 x 10 8 and about 1 x 10 10 The invention relates to a method for producing genetically engineered NK cells of at least 100,000 cells / day, and wherein the subject has less than or equal to 5% of peripheral blasts.

103. A method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells about 7 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells about 7 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1.5 x 10 9 genetically engineered NK cells; and wherein the subject has less than or equal to 5% peripheral blasts and no evidence of extramedullary disease.

104. A method of treating cancer in a subject, the method comprising administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer cell group 2 D (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) administering to the subject a second dose of the genetically engineered NK cells about 7 days after administering the first dose of the genetically engineered cells; and (iii) administering to the subject a third dose of genetically engineered NK cells about 7 days after administering the second dose of the genetically engineered NK cells, Each dose of the genetically engineered NK cells contains approximately 1.5 x 10 9 Genetically engineered NK cells; wherein the subject has less than or equal to 5% peripheral blasts; and Before administering the first dose of the genetically engineered NK cells, the subject has been administered approximately 20 mg / m 2 The dose of decitabine was continued for five days.

Images