Combination Therapy Dosing Regimen

Abstract

Some embodiments of the methods and compositions disclosed herein relate to immune cells engineered to express a cytotoxic chimeric receptor and various dosing regimens for administering such cells. In some embodiments, the immune cells express a chimeric receptor that targets a ligand for NKG2D on tumor cells. In some embodiments, the cancer is a blood cancer, such as acute myeloid leukemia (e.g., relapsed / refractory acute myeloid leukemia) or myelodysplastic syndrome. In some embodiments, the tumor is a solid tumor, such as breast cancer, cervical cancer, colon cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, lung cancer, melanoma, intrahepatic cholangiocarcinoma, or other liver tumor, e.g., secondary metastasis from colon cancer. In some embodiments, the immune cells are administered in combination with a therapeutic agent, such as an additional anticancer agent.

Description

[Technical Field]

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

[0002] Field Some embodiments disclosed herein relate to methods and compositions comprising genetically engineered cells for cancer immunotherapy. In some embodiments, the disclosure relates to cells engineered to express a cytotoxic receptor complex and the administration of such cells according to a specific dosing regimen for successful cancer immunotherapy. [Background technology]

[0003] As knowledge about different cancers and the characteristics of cancer cells that can be used to specifically distinguish them from healthy cells increases, therapies that exploit the distinct characteristics of cancer cells are being developed. Immunotherapy, which employs engineered immune cells, is one approach to treating cancer.

[0004] Incorporation by reference of items contained in the sequence listing file This application incorporates by reference the items set forth in the sequence listing contained in the following XML file submitted concurrently with the application: File name: NKT096WO_ST26.xml, Creation date: November 15, 2023, Size: 46,520 bytes. Summary of the Invention [Means for solving the problem]

[0005] Immunotherapy represents a new technological advance in disease treatment, in which immune cells are engineered to express specific target and / or effector molecules that specifically identify and react to diseased or damaged cells. This represents a promising advance, at least in part, due to the possibility of specifically targeting diseased or damaged cells, as opposed to more traditional approaches such as chemotherapy, in which all cells are affected and the desired outcome is for a sufficient number of healthy cells to survive for the patient to survive. One immunotherapy approach is the recombinant expression of chimeric receptors in immune cells, which achieves targeted recognition and destruction of the desired abnormal cells.

[0006] Provided herein, in some embodiments, are methods for treating cancer in a subject, the methods 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 group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. In some embodiments, each dose of genetically engineered NK cells comprises about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 In some embodiments, the method comprises administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

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

[0008] Provided herein, in some embodiments, are methods for treating cancer in a subject, the methods 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 group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. In some embodiments, each dose of genetically engineered NK cells comprises about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 In some embodiments, the method comprises administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

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

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

[0011] In some embodiments, prior to administering the genetically engineered NK cells to the subject, an additional evaluation of the subject is performed, which includes (a) determining the percentage of peripheral blasts in the subject, and (b) if the subject has 5% or less peripheral blasts, the subject is selected for treatment.

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

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

[0014] In some embodiments, lymphodepleting therapy comprises administration of fludarabine (Flu). In some embodiments, lymphodepleting therapy comprises administration of three doses of Flu. In some embodiments, each dose of Flu is about 10 mg / m 2 ~about 60mg / m 2 In some embodiments, the lymphodepletion therapy comprises administration of Flu and cyclophosphamide (Cy). In some embodiments, the lymphodepletion therapy comprises administration of three doses of Cy. In some embodiments, a first dose of Flu and Cy is each given 5 days prior to the start of an administration cycle, a second dose of Flu and Cy is each given 4 days prior to the start of an administration cycle, and a third dose of Flu and Cy is each given 3 days prior to the start of an administration cycle. In some embodiments, each dose of Cy is about 200 mg / m 2 ~about 600mg / m 2 In some embodiments, each dose of Flu is about 30 mg / m 2each dose of Cy is about 300 mg / m 2 In a further embodiment, each dose of Flu comprises about 30 mg / m 2 each dose of Cy is about 500 mg / m 2 Includes.

[0015] In some embodiments, the lymphodepleting therapy comprises the administration of five doses of Flu. In some embodiments, the lymphodepleting therapy comprises the administration of Flu and cytosine arabinoside (Ara-C). In some embodiments, the lymphodepleting therapy comprises the administration of five doses of Ara-C. In some embodiments, a first dose of Flu and Ara-C is each given 7 days prior to the start of a dosing cycle, a second dose of Flu and Ara-C is each given 6 days prior to the start of a dosing cycle, a third dose of Flu and Ara-C is each given 5 days prior to the start of a dosing cycle, a fourth dose of Flu and Ara-C is each given 3 days prior to the start of a dosing cycle, and a fifth dose of Flu and Ara-C is each given 4 days prior to the start of a dosing cycle. In some embodiments, each dose of Ara-C is about 1 g / m 2 ~approx. 4g / m 2 In some embodiments, each dose of Flu is about 30 mg / m 2 and each dose of Ara-C is about 2 g / m 2 Includes.

[0016] In some embodiments, a method is provided for selecting a subject with 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 group 2D (NKG2D), the method comprising: (a) assessing the level or amount of an NKG2D ligand in a biological sample from the subject with cancer, where 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) 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 if the level or amount of the NKG2D ligand exceeds a reference value; and (c) administering the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject prior to administration of the genetically engineered NK cells.

[0017] In some embodiments, the genetically engineered NK cells are administered to the subject in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. In some embodiments, each dose of genetically engineered NK cells comprises about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 Contains genetically engineered NK cells.

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

[0019] 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%.

[0020] 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 and / or MICB. In some embodiments, the NKG2D ligand comprises ULBP1 and / or ULBP3.

[0021] In some embodiments, the first, second, and third doses of genetically engineered NK cells each comprise about 1 x 10 9 genetically engineered NK cells or approximately 1.5 x 10 9 Contains genetically engineered NK cells.

[0022] In some embodiments, the administration cycle is about 14 days to about 35 days. In some embodiments, the administration cycle is about 21 days. In some embodiments, the administration cycle is about 28 days. In some embodiments, the method includes administering an additional administration cycle.

[0023] In some embodiments, if the subject exhibits a clinical response after a dosing cycle, as appropriate, a partial response (PR), a complete response with incomplete hematologic recovery (CRi), or a complete response (CR), the method includes administering an additional dosing cycle as consolidation treatment.

[0024] In some embodiments, if the subject exhibits a clinical response after a dosing cycle and then exhibits disease progression, the method comprises administering an additional dosing cycle as re-treatment.

[0025] In some embodiments, the method comprises administering 1 to 5 administration cycles, hi some embodiments, the subject is administered lymphodepleting therapy prior to each cycle.

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

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

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

[0029] In some embodiments, the therapeutic agent comprises a monoclonal antibody, 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.

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

[0031] In some embodiments, the cancer being treated is carcinoma, sarcoma, or melanoma. In some embodiments, the cancer being treated is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon 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 some embodiments, the cancer being treated comprises liver cancer or colon cancer, hi some embodiments, the therapeutic agent comprises capecitabine, cisplatin, doxorubicin, regorafenib, or sorafenib.

[0033] 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.

[0034] 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.

[0035] 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.

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

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

[0038] 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 90%, 91%, 92%, 93%, 94%, 95%, or more sequence identity to 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 CD3 zeta. In some embodiments, the costimulatory domain comprises an OX40 domain. In some 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 some embodiments, the engineered NK cells express membrane-bound interleukin-15 (mbIL15). In some embodiments, mbIL15 has at least 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:40.

[0039] In some embodiments, the population of engineered NK cells is allogeneic to the subject, hi some embodiments, the population of engineered NK cells is derived from a subject not afflicted with cancer.

[0040] In some embodiments, at least one dose of the genetically engineered NK cells is administered to the subject on an outpatient basis, and optionally, each dose of the engineered NK cells is administered to the subject on an outpatient basis.

[0041] Also provided herein is the use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D) to treat cancer in a subject, wherein the genetically engineered NK cells are administered to the subject with cancer in a dosing cycle including: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. In some embodiments, the cancer is a solid tumor. In some embodiments, each dose of genetically engineered NK cells is administered to a subject with cancer in a dose cycle including: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells; ... 8 genetically engineered NK cells ~ approximately 1 x 10 10 The subject comprises a first dose of genetically engineered NK cells. 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 a lymphodepletion therapy prior to administering the first dose of 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 a ligand of natural killer cluster 2D (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 including: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10(b) 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, and prior to administering the first dose of the genetically engineered NK cells to the subject, the subject is subjected to lymphodepletion therapy.

[0043] In some embodiments, the Therapeutic Agent increases expression of an NKG2D ligand in a subject.

[0044] Provided herein is a use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer group 2D (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 including: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein the cancer is a solid tumor, and each dose of genetically engineered NK cells is administered to the subject about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 (b) the subject is administered a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0045] Also provided herein is the use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer group 2D (NKG2D) to treat cancer in a subject, wherein the genetically engineered NK cells are administered to the subject with cancer in an administration cycle including: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. In some embodiments, each dose of genetically engineered NK cells is administered to the subject in an amount of about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 In some embodiments, the subject is administered a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0046] Also provided herein is the use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cluster 2D (NKG2D) for treating cancer in a subject, wherein the genetically engineered NK cells are administered to the subject having cancer in an administration cycle including: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 the subject has 5% or less peripheral blasts.

[0047] In some embodiments, the genetically engineered population of NK cells 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 a subject, and any combination thereof.

[0048] In some embodiments, the subject has 5% or less peripheral blasts. In some embodiments, the subject has less than 5% peripheral blasts. In some embodiments, the subject has no evidence of extramedullary disease.

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

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

[0051] Also provided herein is the use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand for natural killer cluster 2D (NKG2D) for treating cancer in a subject, wherein the genetically engineered NK cells are administered in an administration cycle including: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1.5 x 10 9 the subject has 5% or less peripheral blasts, and / or the subject has no evidence of extramedullary disease.

[0052] Also provided is the use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand for natural killer group 2D (NKG2D) to treat cancer in a subject, wherein the genetically engineered NK cells are administered in an administration cycle including: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1.5×10 9 the subject receives a dose of about 20 mg / m of genetically engineered NK cells prior to receiving the first dose of genetically engineered NK cells. 2 daily for 5 days. In some embodiments, the subject has 5% or less peripheral blasts, and optionally the subject has no evidence of extramedullary disease.

[0053] In some embodiments of the provided use, the subject is administered an additional administration cycle. In some embodiments of the provided use, if the subject shows a clinical response after an administration cycle, as appropriate, a partial response (PR), a complete response with incomplete hematological recovery (CRi), or a complete response (CR), an additional administration cycle is administered to the subject as consolidation treatment. In some embodiments of the provided use, if the subject shows a clinical response after an administration cycle and then shows disease progression, the subject is administered an additional administration cycle as retreatment.

[0054] In some embodiments of the provided uses, the subject receives 1 to 5 administration cycles. In some embodiments of the provided uses, the subject receives lymphodepletion therapy before each cycle. In some embodiments, the cancer is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), and optionally, the cancer is relapsed / refractory (r / r) AML or very high-risk MDS. In some embodiments, a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis, and optionally, each dose of the genetically engineered NK cells is administered to the subject on an outpatient basis.

[0055] Also provided herein are methods for treating cancer in a subject, the methods comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle including: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein the cancer is a solid tumor, and each dose of genetically engineered NK cells is administered in an amount of about 1×10 8 genetically engineered NK cells ~ approximately 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 prior to administering the first dose of the engineered NK cells to the subject, the subject is administered lymphodepletion therapy.

[0056] Also provided in some embodiments are methods for 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 a ligand of natural killer cluster 2D (NKG2D), wherein the genetically engineered NK cells are administered in an administration cycle including: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1 x 10 8 genetically engineered NK cells ~ approximately 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 prior to administering the first dose of the genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy. In some embodiments, the therapeutic agent increases expression of an NKG2D ligand in the subject.

[0057] In some embodiments, a method for 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 a ligand of natural killer cluster 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein the cancer is a solid tumor, and each dose of genetically engineered NK cells is administered to the subject about 1 x 10 8genetically engineered NK cells ~ approximately 1 x 10 10 (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0058] Further provided herein is a method for 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 a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 the subject has 5% or less peripheral blasts.

[0059] Further provided herein is a method for 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 a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1.5×10 9 The patient will have 5% or less peripheral blasts and no evidence of extramedullary disease.

[0060] Also provided herein is a method for 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 a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1.5×10 9 the subject has 5% or less peripheral blasts, and the subject receives a dose of about 20 mg / m prior to receiving the first dose of the genetically engineered NK cells. 2 The patient is being given decitabine daily for five days.

[0061] Further provided herein are methods for 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 a ligand of natural killer group 2D (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 a first dose of the genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy.

[0062] Also provided herein is a method for 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 group 2D (NKG2D), wherein the genetically engineered NK cells are administered in administration cycles, and 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 a first dose of the genetically engineered NK cells to the subject, the subject is subjected to lymphodepletion therapy.

[0063] Also provided herein are methods for treating cancer in a subject, the methods 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 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 group 2D (NKG2D), and the engineered NK cells are administered in dosing cycles; and prior to administering a first dose of the engineered NK cells to the subject, the subject is subjected to lymphodepletion therapy.

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

[0065] In some embodiments, the Therapeutic Agent increases expression of an NKG2D ligand in a subject.

[0066] Also provided herein are methods for treating cancer in a subject, the methods including: (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 group 2D (NKG2D), wherein the genetically engineered NK cells are administered in administration cycles; and (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0067] Also provided herein is a method for 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 group 2D (NKG2D), wherein the genetically engineered NK cells are administered in administration cycles, and the subject is being 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 are methods for treating cancer in a subject, the methods comprising administering to the subject a therapeutic agent that increases expression of an NKG2D ligand, wherein the subject is being treated 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 group 2D (NKG2D), and the genetically engineered NK cells are administered in dosing cycles.

[0069] Also provided herein is a method for 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 a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 the subject has 5% or less peripheral blasts.

[0070] In some embodiments, the method includes 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 an NKG2D ligand in the subject, and any combination thereof.

[0071] In some embodiments, the subject has 5% or less peripheral blasts. In some embodiments, the subject has less than 5% peripheral blasts. In some embodiments, the subject has no evidence of extramedullary disease. In some embodiments, the percentage of peripheral blasts is determined in the subject prior to administering the population of NK cells to the subject. In some embodiments, if the subject is determined to have 5% or less peripheral blasts prior to administering the population of NK cells to the subject, the subject is selected for treatment. In some embodiments, the percentage of peripheral blasts is determined in the subject prior to administering the population of NK cells to the subject, and if the subject is determined to have 5% or less peripheral blasts, the subject is selected for treatment. In some embodiments, the method comprises determining the percentage of peripheral blasts in the subject prior to administering the population of NK cells to the subject. In some embodiments, if the subject is determined to have 5% or less peripheral blasts prior to administering the population of NK cells to the subject, the method comprises selecting the subject for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the method includes (a) determining the percentage of peripheral blasts in the subject, and (b) selecting the subject for treatment if the subject is determined to have 5% or less peripheral blasts.

[0072] In some embodiments, the administration cycle includes (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of 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 group 2D (NKG2D), the method comprising: (a) assessing the level or amount of an NKG2D ligand in a biological sample from the subject having cancer, where 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) selecting the subject for treatment with the population of NK cells genetically engineered to express a chimeric receptor that binds to a ligand of NKG2D if the level or amount of the NKG2D ligand exceeds a reference value; and (c) administering the genetically engineered NK cells to the subject, where the biological sample is obtained from the subject prior to administration of 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 group 2D (NKG2D), the method comprising administering genetically engineered NK cells to a subject having cancer, wherein (a) the level or amount of NKG2D ligand in a biological sample from the subject is assessed prior to administering the population of genetically engineered NK cells, the level or amount being the level or amount of a protein or polynucleotide encoded by an NKG2D ligand gene; (b) the subject is selected for treatment with the population of NK cells genetically engineered to express a chimeric receptor that binds to a ligand of NKG2D based on the level or amount of NKG2D ligand exceeding a reference value; and (c) the biological sample is obtained from the subject prior to administering the population of genetically engineered NK cells.

[0075] In some embodiments, the biological sample comprises a bone marrow sample. In some embodiments, the biological sample is a bone marrow sample. In some embodiments, the cancer 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 an administration cycle comprising: (i) a first dose of genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1 x 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 Contains genetically engineered NK cells.

[0077] In some embodiments, the reference value is within 25%, 20%, 15%, 10%, or 5% of the mean level or amount of NKG2D ligand in a population of subjects administered the genetically engineered NK cells who have cancer and who have failed to demonstrate a clinical response after administration of the genetically engineered NK cells. In some embodiments, the reference value is the mean level or amount of NKG2D ligand in a population of subjects administered the genetically engineered NK cells who have cancer and who have failed to demonstrate 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, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 14 days, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administering lymphodepletion therapy to the subject.

[0079] In some embodiments, the percentage of blasts in the subject's bone marrow is about 0% to about 60%, about 0% to about 50%, about 0% to about 40%, about 0% to about 30%, or about 0% to about 20%. In some embodiments, the percentage of blasts in the subject's peripheral blood is about 0% to about 5%, about 0% to about 10%, about 0% to about 20%, about 0% to about 30%, about 0% to about 40%, about 0% to about 50%, about 0% to about 60%, about 0% to about 70%, about 0% to about 80%, or about 0% to 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 engineered NK cells comprises about 1 x 10 8 NK cells ~ approximately 1 x 10 10In some embodiments, each of the first, second, and third doses of 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, second, and third doses of engineered NK cells comprises about 1 x 10 9 In some embodiments, each of the first, second, and third doses of engineered NK cells comprises about 1.5 x 10 9 In some embodiments, each of the first, second, and third doses of engineered NK cells comprises 2 x 10 9 NK cells, 3 x 10 9 NK cells, 4 x 10 9 NK cells, or 5 x 10 9 In some embodiments, each of the first, second, and third doses of engineered NK cells comprises 2 x 10 9 In some embodiments, each of the first, second, and third doses of engineered NK cells comprises 3 x 10 9 In some embodiments, each of the first, second, and third doses of engineered NK cells comprises 4 x 10 9 In some embodiments, each of the first, second, and third doses of engineered NK cells comprises 5 x 10 9 In some embodiments, the NK cells are genetically engineered NK cells.

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

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

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

[0086] In some embodiments, the administration cycle is about 14 days to about 35 days. In some embodiments, the administration cycle is about 21 days. In some embodiments, the administration cycle is about 28 days. In some embodiments, the first, second, and third doses of genetically engineered NK cells are administered to the subject within about 21 days of the first dose. In some embodiments, the first, second, and third doses of genetically engineered NK cells are administered to the subject within about 14 days of the first dose.

[0087] Also provided herein is a method for 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 a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1.5×10 9The subject has no more than 5% peripheral blasts, and the subject has no evidence of extramedullary disease.

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

[0089] In some embodiments, if the subject shows a clinical response after a dosing cycle and then shows disease progression, the method comprises administering an additional dosing cycle. In some embodiments, if the subject shows a clinical response after a dosing cycle and then shows disease progression, the method comprises administering an additional dosing cycle as retreatment. In some embodiments, the method comprises administering 1 to 5 dosing cycles. In some embodiments, the method comprises administering 1 dosing cycle. In some embodiments, the method comprises administering 2 dosing cycles. In some embodiments, the method comprises administering 3 dosing cycles. In some embodiments, the method comprises administering 4 dosing cycles. In some embodiments, the method comprises administering 5 dosing cycles. In some embodiments, the method comprises administering 5 or fewer dosing cycles. In some embodiments, the subject is administered lymphodepleting therapy before each cycle.

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

[0091] In some embodiments, the first administration cycle is initiated after the subject has been administered lymphodepleting therapy. In some embodiments, the first administration cycle is followed by additional administration cycles. In some embodiments, the first administration cycle is followed by a second, third, fourth, or more additional administration cycles. In some embodiments, additional cycle(s) are administered depending on the state of the cancer in the subject, for example, in the event of additional cancer progression or onset. In some embodiments, additional cycle(s) are administered as re-treatment in the event of disease progression. In some embodiments, if a subject shows a clinical response to an administration cycle and then shows disease progression, additional cycle(s) are administered as re-treatment. In some embodiments, after a subject shows a clinical response to a previous cycle, additional cycle(s) are administered as consolidation treatment. In some embodiments, if a subject shows a response (e.g., a complete response), additional cycles are not required.

[0092] In some embodiments, the lymphodepleting therapy comprises administration of fludarabine (Flu). In some embodiments, the lymphodepleting therapy comprises administration of three doses of Flu. In some embodiments, each dose of Flu is about 10 mg / m 2 ~about 60mg / m 2 In some embodiments, each dose of Flu is about 30 mg / m 2In some embodiments, the lymphodepletion therapy comprises administration of Flu and cyclophosphamide (Cy). In some embodiments, the lymphodepletion therapy comprises administration of three doses of Cy. In some embodiments, a first dose of Flu and Cy is each given 5 days before the start of an administration cycle; a second dose of Flu and Cy is each given 4 days before the start of an administration cycle; and a third dose of Flu and Cy is each given 3 days before the start of an administration cycle. In some embodiments, each dose of Cy is about 200 mg / m 2 ~about 600mg / m 2 In some embodiments, each dose of Cy is about 300 mg / m 2 In some embodiments, each dose of Cy is about 500 mg / m 2 In some embodiments, each dose of Flu is about 30 mg / m 2 and each dose of Cy is about 300 mg / m 2 In some embodiments, each dose of Flu is about 30 mg / m 2 each dose of Cy is about 500 mg / m 2 In some embodiments, 30 mg / m 2 of Flu and 500mg / m 2 Cy are given 5, 4, and 3 days, respectively, before the start of the administration cycle. In some embodiments, a lapse of about 2 days is allowed between the third administration of cyclophosphamide and fludarabine and the start of the administration cycle.

[0093] In some embodiments, lymphodepleting therapy comprises administration of fludarabine (Flu). In some embodiments, lymphodepleting therapy comprises administration of 3 to 5 doses of Flu. In some embodiments, each dose of Flu is about 10 mg / m 2 ~about 60mg / m 2 In some embodiments, each dose of Flu is about 30 mg / m 2In some embodiments, the lymphodepleting therapy comprises administering Flu and cytosine arabinoside (Ara-C). In some embodiments, the lymphodepleting therapy comprises five doses of Ara-C. In some embodiments, the lymphodepleting therapy comprises five doses of Ara-C per day and five doses of fludarabine per day, wherein the first dose of Ara-C and fludarabine is administered 7 days before the start of the administration cycle. In some embodiments, the first dose of Flu and Ara-C is each administered 7 days before the start of the administration cycle; the second dose of Flu and Ara-C is each administered 6 days before the start of the administration cycle; the third dose of Flu and Ara-C is each administered 5 days before the start of the administration cycle; the fourth dose of Flu and Ara-C is each administered 4 days before the start of the administration cycle; and the fifth dose of Flu and Ara-C is each administered 3 days before the start of the administration cycle. In some embodiments, about 2 days are allowed to lapse between the fifth dose of Ara-C and fludarabine and the start of the administration cycle. In some embodiments, each dose of Ara-C is about 1 g / m 2 ~approx. 4g / m 2 In some embodiments, each dose of Ara-C comprises about 2 g / m 2 In some embodiments, each dose of Flu is about 30 mg / m 2 and each dose of Ara-C is about 2 g / m 2 In some embodiments, about 30 mg / m 2 of Flu and approximately 2g / m 2 Ara-C are given 7, 6, 5, 4, and 3 days, respectively, before the start of the administration cycle.

[0094] Depending on the embodiment, other lymphodepleting agents, such as, 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, hi some embodiments, the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly(ADP-ribose) polymerase (PARP), or any combination thereof.

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

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

[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 an antimitotic agent. In some embodiments, the antimitotic agent comprises vincristine, vinblastine, vinorelbine, docetaxel, paclitaxel, eribulin, isabepilone, epothilone, or any combination thereof. In some embodiments, the therapeutic agent comprises vincristine. In some embodiments, the therapeutic agent comprises vinblastine. In some embodiments, the therapeutic agent comprises paclitaxel. In some embodiments, the therapeutic agent comprises docetaxel.

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

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

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

[0104] In some embodiments, the therapeutic agent comprises a monoclonal antibody. In some embodiments, the monoclonal antibody comprises an anti-CD20 antibody, an anti-CTLA4 antibody, an anti-EGFR antibody, an anti-HER2 / neu antibody, an anti-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 the NK cell and an antigen expressed by the cancer cell, hi some embodiments, the activating receptor is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof.

[0106] In some embodiments, the therapeutic agent is decitabine. In some embodiments, a dose of decitabine is administered to the subject daily for 5 days prior to the start of an administration cycle. In some embodiments, the dose of decitabine is about 20 mg / m 2 Includes.

[0107] Also provided herein is a method for 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 a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells, wherein each dose of genetically engineered NK cells is about 1.5×10 9the subject receives a dose of about 20 mg / m prior to administration of the first dose of genetically engineered NK cells. 2 The patient is being given decitabine daily for five days.

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

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

[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 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 the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer. In some embodiments, the cancer is 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 colon cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is gastric 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 relapses after hematopoietic cell transplantation (HCT). In some embodiments, the cells of the cancer 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 1, 2, 3, or 4 lines of prior therapy. In some embodiments, the subject has been treated with 1 line of prior therapy. In some embodiments, the subject has been treated with 2 lines of prior therapy. In some embodiments, the subject has been treated with 3 lines of prior therapy. In some embodiments, the subject has been treated with 4 lines of prior therapy. 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 4 lines of prior therapy. 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 CD3 zeta. 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 some 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 some embodiments, the chimeric receptor has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 39.

[0122] In some embodiments, the genetically engineered NK cells express membrane-bound interleukin-15 (mbIL15). In some embodiments, mbIL15 has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, mbIL15 has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, mbIL15 has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the genetically engineered population of NK cells is allogeneic to the subject. In some embodiments, the genetically engineered population of cells is derived from a subject not afflicted with cancer.

[0123] In some embodiments, a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis. In some embodiments, each dose of the genetically engineered NK cells is administered to the subject on an outpatient basis.

[0124] Also provided herein is the 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 of natural killer group 2D (NKG2D), and (b) a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof; the genetically engineered NK cells are administered to the subject in administration cycles; and prior to administering a first dose of the genetically engineered NK cells to the subject, the subject is subjected to lymphodepletion therapy.

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

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

[0127] In some embodiments, the Therapeutic Agent increases expression of an NKG2D ligand in a 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 for natural killer group 2D (NKG2D), and (b) a therapeutic agent that increases expression of an NKG2D ligand in the subject.

[0129] Also provided herein is the use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D), for treating cancer in a subject, wherein the subject is being treated with or is a candidate for treatment with a therapeutic agent that increases expression of the NKG2D ligand in the subject.

[0130] Also provided herein is the use of a therapeutic agent that increases expression of an NKG2D ligand in a subject to treat cancer in the subject, wherein the subject is being 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 group 2D (NKG2D).

[0131] In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, an NK cell engager, and any combination thereof. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a monoclonal antibody. In some embodiments, the therapeutic agent is an NK cell engager. In some embodiments, prior to administering the first dose of engineered NK cells to the subject, the subject is subjected to lymphodepletion therapy.

[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 genetically engineered NK cells, (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells, and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. In some embodiments, each dose of genetically engineered NK cells comprises about 1 x 108 NK cells ~ approximately 1 x 10 10 In some embodiments, each dose of engineered NK cells comprises about 1 x 10 9 In some embodiments, each dose of engineered NK cells comprises about 1.5 x 10 9 Contains NK cells.

[0134] In some embodiments, the first administration cycle is initiated after the subject has been administered lymphodepleting therapy. In some embodiments, the first administration cycle is followed by additional administration cycles. In some embodiments, the first administration cycle is followed by a second, third, fourth or more additional administration cycles. In some embodiments, the additional cycle(s) are administered depending on the state of the cancer in the subject, for example, in the event of progression or onset of additional cancer. In some embodiments, if the subject shows a response (e.g., a complete response), additional cycles are not required. [Brief explanation of the drawings]

[0135] [Figure 1A] 1A-1B show non-limiting schematic diagrams of polynucleotides encoding cytotoxic receptor constructs that contain a binding moiety for a ligand of NKG2D and either encode mbIL15 (receptor B) or not (receptor A). [Figure 1B] Same as above.

[0136] [Figure 2A] 2A-2B show non-limiting schematic diagrams of polynucleotides encoding cytotoxic receptor constructs that include the NKG2D receptor domain (e.g., fragments) and either encode (receptor B) or not (receptor A) mbIL15. [Figure 2B] Same as above.

[0137] [Figure 3A]3A-3B show non-limiting schematic diagrams of administration cycles according to embodiments disclosed herein: Fig. 3A shows a 28-day cycle including three administration events; Fig. 3B shows a 28-day cycle including two administration events. [Figure 3B] Same as above.

[0138] [Figure 4A] Figure 4A shows the 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 μg / mL cetuximab or an isotype control antibody.

[0139] [Figure 4B] Figure 4B shows 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 of anti-CD16 neutralizing antibody (anti-CD16) or isotype control antibody (iso).

[0140] [Figure 5A] Figure 5A shows IC50 curves obtained from the specific killing of A-431 cells treated with NK cells expressing NKG2D chimeric receptors from donors with the indicated CD16 phenotype in combination with 10 μg / mL cetuximab or an isotype control antibody.

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

[0142] [Figure 6A] 6A-6B show the HSA and Loewe synergy scores, respectively, for the combination of NK cells expressing NKG2D chimeric receptors with cetuximab for all six donors. [Figure 6B] Same as above.

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

[0144] [Figure 8A] 8A, 8B, and 8C 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. [Figure 8B] Same as above. [Figure 8C] Same as above.

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

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

[0147] Some embodiments of the methods and compositions provided herein relate to engineered immune cells, therapeutic agents, and combinations thereof for use in immunotherapy. In some embodiments, the engineered cells are engineered in multiple ways, for example, to express a receptor complex that induces cytotoxicity. As used herein, the term "cytotoxic receptor complex" is given its ordinary meaning and (unless otherwise specified) also refers to a chimeric antigen receptor (CAR), a chimeric receptor (also referred to as an activating chimeric receptor in the case of an NKG2D chimeric receptor). In some embodiments, the cells are further engineered to achieve modified cellular responsiveness to non-tumor tissue and / or other therapeutic cells.

[0148] I. Cell Types Some embodiments of the methods and compositions provided herein relate to cells, such as immune cells. For example, immune cells, such as NK cells or T cells, can be engineered to contain chimeric receptors, such as NKG2D-ligand-directed chimeric receptors, as described herein, or can be engineered to contain nucleic acids encoding said chimeric receptors. Additional embodiments relate to engineering a second set of cells to express another cytotoxic receptor complex, such as the NKG2D-chimeric receptor complex disclosed herein.

[0149] Traditional anti-cancer therapies have relied on surgical approaches, radiation therapy, chemotherapy, or a combination of these methods. As research has led to a deeper understanding of some of the mechanisms of specific cancers, this knowledge has been utilized to develop targeted cancer therapies. Targeted therapy is a cancer treatment that employs specific drugs to target specific genes or proteins found in cancer cells or cells that support cancer growth (such as vascular cells) and reduce or stop cancer cell growth. More recently, genetic engineering has made it possible to develop approaches that utilize specific 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. As described in more detail below, various types of immune cells can be used, such as T cells, natural killer (NK) cells, or a combination thereof. 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 T cells and NK cells.

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

[0151] Engineered cells for immunotherapy In some embodiments, immune system cells are engineered to have enhanced cytotoxic effects against target cells, such as tumor cells. For example, immune system cells can be engineered to contain a tumor-targeting chimeric receptor and / or a tumor-targeting CAR, as described herein. In some embodiments, white blood cells or leukocytes are used because their primary function is to defend the body against abnormal cell proliferation and infectious diseases. There are various types of leukocytes that play specific roles in the human immune system and are therefore a preferred starting point for engineering the cells disclosed herein. Leukocytes include granulocytes and agranulocytes (with or without granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Granulocytes include lymphocytes and monocytes. Cells as described herein or otherwise can be engineered to contain a chimeric antigen receptor, such as an NKG2D ligand-targeting chimeric receptor, or a nucleic acid encoding the chimeric receptor. In some embodiments, immune cells engineered to express a chimeric receptor are also engineered to express (e.g., bicistronic) a membrane-bound interleukin-15 (mbIL15) domain. As discussed in more detail below, in some embodiments, the therapeutic cells are further genetically modified to enhance the cytotoxicity and / or persistence of the cells. In some embodiments, the genetic modification enhances the ability of the cells to resist signals emanating from the tumor microenvironment that would otherwise cause a decrease in efficacy or shortened lifespan of the therapeutic cells.

[0152] Monocytes for immunotherapy In some embodiments, the 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 the primary functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of engulfing cellular material or whole cells, followed by digestion and destruction of the engulfed cellular material. In some embodiments, monocytes are used in conjunction with one or more additional engineered cells disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express activating chimeric receptors that target ligands on tumor cells, such as 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) the membrane-bound interleukin-15 (mbIL15) domain. In some embodiments, the monocytes are autologous cells. In some embodiments, the monocytes are allogeneic cells.

[0153] Lymphocytes for immunotherapy In some embodiments, immune cells comprise 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, immune cells comprise T cells. Thus, in some embodiments, immune cells comprise NK cells. In some embodiments, immune cells comprise B cells. While B cells are engineered according to some embodiments disclosed herein, some embodiments also relate to engineered T cells or engineered NK cells (mixtures of T cells and NK cells are used in some embodiments, either from the same donor or different donors). Some embodiments of the methods and compositions disclosed herein relate to 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, lymphocytes engineered to express a chimeric receptor are also engineered to express (e.g., bicistronic) a membrane-bound interleukin-15 (mbIL15) domain. In some embodiments, the lymphocytes are autologous. In some embodiments, the lymphocytes are allogeneic.

[0154] T Cells for Immunotherapy 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 the cell surface. T cells are divided into a variety of different subtypes, such as effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated non-mutated T cells, and gamma delta T cells. In some embodiments, specific subtypes of T cells are engineered. In some embodiments, CD4+ T cells are engineered. In some embodiments, CD8+ T cells are engineered. In some embodiments, regulatory T cells are engineered. In some embodiments, gamma delta T cells are engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. For example, in some embodiments, CD4+ and CD8+ T cells are engineered. In some embodiments, there is no specific selection of the type of T cells engineered to express the cytotoxic receptor complexes disclosed herein. In some embodiments, certain techniques, such as the use of cytokine stimulation, are used to enhance the expansion / recovery of T cells with specific marker profiles. For example, in some embodiments, activation of specific human T cells, e.g., CD4+ T cells, CD8+ T cells, is achieved by using CD3 and / or CD28 as stimulatory molecules. In some embodiments, methods are provided for treating or preventing cancer or infectious diseases, comprising administering a therapeutically effective amount of T cells expressing a cytotoxic receptor complex and / or homing moiety described herein. In some embodiments, the T cells are autologous. In some embodiments, the T cells are allogeneic. Some embodiments of the methods and compositions disclosed herein relate to T cells 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, T cells engineered to express chimeric receptors are also engineered to express (e.g., bicistronic) the membrane-bound interleukin-15 (mbIL15) costimulatory domain.

[0155] NK cells for immunotherapy In some embodiments, the immune cells comprise NK cells. Thus, in some embodiments, methods are provided for treating or preventing cancer or infectious disease, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex and / or homing moiety described herein. In some embodiments, methods are provided for treating cancer, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex. In some embodiments, the NK cells are autologous cells. In some embodiments, the NK cells are allogeneic cells. In some embodiments, NK cells are preferred due to their relatively high natural cytotoxic capacity. In some embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, resulting in more effective activity against target cells (e.g., tumor cells or other diseased cells). Some embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, NK cells engineered to express chimeric receptors are also engineered to express (e.g., bicistronic) a membrane-bound interleukin-15 (mbIL15) domain. Thus, in some embodiments, NK cells are engineered to express (e.g., bicistronic) a chimeric receptor (e.g., NKG2D ACR) and mbIL15.

[0156] In some embodiments, immortalized NK cells are used and engineered as disclosed herein. In some embodiments, the NK cells are derived from the cell line NK-92. NK-92 cells are derived from NK cells, but while retaining most of the activating receptors, they lack the major inhibitory receptors exhibited by normal NK cells. Some embodiments of the NK-92 cells described herein relate to NK-92 cells engineered to silence specific additional inhibitory receptors, such as SMAD3, allowing for upregulation of interferon-γ (IFNγ), granzyme B, and / or perforin production. Additional information regarding the NK-92 cell line is disclosed in International Publication No. WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, which are incorporated herein by reference in their entireties. In some embodiments, NK-92 cells are used in combination with one or more of the other cell types disclosed herein. For example, in one embodiment, NK-92 cells are used in combination with the NK cells disclosed herein. In an additional embodiment, NK-92 cells are used in combination with the T cells disclosed herein.

[0157] Hematopoietic stem cells for cancer immunotherapy 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 some embodiments, the cells are engineered to express homing and / or cytotoxic receptor complexes. HSCs are used in some embodiments to exploit their engraftment capacity for long-term blood cell production, and can serve as a sustained source of targeted anti-cancer effector cells, for example, to combat cancer remission. In some embodiments, this continuous generation helps to offset anergy or depletion of other cell types, for example, by the tumor microenvironment. In some embodiments, allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In some embodiments, HSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, HSCs engineered to express a chimeric receptor are also engineered to express (eg, bicistronic) a membrane-bound interleukin-15 (mbIL15) domain.

[0158] induced pluripotent stem cells 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. iPSCs are used in some embodiments to exploit their ability to differentiate and derive into non-pluripotent cells, such as, but not limited to, CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem cells and progenitor cells), hematopoietic pluripotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells, by differentiating iPSCs or less differentiated cells containing the same genetic modifications at the same selected sites. In some embodiments, iPSCs are used to generate iPSC-derived NK cells or T cells. In some embodiments, the cells are engineered to express homing and / or cytotoxic receptor complexes. In some embodiments, iPSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, iPSCs engineered to express chimeric receptors are also engineered to express (e.g., bicistronic) the membrane-bound interleukin-15 (mbIL15) costimulatory domain. In some embodiments, engineered iPSCs are differentiated into NK, T, or other immune cells, such as for use in the compositions or methods provided herein.

[0159] II. Extracellular Domain (Tumor Binding Agent) Some embodiments of the compositions and methods described herein relate to chimeric receptors comprising an extracellular domain that includes a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain) described herein. Some embodiments of the compositions and methods described herein relate to chimeric receptors (also referred to as activating chimeric receptors) comprising an extracellular domain that includes a ligand-binding domain that binds to 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 the wild-type or non-wild-type sequence of an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), vH or vL domain, camelid VHH domain, or a non-immunoglobulin scaffold such as a DARPIN, affibody, affilin, adnectin, affitin, repebody, fynomer, alphabody, avimer, atrimer, centilin, pronectin, anticalin, Kunitz domain, armadillo repeat protein, autoantigen, receptor, or ligand. In some embodiments, the tumor-binding domain comprises more than one antigen-binding domain.

[0161] antigen-binding proteins In some embodiments, an antigen-binding protein is provided. As used herein, the term "antigen-binding protein" is given its ordinary meaning and also refers to a protein comprising an antigen-binding fragment that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding fragment to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of the antibody that binds the antigen or all three CDRs from the light chain of the antibody that binds the antigen. Further, in some embodiments, the antigen-binding fragment comprises all six CDRs (three from the heavy chain and three from the light chain) of an antibody that binds the antigen. In some embodiments, the antigen-binding fragment comprises one, two, three, four, five, or six CDRs from an antibody that binds the antigen, and in some embodiments, the CDRs can be any combination of heavy 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. More 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 fragments), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments. These molecules can be derived from mammals such as humans, mice, rats, rabbits, pigs, dogs, or camels. Antibody fragments may compete with intact (e.g., native) antibodies for target antigen binding, and fragments can be generated by modification of intact antibodies (e.g., enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technology or peptide synthesis. Antigen-binding proteins can include, for example, alternative protein scaffolds or artificial scaffolds grafted with CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds containing, for example, biocompatible polymers. Additionally, peptide antibody mimics ("PAMs") can be used, as can antibody mimic-based scaffolds that utilize fibronectin components as the scaffold.

[0163] In some embodiments, the antigen-binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or a set of multiple polypeptides. For example, antigen-binding proteins may include, but are not limited to, diabodies, intrabodies, domain antibodies (a single VL or VH domain, or two or more VH domains connected by a peptide linker), maxibodies (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" is given its ordinary meaning and refers to a tetrameric molecule in which each tetramer contains two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

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

[0166] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. From the N-terminus to the 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 and lambda light chains. The "light chain" of an antibody refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformation. Kappa (K) light chains and lambda (λ) light chains refer to the two major antibody light chain isotypes. A light chain can comprise a polypeptide that, from the amino terminus to the carboxyl terminus, contains 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), which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The "heavy chain" of an antibody refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations and typically determines the class to which the antibody belongs. A heavy chain can comprise, from the amino terminus to the carboxyl terminus, a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and, optionally, immunoglobulin heavy chain constant domain 4 (CH4).

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

[0170] In some embodiments, the antigen-binding protein is an antibody. The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be monoclonal, polyclonal, multi- or single-chain, or intact immunoglobulins and can be naturally or recombinantly derived. Antibodies can be tetramers of immunoglobulin molecules. Antibodies can be "humanized," "chimeric," or non-human. Antibodies can include intact immunoglobulins of any isotype, including, for example, chimeric antibodies, humanized antibodies, human antibodies, and bispecific antibodies. Intact antibodies generally contain 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," i.e., different portions of the antibody can be derived from two different species, as described further below. Unless otherwise specified, the term "antibody" also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains, provided that the antibody retains the same or similar binding and / or function as an antibody composed of two full-length light and heavy chains. For example, antibodies with one, two, three, four, or five amino acid residue substitutions, insertions, or deletions at the N-terminus and / or C-terminus of the heavy and / or light chains are included in the definition, provided that the antibody retains the same or similar binding and / or function as an antibody comprising two full-length heavy chains and two full-length light chains. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies. In some embodiments, monoclonal and polyclonal antibodies are provided. As used herein, the term "polyclonal antibody" is given its ordinary meaning and typically refers to a population of antibodies that differ greatly in composition and binding specificity. As used herein, the term "monoclonal antibody" ("mAb") is given its ordinary meaning and refers to one or more of a population of antibodies having identical sequence. A monoclonal antibody binds to an 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 epitope of an antigen (e.g., by 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 Fvs (sdFv), Fd fragments consisting of a VH domain and a CHI domain, linear antibodies, single-domain antibodies (either VL or VH) such as sdAb, camelid VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, isolated CDRs, or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds 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 containing 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, Fab fragments are provided. Fab fragments are monovalent fragments containing VL, VH, CL, and CH1 domains; F(ab')2 fragments are bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments contain VH and CH1 domains; Fv fragments contain the VL and VH domains of a single antibody arm; and dAb fragments contain the VH domain, VL domain, or antigen-binding fragments of the VH or VL domain. In some embodiments, these antibody fragments can be incorporated into single-domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. In some embodiments, the antibody comprises at least one CDR described herein.

[0173] Also provided herein in some embodiments are single-chain variable fragments. As used herein, the term "single-chain variable fragment" ("scFv") is given its ordinary meaning and refers to a fusion protein in which a VL domain and a VH domain are linked via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, the linker being long enough to allow the protein chain to fold back on itself and form a monovalent antigen-binding site. For clarity, unless otherwise indicated, a "single-chain variable fragment" is not an antibody or antibody fragment as defined herein. Diabodies are bivalent antibodies comprising two polypeptide chains, each of which comprises a VH domain and a VL domain connected by a linker configured to reduce or prevent pairing between the two domains on the same chain, such that each domain can pair with a complementary domain on another polypeptide chain. According to some embodiments, if the two polypeptide chains of a diabody are identical, the diabody resulting from their pairing has two identical antigen-binding sites. Diabodies can be made with two different antigen-binding sites using polypeptide chains with different sequences. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which can be the same or different.

[0174] In some embodiments, an antigen-binding protein comprises one or more CDRs. As used herein, the term "CDR" is given its ordinary meaning and also refers to the complementarity-determining regions (also called "minimal recognition units" or "hypervariable regions") within an antibody variable sequence. CDRs enable 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 particular epitope or domain on the target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically follow the following order of these elements: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. For the heavy chain variable region, the order is typically, from N-terminus to C-terminus, FW-H1, CDR-H1, FW-H2, CDR-H2, FW-H3, CDR-H3, and FW-H4. For the light chain variable region, the order is typically, from N-terminus to C-terminus, FW-L1, CDR-L1, FW-L2, CDR-L2, FW-L3, CDR-L3, and FW-L4. A numbering system has been devised to number the amino acids occupying each position in these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system.Other numbering systems for amino acids of immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). The binding domains disclosed herein can utilize CDRs defined according to any of these systems. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or conformational definitions, or any combination of the foregoing. Any CDR, individually or within the context of a variable domain, can be appropriately interpreted according to any of these numbering systems by one of skill in the art. One or more CDRs can be incorporated covalently or noncovalently into a molecule to form an antigen-binding protein.

[0175] In some embodiments, the antigen binding proteins provided herein comprise one or more CDR(s) as part of a larger polypeptide chain. In some embodiments, the antigen binding protein covalently links one or more CDR(s) to another polypeptide chain. In some embodiments, the antigen binding protein non-covalently incorporates one or more CDR(s). In some embodiments, the antigen binding protein may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support, or framework, or scaffold, capable of displaying one or more sequences of antigen-binding amino acids (e.g., CDRs, variable regions, etc.) at localized surface regions. Such a structure may be a naturally occurring polypeptide or polypeptide "fold" (structural motif), or may have one or more modifications 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 a variety of 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 structure is based on a protein scaffold or skeleton other than an immunoglobulin domain, hi some such embodiments, the framework structure is based on fibronectin, ankyrin, lipocalin, neocarzinostein, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and / or tendamistat domain.

[0177] Also provided in some embodiments are antigen-binding proteins with more than one binding site. In some embodiments, the binding sites are identical to one another, while in some embodiments, the binding sites are different from one another. For example, while antibodies typically have two identical binding sites, "bispecific" or "bifunctional" antibodies have two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes, which may be present on the same or different protein targets. In some embodiments, this is particularly advantageous because bispecific chimeric antigen receptors can confer the engineered cells the ability to target multiple tumor markers. For example, bispecific antibodies can bind to additional tumor markers, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others, as well as additional tumor markers, such as CD70, CD123, CD19, Her2, mesothelin, claudin 6, BCMA, EGFR, or other markers disclosed herein or recognized in the art as tumor-specific or tumor-associated antigens.

[0178] Natural killer group domains that bind tumor ligands In some embodiments, engineered immune cells, such as NK cells, are exploited for their ability to recognize and destroy tumor cells. NK cells express both inhibitory and activating receptors on their cell surface. Inhibitory receptors bind to self-molecules expressed on the surface of healthy cells (thus preventing an immune response against "self" cells), while activating receptors bind to ligands expressed on abnormal cells, such as tumor cells. When the balance of activation between inhibitory and activating receptors is in favor of activating receptors, NK cell activation occurs and target (e.g., tumor) cells are lysed.

[0179] Natural killer group 2 member D (NKG2D) is an NK cell-activating receptor that recognizes various ligands expressed on cells. Surface expression of various NKG2D ligands is generally low on 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 function of NK cells. In some embodiments, T cells are engineered to express an extracellular domain that binds to one or more tumor ligands and activates the T cells. For example, in some embodiments, T cells are engineered to express an NKG2D receptor as a binding / activating moiety. In some embodiments, NK cells are engineered to express an extracellular domain that binds to one or more tumor ligands and activates the NK cells. For example, in some embodiments, NK cells are engineered to express an NKG2D receptor as a binding / activating moiety. In some embodiments, the engineered cells disclosed herein are engineered to express other members of the NKG2 family, such as NKG2A, NKG2C, and / or NKG2E. Combinations of such receptors are engineered in some embodiments. Additionally, in some embodiments, other receptors, such as killer cell immunoglobulin-like receptors (KIRs), are expressed.

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

[0181] In some embodiments, cells are engineered to express a cytotoxic receptor complex comprising a functional fragment of NKG2D as an extracellular component to recognize a ligand on the surface of tumor cells or other diseased cells. In one embodiment, the functional fragment of NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the fragment of NKG2D has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to full-length wild-type NKG2D. In some embodiments, the nucleic acid sequence encoding the fragment can have one or more additional mutations from SEQ ID NO: 25 but retain ligand-binding function, or in some embodiments, have enhanced ligand-binding function. In some embodiments, the functional fragment of NKG2D has the amino acid sequence of SEQ ID NO: 26. In some embodiments, the NKG2D fragment is provided as a dimer, trimer, or other linked form; such embodiments provide enhanced ligand-binding activity. In some embodiments, the sequence encoding the NKG2D fragment is fully or partially codon-optimized, as appropriate. In one embodiment, the sequence encoding the codon-optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28. Advantageously, according to some embodiments, the functional fragment lacks its original transmembrane or intracellular domain, but retains the ability of NKG2D to bind to a ligand and to transmit an activation signal upon ligand binding. A further advantage of such an NKG2D fragment is that expression of DAP10 is not required to localize NKG2D to the cell membrane. Thus, in some embodiments, the cytotoxic receptor complex encoded by the polypeptide disclosed herein does not include DAP10.In some embodiments, immune cells, e.g., NK cells or T cells (non-alloreactive T cells engineered according to embodiments disclosed herein), are engineered to express one or more chimeric receptors targeting, e.g., CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, EGFR, and NKG2D ligands, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. Such cells, in some embodiments, also express (e.g., bicistronic) mbIL15.

[0182] In some embodiments, the cytotoxic receptor complex is configured to dimerize. The dimerization can consist of a homodimer or a heterodimer, depending on the embodiment. In some embodiments, dimerization results in improved ligand recognition by the cytotoxic receptor complex (and thus the receptor-expressing NK cells), resulting in a reduction (or absence) of adverse toxic effects. In some embodiments, the cytotoxic receptor complex employs an endodimer, or repeats of one or more component subunits. For example, in some embodiments, the cytotoxic receptor complex can optionally include a first NKG2D extracellular domain coupled to a second NKG2D extracellular domain, and a transmembrane / signaling region (or a separate transmembrane region with a separate signaling region).

[0183] In some embodiments, the various domains / subdomains are separated by a linker, for example, a GS3 linker (SEQ ID NOS: 15 and 16, nucleotide and protein, respectively) is used (or a GSn linker). In some embodiments, the various domains / subdomains are separated by a linker comprising the sequence of SEQ ID NO: 44. Other linkers for use in accordance with various embodiments disclosed herein include, but are not limited to, those encoded by SEQ ID NO: 17, 19, 21, or 23. In some embodiments, other linkers comprise the peptide sequence of one of SEQ ID NOs: 18, 20, 22, and 24. This provides the possibility of separating the various component parts of the receptor complex along a polynucleotide, which can enhance the expression, stability, and / or functionality of the receptor complex.

[0184] III. Cytotoxic signaling complex Some embodiments of the compositions and methods described herein relate to chimeric receptors, e.g., chimeric receptors directed against NKG2D ligands, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6, including cytotoxic signaling complexes. As disclosed herein, according to some embodiments, the provided chimeric receptors comprise one or more transmembrane and / or intracellular domains that initiate a cytotoxic signaling cascade when the extracellular domain(s) bind to a ligand on the surface of a target cell. 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., CD3 zeta) and a costimulatory signaling domain.

[0185] In some embodiments, a 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 moiety comprises a given domain—for example, a costimulatory domain may comprise two subdomains. Furthermore, in some embodiments, a domain can perform multiple functions, for example, a transmembrane domain can also perform a signaling function.

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

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

[0188] In some embodiments, the transmembrane domain comprises a "hinge," e.g., a CD8a hinge. In some embodiments, the hinge of CD8α has the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the hinge of CD8α 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 a CD8α hinge having the sequence of SEQ ID NO: 1. In some embodiments, the hinge of CD8α comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD8α hinge may be truncated or modified to have 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: 2.

[0189] In some embodiments, the transmembrane domain comprises a CD8α transmembrane region. In some embodiments, the CD8α transmembrane region is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 3. In some 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 some embodiments, the CD8α transmembrane region comprises the amino acid sequence of SEQ ID NO: 4. In some 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 a CD8α having the sequence of SEQ ID NO: 4.

[0190] Taking some embodiments together, the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the CD8 transmembrane domain is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the CD8 transmembrane domain sequence of SEQ ID NO: 13. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 14. In some 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%, or 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 some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 30. In some 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%, or at least 95% sequence identity to the sequence of SEQ ID NO: 30.

[0192] V. Stimulating molecules In some embodiments, the intracellular signaling domain of the chimeric receptor provided herein comprises a stimulatory molecule. Accordingly, some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors) that comprise a stimulatory molecule. In addition to various transmembrane and signaling domains (and transmembrane / signaling domain combinations), in some embodiments, additional stimulatory molecules may be provided. These may be, for example, certain molecules that further enhance the activity of immune cells. Cytokines may be used in some embodiments. For example, certain interleukins, such as IL-2 and / or IL-15, are used as non-limiting examples. In some embodiments, therapeutic immune cells are engineered to express such molecules in a secreted form. In additional embodiments, such stimulatory molecules are engineered to be membrane-bound and act as autocrine stimulatory molecules (or even as paracrine stimulatory molecules for neighboring cells).

[0193] In some embodiments, the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15). In some embodiments, IL15 is expressed from a separate cassette on a construct comprising any one of the CARs disclosed herein. In some embodiments, IL15 is expressed from the same cassette as any one of the CARs disclosed herein. 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 autocleaving 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, the IL15 is membrane-bound IL15 (mbIL15). In some embodiments, the mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain. In some embodiments, the native IL15 sequence is encoded by a sequence having 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 having 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-associated by coupling to at least one transmembrane domain. In some embodiments, the at least one transmembrane domain comprises a CD8 transmembrane domain (e.g., SEQ ID NO: 4). In some embodiments, mbIL15 may comprise additional components such as a leader sequence and / or a hinge sequence. In some embodiments, the leader sequence is a CD8 leader sequence. In some embodiments, the hinge sequence is a CD8 hinge sequence (e.g., SEQ ID NO: 14). In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain (e.g., SEQ ID NO: 4) and a CD8 hinge (e.g., SEQ ID NO: 2). In some embodiments, 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 can be recognized and cleaved by cytoplasmic proteases, resulting in the separation (and separate expression) of the various components 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, such as 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, various components of the engineered cytotoxic receptor complex can be delivered to NK cells or T cells in a single vector or by multiple vectors. Thus, as shown schematically in the figures, the construct can be encoded by a single polynucleotide but can also include cleavage sites (such as in some embodiments of IL-15) so that downstream elements of the construct are expressed by the cell as separate proteins. In some embodiments, a T2A cleavage site is used. In some embodiments, the T2A cleavage site is encoded by the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the T2A cleavage site may be truncated or modified to be 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:9. In some embodiments, the T2A cleavage site comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the T2A cleavage site comprises the amino acid sequence of SEQ ID NO:45. In some 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 some embodiments, NK cells are engineered to express membrane-bound interleukin-15 (mbIL15). In such embodiments, mbIL15 expression on NK enhances the cytotoxic effect of the engineered NK cells by enhancing the proliferation and / or lifespan of the NK cells. In some embodiments, mbIL15 is encoded by the same polynucleotide as the CAR. In some embodiments, mbIL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 11. In some embodiments, mbIL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 11 and a sequence 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 operably coupled to the amino acid sequence of a transmembrane domain. In some embodiments, mbIL15 is encoded by the nucleic acid sequence of SEQ ID NO: 35. In some 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 some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 40. In some 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 explored in PCT Application Publication Nos. WO2018 / 183385 and WO2020 / 056045, each of which is expressly incorporated by reference in its entirety and relates to membrane-bound IL15 sequences.

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

[0199] In some embodiments, unexpectedly enhanced signaling is achieved through the use of multiple signaling domains whose activities act synergistically. For example, in some embodiments, the intracellular signaling domain further comprises an OX40 domain. In some embodiments, the OX40 domain is an intracellular signaling domain. In some embodiments, the OX40 intracellular signaling domain is encoded by a nucleic acid comprising the sequence of SEQ ID NO:5. In some embodiments, the OX40 intracellular signaling domain can be truncated or modified to be 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 some embodiments, the OX40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:6. In some 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%, or at least 95% sequence identity to SEQ ID NO:6. In some embodiments, OX40 is used as the only signaling domain in a chimeric receptor construct; however, in some embodiments, OX40 can be used in conjunction 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 a further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0200] In some embodiments, the signaling domain comprises a 4-1BB domain. In some embodiments, the 4-1BB domain is an intracellular signaling domain. In some embodiments, the 4-1BB domain is encoded by the nucleic acid sequence of SEQ ID NO:29. In some embodiments, the 4-1BB domain may be truncated or modified so as to be 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:29. In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the 4-1BB intracellular signaling domain is truncated or modified. In some 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 to a 4-1BB intracellular signaling domain having the sequence of SEQ ID NO:30. In some embodiments, 4-1BB is used as the only signaling domain in a chimeric receptor construct; however, in some 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 CD3 zeta is used. For example, in some embodiments, the intracellular signaling domain comprises CD3 zeta and 4-1BB, or a signaling portion thereof. In some embodiments, the intracellular signaling domain comprises CD3 zeta and 4-1BB. As a further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0201] In some embodiments, the signaling domain comprises a CD28 domain. In some embodiments, the CD28 domain is an intracellular signaling domain. In some embodiments, the CD28 intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the CD28 intracellular signaling domain may be truncated or modified to be 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: 32. In some embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CD28 intracellular signaling domain is truncated or modified. In some 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 some embodiments, CD28 is used as the only signaling domain in the construct, although in some embodiments, CD28 may 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 a further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0202] VII. Cytotoxic Receptor Complex Constructs Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as activating chimeric receptors (ACRs), that target ligands for NKG2D. Expression of these cytotoxic receptor complexes in immune cells, such as genetically engineered non-responsive 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 some embodiments, a polynucleotide encoding a tumor-binding agent / CD8 hinge-CD8™ / OX40 / CD3 zeta chimeric receptor complex is provided (see Figure 2A, 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 CD3 zeta domain. In some embodiments, the polynucleotide further encodes a 2A cleavage site and an mbIL-15 domain, as described herein (see Figure 2A, Chimeric Receptor B, which depicts a polynucleotide structure in which a single polynucleotide encodes both the receptor and mbIL15). In some embodiments, the receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, as included herein by way of example of a constituent portion. In some 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 combining one or more SEQ ID NOs described herein. It is understood that certain sequence variability, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of ease or efficiency in cloning (e.g., for creation of restriction sites). In some embodiments, the chimeric receptor comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to one or more of the sequences of the SEQ ID NOs provided herein, or a portion thereof (e.g., a portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence), or a range defined by any two of the foregoing percentages.

[0204] In some embodiments, a polynucleotide encoding an NKG2D / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta activating chimeric receptor complex is provided (see FIG. 2B, NKG2D ACR A). The polynucleotide comprises or consists of a fragment of the NKG2D receptor capable of binding to a ligand of the NKG2D receptor described herein, a CD8 alpha hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain. In some 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 differs from SEQ ID NO: 33 but can be encoded by a nucleic acid sequence that remains expressed as an amino acid sequence 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 some embodiments, the chimeric receptor amino acid sequence may differ from SEQ ID NO: 40, but the chimeric receptor retains NK cell activation and / or cytotoxicity function, or in some embodiments, has enhanced NK cell activation and / or cytotoxicity function. Additionally, in some embodiments, the construct may be co-expressed with mbIL15, such as mbIL15 encoded by SEQ ID NO: 35 or 37, as appropriate (FIG. 2B, NKG2D ACR B, represents a polynucleotide structure in which a single polynucleotide encodes both the receptor and mbIL15). In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 36, 38, or 40. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 38. In some 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 remains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical, depending on the embodiment, 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 remains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical, depending on the embodiment, to SEQ ID NO: 36. In some embodiments, the amino acid sequence of mbIL15 may differ from SEQ ID NO: 38, but remains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical, depending on the embodiment, to SEQ ID NO: 38. In some embodiments, the amino acid sequence of mbIL15 may differ from SEQ ID NO: 40, but remain 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.

[0205] Additional information regarding chimeric receptors for use in the presently disclosed methods and compositions can be found in PCT Patent Publication No. WO2018 / 183385, filed March 27, 2018, which is incorporated herein by reference in its entirety.

[0206] VIII. Treatment Methods Some embodiments relate to methods of treating, ameliorating, inhibiting, or preventing cancer using cells or immune cells comprising a chimeric antigen receptor and / or an activated chimeric receptor disclosed herein. Some embodiments relate to methods of treating, ameliorating, inhibiting, or preventing cancer using cells or immune cells comprising a chimeric receptor provided herein in combination with a therapeutic agent. In some embodiments, the method comprises treating or preventing cancer. In some embodiments, the method comprises administering a therapeutically effective amount of immune cells expressing a tumor-targeting chimeric antigen receptor and / or a tumor-targeting chimeric receptor described herein. Examples of types of cancer that can be treated in this way are described herein.

[0207] Provided herein is a method for treating cancer in a subject.In some embodiments, the method comprises administering to the subject the NKG2D ligand binding domain disclosed herein, any one of the chimeric receptors disclosed herein, or any one of the cells disclosed herein, or any combination thereof, and a therapeutic agent.

[0208] In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, 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 expression of an NKG2D ligand in the subject.

[0209] In some embodiments, a therapeutic agent is administered before, 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 but prior to administration of the genetically engineered cells. In some embodiments, the therapeutic agent is administered simultaneously 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 the genetically engineered cell(s) and therapeutic agents described herein achieves, for example, one, two, three, four, or more of the following effects: (i) reduction or amelioration of the severity of a disease or symptoms associated therewith; (ii) reduction in the duration of symptoms associated with a disease; (iii) protection against progression of a disease or symptoms associated therewith; (iv) regression of a disease or symptoms associated therewith; (v) protection against the onset or occurrence of symptoms associated with a disease; (vi) protection against recurrence of symptoms associated with a disease; (vii) reduction in hospitalization of a subject; (viii) reduction in length of hospitalization; (ix) increase in survival of a subject with a disease; (x) reduction in the number of symptoms associated with a disease; and (xi) enhancement, improvement, supplement, complement, or augmentation of the prophylactic or therapeutic effect 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 by a variety of routes, including but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal, and / or localized delivery to the affected tissue. Administration of therapeutic agents can be by a variety of routes, including but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal, and / or localized delivery to the affected tissue.

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

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

[0215] IX. Subject Selection Provided herein is a method for selecting a subject for treatment.In some aspects, the method for selecting a subject is used to identify the subject who is likely to show clinical response to treatment (for example, partial response (PR), complete response with incomplete hematological recovery (CRi) or complete response (CR)).In some embodiments, the identified subject is selected for treatment and / or is administered treatment.

[0216] NKG2D ligand(s) In some embodiments, the method includes selecting a subject having a level or amount of an NKG2D ligand above a reference value. For example, in some cases, a subject having a level or amount of an NKG2D ligand above the reference value is predicted to show a clinical response to the treatment. Conversely, a subject having an expression level or amount of an NKG2D ligand below the reference value is predicted to not show a clinical response to the 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 a ligand of NKG2D.

[0217] In some embodiments, comparing the level or amount of an NKG2D ligand with a reference value for the NKG2D ligand allows for the assessment, measurement, and / or determination of the probability and / or likelihood of a clinical outcome (e.g., CR, PR, or PD) following administration of a treatment and / or associated with the 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 a value for 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 transformation thereof, that is a boundary or threshold separating an amount or level of an NKG2D ligand gene or protein product, or a transformation thereof, that indicates the likelihood of a clinical response (e.g., CR or PR) and / or an increased, elevated, or high probability of a clinical response (e.g., CR or PR) after administration of treatment, from a value or measurement of an 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) after administration of treatment. In some embodiments, the reference value is a boundary value, dividing value, and / or threshold value between the amount or level of an NKG2D ligand gene or protein product at which a majority of one or more clinical responses will occur or have occurred and the amount or level of an NKG2D ligand gene or protein product at which a minority of one or more clinical responses will occur or have occurred.

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

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

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

[0221] In some embodiments, the reference value is within 25%, 20%, 15%, 10%, or 5% of the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who do not show a clinical response after receiving the treatment. In some embodiments, the reference value is within 25% of the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who do not show a clinical response after receiving the treatment. In some embodiments, the reference value is within 20% of the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who do not show a clinical response after receiving the treatment. In some embodiments, the reference value is within 15% of the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who do not show a clinical response after receiving the treatment. In some embodiments, the reference value is within 15% of the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who do not show a clinical response after receiving the treatment. In some embodiments, the reference value is within 10% of the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who have not shown a clinical response after receiving the treatment. In some embodiments, the reference value is within 5% of the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who have not shown a clinical response after receiving the treatment. In some embodiments, the reference value is the mean level or amount of NKG2D ligand in a population of treated subjects who have cancer and who have not shown a clinical response after receiving the treatment.

[0222] In some embodiments, if the amount or level of NKG2D ligand in a biological sample obtained from a subject is greater than a reference value, the subject is identified as likely to show 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 greater than 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 greater than a reference value, the subject is administered treatment.

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

[0225] In some embodiments, the biological sample is obtained from the subject within about 14 days, 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 engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 14 days prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 10 days prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 7 days prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 6 days prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 5 days prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 4 days prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 3 days prior to administering the engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about two days prior to administering the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about one 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 ligands include 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 an NKG2D ligand protein product are known in the art. Suitable methods for assessing, measuring, determining, and / or quantifying the level, amount, or concentration of one or more protein products include, but are not limited to, immunoassays, nucleic acid- or protein-based aptamer technology, high-precision liquid chromatography (HPLC), peptide sequencing, and detection by microarray adaptations of any of the foregoing (including nucleic acid, antibody, or protein-protein (i.e., non-antibody) arrays). In some embodiments, the immunoassay is or includes a method or assay that detects a protein based on an immunological reaction, for example, by detecting the binding of an antibody or antigen-binding antibody fragment to a gene product. Immunoassays include, but are not limited to, quantitative immunocytochemistry or immunohistochemistry, ELISA (including direct, indirect, sandwich, competitive, multiplex, and portable ELISA), Western blotting (including one-, two-, or more-dimensional blotting or other chromatographic means, optionally including peptide sequencing), enzyme immunoassays, radioimmunoassays, 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 an NKG2D ligand protein product is determined by the expression intensity of the NKG2D ligand protein product in cells in a biological sample that are positive for the NKG2D ligand protein product. 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 and the expression intensity of the NKG2D ligand protein product in cells in a biological sample that are positive for the NKG2D ligand protein product.

[0228] Methods for determining the amount or level of NKG2D ligand gene product are also known in the art.Suitable methods for evaluating, 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), such as reverse transcriptase (rt) PCR, droplet digital PCR, real-time and quantitative PCR (qPCR) method, Northern blot; Southern blot, such as Southern blot of reverse transcription product and derivative; array-based method, such as blotted array, microarray, or in situ synthesized array; sequencing, such as 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 NKG2D ligand gene product is determined by the number or percentage of cells that are positive for NKG2D ligand gene product in biological sample. In some embodiments, the level or amount of the NKG2D ligand gene product is determined by the expression intensity of the NKG2D ligand gene product in cells in the 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 expression intensity of the NKG2D ligand gene product in cells in the biological sample that are positive for the NKG2D ligand gene product.

[0229] Peripheral blasts In some embodiments, the method includes selecting for treatment subjects with bone marrow-restricted disease (also known as bone marrow-confined disease) and / or a certain percentage of peripheral blasts (blasts in the peripheral blood) or less. For example, in some cases, subjects with 5% or less peripheral blasts (e.g., no evidence of extramedullary disease) are predicted to show a clinical response to treatment. Conversely, subjects with more than 5% peripheral blasts are predicted to show no 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 after HCT. In some embodiments, the 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, prior to administering the population of NK cells to the subject, the method comprises determining whether the subject has a bone marrow restricted disease and / or selecting a subject with a bone marrow restricted disease for treatment. In some embodiments, prior to administering the population of NK cells to the subject, the method comprises determining whether the subject has a bone marrow restricted disease. In some embodiments, prior to administering the population of NK cells to the subject, the method comprises selecting a subject for treatment with a bone marrow restricted disease. In some embodiments, prior to administering the population of NK cells to the subject, the method comprises determining whether the subject has a bone marrow restricted disease and, if the subject has a bone marrow restricted disease, selecting the subject for treatment.

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

[0233] In some embodiments, the subject has bone marrow restricted disease (also known as bone marrow confined disease). In some embodiments, bone marrow restricted disease is defined as 5% or less peripheral blasts with no evidence of extramedullary disease. Thus, in some embodiments, the subject has 5% or less peripheral blasts. Thus, in some embodiments, the subject has 5% or less peripheral blasts with no evidence of extramedullary disease. In some embodiments, the subject has less than 5% peripheral blasts. In some embodiments, the subject has less than 5% peripheral blasts with no evidence of extramedullary disease. In some embodiments, upon administration of lymphodepleting therapy, the subject has 5% or less peripheral blasts. In some embodiments, upon administration of the first cycle of doses, the subject has less than 5% peripheral blasts. In some embodiments, upon administration of lymphodepleting therapy, the subject has less than 5% peripheral blasts. In some embodiments, upon administration of the first cycle of doses, the subject has less than 5% peripheral blasts.

[0234] X. Administration and Dosage Further provided herein are methods for treating a subject with cancer, comprising administering to the subject a composition comprising immune cells (such as NK cells and / or T cells) engineered to express the cytotoxic receptor complex 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 the use of (a) a tumor-targeting chimeric antigen receptor and / or a tumor-targeting chimeric receptor, or a cell expressing a tumor-targeting chimeric antigen receptor and / or a tumor-targeting chimeric receptor; and (b) a therapeutic agent to treat a cancer patient. Methods for using such engineered immune cells and therapeutic agents to treat cancer are also provided.

[0235] In certain embodiments, treatment of a subject with the genetically engineered cell(s) and therapeutic agents described herein achieves, for example, one, two, three, four, or more of the following effects: (i) reduction or amelioration of the severity of a disease or its associated symptoms; (ii) reduction in the duration of symptoms associated with the disease; (iii) protection against progression of the disease or its associated symptoms; (iv) regression of the disease or its associated symptoms; (v) protection against the onset or occurrence of symptoms associated with the disease; (vi) protection against recurrence of symptoms associated with the disease; (vii) reduction in the subject's hospitalization; (viii) reduction in the length of hospitalization; (ix) increase in survival of a subject with the disease; (x) reduction in the number of symptoms associated with the disease; and (xi) enhancement, improvement, complement, supplement, or augmentation of the prophylactic or treatment effect(s) of another therapy. Each of these comparisons is relative to a different therapy for the disease, including, for example, cell-based immunotherapy for the disease using cells that do not express the constructs disclosed herein. Advantageously, the non-alloreactive engineered T cells disclosed herein further enhance one or more of the above.

[0236] Administration can be by a variety of routes, including, 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 dose of immune cells, such as NK cells and / or T cells, can be readily determined for a given subject based on their body weight, the type and state of disease, and the desired aggressiveness of treatment, but may range from about 10 per kg depending on the embodiment. 5 Approximately 10 cells per kg 12 ranges from 10 cells to 10 5 ~10 7 , 10 7 ~10 10 , 10 10 ~10 12 In one embodiment, a dose-escalation regimen is used. In some embodiments, immune cells, such as NK cells and / or T cells, are administered at a dose of, for example, about 1 x 10 6 cells / kg ~ approx. 1×10 8 The dose ranges from 0.1 to 1000 cells / kg.

[0238] In some embodiments, 1×10 8 NK cells will be administered in three 28-day cycles (2 × 10 for subjects weighing less than 50 kg). 6 In some embodiments, 3×10 8 In some embodiments, 1 x 10 NK cells are administered in three 28-day cycles. 9 NK cells are administered three times in 28-day cycles.

[0239] In some embodiments, 1.5×10 8 NK cells will be administered in two 28-day cycles (3 × 10 for subjects weighing less than 50 kg). 6 In some embodiments, 4.5×10 8NK cells are administered twice in a 28-day cycle. In some embodiments, 1.5 x 10 9 NK cells are administered twice in 28-day cycles.

[0240] In some embodiments, 1.5×10 9 NK cells will be administered in three 28-day cycles (3 × 10 for subjects weighing less than 50 kg). 7 In some embodiments, 3×10 9 NK cells are administered in three 28-day cycles. In some embodiments, 1.5 x 10 10 In some embodiments, at least 4.5 x 10 NK cells are administered in three 28-day cycles. 9 NK cells are administered throughout the cycle.

[0241] In some embodiments, subjects who show at least a partial response receive at least one additional administration cycle. The administration cycles may continue, depending on the embodiment, as long as the subject shows an anti-tumor response and tolerates the engineered NK cells. In some embodiments, if the subject does not respond (e.g., there is no tumor response) and / or does not tolerate the treatment, the subject does not receive additional administration cycles. However, as discussed herein, in some embodiments, the disclosed administration regimens have limited or no adverse effects or toxicity. In some embodiments, the decision to receive / administer additional administration cycles is made at the evaluation 30 days after the start of the administration cycle (whether it is the first administration cycle or a subsequent cycle). In some embodiments, the subject is given no more than five additional cycles.

[0242] In some embodiments, one dose of NK cells in an administration cycle is administered on an outpatient basis. In some embodiments, two doses of NK cells in an administration cycle are administered on an outpatient basis. In some embodiments, each dose of NK cells in an administration cycle is administered on an outpatient basis.

[0243] In some embodiments, administration of the engineered NK cells is preceded by one or more preparatory treatments. In some embodiments, administration of the engineered NK cells is preceded by lymphodepletion therapy (also referred to as "lymphodepletion"). In some embodiments, each administration cycle is preceded by lymphodepletion. In some embodiments, a combination of chemotherapeutic agents is used for lymphodepletion. In some embodiments, a single chemotherapeutic agent is used for lymphodepletion. In some embodiments where a combination of chemotherapeutic agents is used, agents with different mechanisms of action are optionally used. In some embodiments, agents from different classes are optionally used. In some embodiments, antimetabolites are used. In some embodiments, antimetabolites inhibit and / or prevent cell replication. In some embodiments, antimetabolites are nucleotide substitutes that disrupt DNA replication and are effective in targeting rapidly dividing tumor cells.

[0244] In some embodiments, cytosine arabinoside (Ara-C) is used. In some embodiments, about 0.2 to about 10 g / m 2 A dose of Ara-C was administered, approximately 0.2 g / m 2 , about 0.5g / m 2 , about 1.0g / m 2 , about 1.5g / m 2 , about 2.0g / m 2 , about 2.5g / m 2 , about 3.0g / m 2 , about 3.5g / m 2 , about 4.0g / m 2 , about 5.0g / m 2 , about 6.0g / m 2 , about 7.0g / m 2 , about 8.0g / m 2 , about 9.0g / m 2 , about 10.0g / m 2 , about 1.5g / m 2 or any dose between the recited doses. In some embodiments, about 2 g / m 2In some embodiments, a dose 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 dose of Ara-C is given daily for about 5 days. In some embodiments, the dose can be divided, for example, given twice daily, if necessary. 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 alpha, ribonucleotide reductase, and / or DNA primase, thereby inhibiting DNA synthesis.

[0245] In some embodiments, the additional agent is fludarabine. In some embodiments, the additional agent is about 5.0 mg / m 2 ~about 200mg / m 2 Fludarabine is administered at a dose of approximately 5.0 mg / m 2 , about 10.0mg / m 2 , about 15.0mg / m 2 , about 20.0mg / m 2 , about 25.0mg / m 2 , about 30.0mg / m 2 , about 35.0mg / m 2 , about 40.0mg / m 2 , about 45.0mg / m 2 , about 50.0mg / m 2 , about 60.0mg / m 2 , about 70.0mg / m 2 , about 80.0mg / m 2 , about 90.0mg / m 2 , about 100.0mg / m 2 , about 125.0mg / m 2 , about 150.0mg / m 2 , about 175.0mg / m 2 , about 200.0mg / m 2 or any dose between the recited doses. In some embodiments, about 30 mg / m 2In some embodiments, the dose 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 dose of fludarabine is administered daily for about 3 days. In some embodiments, the dose of fludarabine is administered daily for about 5 days. In some embodiments, the dose can be divided, for example, given twice a day, if necessary.

[0246] In some embodiments, the combination of fludarabine and Ara-C is about 20 mg / m 2 ~40mg / m 2 daily dose of fludarabine and approximately 1.5 g / m 2 ~2.5g / m 2 In some embodiments, the combination of fludarabine and Ara-C is used at a daily dose of about 30 mg / m 2 daily dose of fludarabine and approximately 2 g / m 2 In some embodiments, the combination of fludarabine and Ara-C (or other agent or agents disclosed herein) is administered for about 5 days, with administration beginning about 7 days (e.g., day -7 to day -3) prior to the first administration of engineered NK cells. In some embodiments, lymphodepletion begins on day -5 prior to administration of the engineered NK cells. In some embodiments, this combination advantageously functions not only as a lymphodepleting regimen but also as an anti-cancer agent (in addition to the engineered NK cells). In some embodiments, the lymphodepleting regimen works synergistically with the engineered NK cells to provide effective reduction and / or elimination of cancer cells.

[0247] In some embodiments, the additional agent is cyclophosphamide. In some embodiments, the additional agent is about 100 mg / m 2 ~about 100mg / m 2 Fludarabine is administered at a dose of approximately 100.0 mg / m 2 , about 200mg / m 2 , about 300mg / m 2 , about 400mg / m2 , about 500mg / m 2 , about 600mg / m 2 , about 700mg / m 2 , about 800mg / m 2 , about 900mg / m 2 , about 1000mg / m 2 or any dose between the recited doses. In some embodiments, about 300 mg / m 2 In some embodiments, a dose of cyclophosphamide of 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, for example, given twice a day, if necessary.

[0248] In some embodiments, a combination of fludarabine and cyclophosphamide is used. In some embodiments, cyclophosphamide (300 mg / m 2 ) and fludarabine (30 mg / m 2 ) is administered daily for three days. In some embodiments, cyclophosphamide (500 mg / m 2 ) and fludarabine (30 mg / m 2 ) is administered daily for three days. In some embodiments, fludarabine and cyclophosphamide are each administered daily for 5, 4, and 3 days prior to administration of the engineered NK cells.

[0249] In certain embodiments, a dose of the genetically engineered cells(s) or compositions described herein is administered to a subject daily, every other day, every third day, once a week, twice a week, three times a week, or once every two weeks. In other embodiments, two, three, or four doses of the genetically engineered cells(s) or compositions described herein are administered to a subject daily, every third day, every third day, once a week, or once every two weeks. In some embodiments, a dose of the genetically engineered cells(s) or compositions described herein is administered for 2, 3, 5, 7, 14, or 21 days. In certain embodiments, a dose of the genetically engineered cells(s) or compositions described herein is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or more.

[0250] In some embodiments, subjects undergo lymphodepletion at least once prior to administration of the genetically engineered cells disclosed herein. In some embodiments, lymphodepletion occurs before one or more additional doses of the genetically engineered cells are administered. In some embodiments, lymphodepletion is followed by an administration cycle comprising at least two doses of the genetically engineered cells disclosed herein, the two doses being separated by a time interval. In some embodiments, the time interval is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more days (including intervals between times representing a price interval, e.g., 84 hours or 3.5 days, since the last administration). In some embodiments, the administration cycle itself is about 14, 21, 28, 35, 42, or more days. In some embodiments, three doses are administered approximately one week apart. In some embodiments, the two doses are administered about one week apart. In some embodiments, the subject receives a first dose on day 0 of the cycle, a second dose on day 7 of the cycle, and a third dose on day 14 of the cycle. In some such embodiments, a 28-day cycle is used, and the primary endpoint is assessed on day 28 (see, e.g., Figure 3A). In some embodiments, the subject receives a first dose on day 0 of the cycle, and a second dose on day 7 of the cycle. In some such embodiments, a 28-day cycle is used, and the primary endpoint is assessed on day 28 (see, e.g., Figure 3B).

[0251] In some embodiments, if the subject shows clinical response after the administration cycle, the subject is administered additional administration cycle as consolidation treatment.Clinical response can include complete response (CR; for example, complete remission), complete response with incomplete hematological recovery (CRi; for example, CR with residual thrombocytopenia), morphological leukemia-free state (MLFS), and partial response (PR; for example, partial remission).Methods for evaluating clinical response are known in the art (Dohner et al., Blood (2017) 129(4):424-47).

[0252] For example, in some embodiments, if a subject shows a complete response (CR) after a dosing cycle, the subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject shows a complete response with incomplete hematological recovery (CRi) after a dosing cycle, the subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject shows a morphological leukemia-free state (MLFS) after a dosing cycle, the subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject shows a partial response (PR) after a dosing cycle, the subsequent dosing cycle is administered as consolidation treatment.

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

[0254] In some embodiments, if a subject shows a clinical response from a dosing cycle but then shows disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject shows a CR from a dosing cycle but then shows disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject shows a CR from a dosing cycle but then shows disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject shows a MLFS from a dosing cycle but then shows disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject shows a PR from a dosing cycle but then shows disease progression, the subject is administered an additional cycle as retreatment.

[0255] In some embodiments, the dosing regimen comprises from 1 to 5 dosing cycles. In some embodiments, the dosing regimen consists of from 1 to 5 dosing cycles. In some embodiments, the dosing regimen consists of from 1 to 5 dosing cycles. In some embodiments, the dosing regimen consists of 1 dosing cycle. In some embodiments, the dosing regimen consists of 2 dosing cycles. In some embodiments, the dosing regimen consists of 3 dosing cycles. In some embodiments, the dosing regimen consists of 4 dosing cycles. In some embodiments, the dosing regimen consists of 5 dosing cycles. In some embodiments, the subject is administered lymphodepleting therapy prior to each dosing cycle.

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

[0257] In some embodiments, if subsequent administration cycles are required (e.g., if the subject requires further treatment), lymphodepletion is performed before the start of each administration cycle.For example, in some embodiments, the subject undergoes lymphodepletion, receives multiple doses of engineered cells according to the cycle, and is evaluated at the end of the cycle time.If deemed necessary, the subject undergoes a second lymphodepletion, and then a second administration cycle.In such embodiments where multiple administration cycles are used, the first administration cycle and the second administration cycle do not need to be the same (e.g., the first cycle can be two administrations, and the second cycle uses three administrations).Depending on the subject, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administration cycles may be administered.

[0258] Depending on the embodiment, various types of cancer can be treated. In some embodiments, the cancer is a cancer that expresses an NKG2D ligand. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer being treated is acute myeloid leukemia (AML). In some embodiments, the cancer being treated is a myelodysplastic syndrome. In some embodiments, the cancer is a solid tumor. Additional embodiments provided herein include, but are not limited to, treatment or prevention of the following non-limiting examples: 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 astrocytoma, spinal cord tumor, brain stem glioma, glioblastoma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumor, Burkitt's lymphoma, cervical cancer, colorectal cancer, Cancers include chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorders, ductal breast cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the 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] Also provided herein, in some embodiments, are nucleic acid or amino acid sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges thereof) sequence identity and / or homology to the respective nucleic acid or amino acid sequences of SEQ ID NOs: 1-38 (or a combination of two or more of SEQ ID NOs: 1-38), and exhibit, but are not limited to, (i) enhanced proliferation, (ii) enhanced activation, (iii) enhanced cytotoxic activity against cells presenting a ligand bound by NK cells bearing a receptor encoded by the nucleic acid and amino acid sequences, (iv) enhanced tumor suppression. Nucleic acid and amino acid sequences are provided that also exhibit one or more of said functions compared to each of SEQ ID NOS: 1-38 (or combinations of two or more of SEQ ID NOS: 1-38), including: (i) enhanced homing to tumor or infection sites; (ii) reduced off-target cytotoxicity; (iii) enhanced secretion of immunostimulatory cytokines and chemokines (including, but not limited to, IFNg, TNFα, IL-22, CCL3, CCL4, and CCL5); (iv) enhanced ability to stimulate additional innate and adaptive immune responses; and (viii) combinations thereof. Also provided herein in some embodiments are nucleic acid and amino acid sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges thereof) sequence identity and / or homology to the 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), respectively, and that also exhibit one or more of the functions compared to SEQ ID NOs: 1-45 (or a combination of two or more of SEQ ID NOs: 1-45), respectively.

[0260] Additionally, in some embodiments, amino acid sequences corresponding to any of the nucleic acids disclosed herein are provided, taking into account the degeneracy of the nucleic acid code. Additionally, sequences (whether nucleic acid or amino acid) that differ from the sequences explicitly disclosed herein but have functional similarity or equivalence are also contemplated to be within the scope of the present disclosure. This includes mutations, truncations, substitutions, or other types of modifications.

[0261] In some 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 the expression of the cytotoxic receptor complex.

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

[0263] Further provided herein are engineered immune cells (e.g., NK cells and / or T cells) comprising the polynucleotides, vectors, or cytotoxic receptor complexes disclosed herein. Also provided herein are compositions comprising a mixture of engineered immune cells (e.g., NK cells and / or engineered T cells), each population comprising a polynucleotide, vector, or cytotoxic receptor complex disclosed herein.

[0264] XI. Cancer Types and Combination Therapies Some embodiments of the compositions and methods described herein relate to administering immune cells comprising a tumor-targeting chimeric antigen receptor and / or a tumor-targeting chimeric receptor to a subject with cancer. Some embodiments provided herein relate to combination therapy, in which immune cells comprising a tumor-targeting chimeric antigen receptor and / or a tumor-targeting chimeric receptor are administered to a subject with cancer in combination with a therapeutic agent (an additional anti-cancer agent). Some embodiments provided herein relate to combination therapy, in which immune cells comprising a tumor-targeting chimeric antigen receptor and / or a tumor-targeting chimeric receptor are administered to a subject with a solid tumor in combination with a therapeutic agent (an additional anti-cancer agent).

[0265] Cancer type Various embodiments provided herein include treatment or prevention of the following non-limiting examples of cancer: In some embodiments, the cancer is a cancer that expresses an NKG2D ligand. 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. 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, astrocytoma, spinal cord tumor, brain stem glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumor, Burkitt's lymphoma, cervical cancer, colorectal cancer, chronic lymphocytic leukemia ( CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorder, breast ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the 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 some embodiments, acute myeloid leukemia (AML) is treated with genetically engineered immune cells in combination with a therapeutic agent (described in more detail below). In some embodiments, the cancer is AML. In some embodiments, the cancer is r / r AML. In some embodiments, the cancer has relapsed following HCT.

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

[0268] In some embodiments, solid tumors are treated with genetically engineered immune cells in combination with a therapeutic agent (described in more detail below).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0288] In some embodiments, the therapeutic agent comprises an antimitotic agent. In some embodiments, the antimitotic agent comprises vincristine, vinblastine, vinorelbine, docetaxel, paclitaxel, eribulin, isabepilone, epothilone, or any combination thereof. In some embodiments, the therapeutic agent comprises vincristine. In some embodiments, the antimitotic agent 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 antimitotic agent comprises eribulin. In some embodiments, the antimitotic agent comprises isabepilone. In some embodiments, the antimitotic agent comprises epothilone.

[0289] In some embodiments, the therapeutic agent comprises an antibiotic. In some embodiments, the antibiotic comprises bleomycin; actinomycin D; an anthracycline, optionally doxorubicin, daunorubicin, or idarubicin, mitomycin, or any combination thereof. In some embodiments, the therapeutic agent comprises bleomycin. In some embodiments, the therapeutic agent comprises actinomycin D. In some embodiments, the therapeutic agent comprises an anthracycline. In some embodiments, the therapeutic agent comprises doxorubicin. In some embodiments, the therapeutic agent comprises daunorubicin. In some embodiments, the therapeutic agent comprises idarubicin. In some embodiments, the therapeutic agent comprises 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., venetoclax). In some embodiments, the therapeutic agent comprises venetoclax. In some embodiments, the therapeutic agent comprises azacitadine. In some embodiments, the therapeutic agent comprises venetoclax and azacitadine. In some embodiments, the therapeutic agent comprises a glucocorticoid (e.g., prednisone). In some embodiments, the therapeutic agent comprises prednisone.

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

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

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

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

[0296] 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-CTLA4 antibody. In some embodiments, the monoclonal antibody comprises an anti-EGFR antibody (e.g., cetuximab). In some embodiments, the monoclonal antibody comprises an anti-HER2 / neu antibody. In some embodiments, the monoclonal antibody comprises an anti-PD1 antibody. In some embodiments, the monoclonal antibody comprises an anti-PD-L1 antibody. In some embodiments, the monoclonal antibody comprises an anti-VEGF antibody.

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

[0298] In some embodiments, the Therapeutic Agent increases ligands for 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, a dose of decitabine is administered to the subject daily for about 1 day to about 10 days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of decitabine is administered to the subject daily for 5 days prior to administration of the first dose of engineered NK cells. In some embodiments, the dose of decitabine is about 10 mg / m2 ~about 30mg / 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 some embodiments, the therapeutic agent is an NK cell engager (e.g., a molecule that binds to both an antigen expressed by a cell of the cancer and an antigen expressed by an NK cell). In some embodiments, the NK cell engager binds to an activating receptor on an NK cell and an antigen expressed by a cell of the cancer. In some embodiments, the activating receptor on the NK cell is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof. In some embodiments, the NK cell engager binds to CD16. In some embodiments, the NK cell engager binds to NKp30. In some embodiments, the NK cell engager binds to NKp46. In some embodiments, the NK cell engager binds to NKG2D.

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

[0303] Depending on the embodiment, cisplatin is administered intravenously on a weekly basis for a total of three, four, five, or six doses. In some embodiments, cisplatin is administered every three weeks for a total of three doses. In some embodiments, cisplatin is administered daily for five days. In some embodiments, a series of three to four doses constitutes one cycle, and optionally, more than one cycle is administered. In some embodiments, more than one cycle is administered.

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

[0305] In some embodiments, the administered therapeutic agent is sorafenib. In some embodiments, sorafenib is administered orally (e.g., in tablet form). In some embodiments, the dose of sorafenib is in the range of about 200 mg to about 800 mg per day. In some embodiments, the dose of sorafenib is in the range of about 200 mg to about 250 mg per day. In some embodiments, the dose of sorafenib is in the range of about 250 mg to about 300 mg per day. In some embodiments, the dose of sorafenib is in the range of about 300 mg to about 350 mg per day. In some embodiments, the dose of sorafenib is in the range of about 350 mg to about 400 mg per day. In some embodiments, the dose of sorafenib is in the range of about 400 mg to about 450 mg per day. In some embodiments, the dose of sorafenib is in the range of about 450 mg to about 500 mg per day. In some embodiments, the dose of sorafenib is in the range of about 500 mg to about 550 mg per day. In some embodiments, the dose of sorafenib is in the range of about 550 mg to about 600 mg per day. In some embodiments, the dose of sorafenib is in the range of about 600 mg to about 650 mg per day. In some embodiments, the dose of sorafenib is in the range of about 650 mg to about 700 mg per day. In some embodiments, the dose of sorafenib is in the range of about 700 mg to about 750 mg per day. In some embodiments, the dose of sorafenib is in the range of about 750 mg to about 800 mg per day. In some 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 400 mg, a subject would take a 200 mg dose (eg, a tablet) in the morning and a second 200 mg dose in the evening.

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

[0307] In some embodiments, the administered therapeutic agent is regorafenib. In some embodiments, regorafenib is administered orally (e.g., in tablet form). In some embodiments, the dose of regorafenib is in the range of about 100 mg to about 200 mg per day. In some embodiments, the dose of regorafenib is in the range of about 100 mg to about 110 mg per day. In some embodiments, the dose of regorafenib is in the range of about 110 mg to about 120 mg per day. In some embodiments, the dose of regorafenib is in the range of about 120 mg to about 130 mg per day. In some embodiments, the dose of regorafenib is in the range of about 130 mg to about 140 mg per day. In some embodiments, the dose of regorafenib is in the range of about 150 mg to about 160 mg per day. In some embodiments, the dose of regorafenib is in the range of about 160 mg to about 170 mg per day. In some embodiments, the dose of regorafenib is in the range of about 170 mg to about 180 mg per day. In some embodiments, the dose of regorafenib is in the range of about 180 mg to about 190 mg per day. In some embodiments, the dose of regorafenib is in the range of about 190 mg to about 200 mg per day. In some embodiments, the total dose is divided into two administrations per day, for example, 8 to 12 hours apart. For example, if the total daily dose is 160 mg, a subject would take an 80 mg administration (e.g., two 40 mg tablets) in the morning and a second 80 mg administration (e.g., two more 40 mg tablets) in the evening.

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

[0309] In some embodiments, the administered therapeutic agent is atezolizumab. In some embodiments, atezolizumab is administered as a 60-minute IV infusion. If 60 minutes is tolerated by the subject, a shorter 30-minute infusion is used as appropriate. Depending on the embodiment, the dose of atezolizumab varies depending on the amount of time that passes between doses. For example, in some embodiments, a 2-week lapse is allowed between doses. In such embodiments, a lower dose is used based on a higher dosing frequency. In some embodiments, a 3-week lapse is allowed between doses. In such embodiments, a medium dose is used based on a medium dosing frequency. In some embodiments, a 4-week lapse is allowed between doses. In such embodiments, a higher dose is used based on a lower dosing frequency.

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

[0311] In some embodiments employing every 3 weeks dosing, atezolizumab is administered at a dose ranging from about 1000 to about 1400 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1000 to about 1050 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1050 to about 1100 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1100 to about 1150 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1150 to about 1200 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1200 to about 1250 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1250 to about 1300 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1300 to about 1350 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1350 to about 1400 mg.

[0312] In some embodiments employing every 4 weeks dosing, atezolizumab is administered at a dose ranging from about 1500 to about 1800 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1500 to about 1550 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1550 to about 1600 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1600 to about 1640 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1640 to about 1680 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1680 to about 1700 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1700 to about 1750 mg. In some embodiments, atezolizumab is administered at a dose ranging from about 1750 to about 1800 mg.

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

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

[0315] According to some 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 some embodiments, bevacizumab is administered prior to lymphodepletion described herein (e.g., also prior to administration of engineered immune cells). In some embodiments, bevacizumab is administered between lymphodepletion and administration of engineered immune cells. In some embodiments, bevacizumab is administered simultaneously with the engineered immune cells. In some embodiments, bevacizumab is administered after administration of engineered immune cells.

[0317] In some embodiments, the administered therapeutic agent is levantinib. In some embodiments, levantinib is administered orally (e.g., in tablet form). In some embodiments, the dose of levantinib is in the range of about 1 mg to about 12 mg per day. In some embodiments, the dose of levantinib is in the range of about 1 mg to about 2 mg per day. In some embodiments, the dose of levantinib is in the range of about 2 mg to about 3 mg per day. In some embodiments, the dose of levantinib is in the range of about 3 mg to about 4 mg per day. In some embodiments, the dose of levantinib is in the range of about 4 mg to about 5 mg per day. In some embodiments, the dose of levantinib is in the range of about 5 mg to about 6 mg per day. In some embodiments, the dose of levantinib is in the range of about 6 mg to about 7 mg per day. In some embodiments, the dose of levantinib is in the range of about 7 mg to about 8 mg per day. In some embodiments, the dose of levantinib is in the range of about 8 mg to about 9 mg per day. In some embodiments, the dose of levantinib is in the range of about 9 mg to about 10 mg per day. In some embodiments, the dose of levantinib is in the range of about 10 mg to about 11 mg per day. In some embodiments, the dose of levantinib is in the range of about 11 mg to about 12 mg per day. In some embodiments, the total dose is divided into two administrations per day, for example, 8 to 12 hours apart. For example, if the total daily dose is 10 mg, the subject takes a 5 mg administration (e.g., a 5 mg tablet) in the morning and a second 5 mg administration in the evening (e.g., an additional 5 mg tablet).

[0318] In some embodiments, levantinib is administered in combination with pembrolizumab at a dose of about 150 mg to about 300 mg (in a 3-week cycle) or at a dose of about 350 mg to about 500 mg (in a 6-week cycle), as appropriate. In some embodiments, the dose of pembrolizumab ranges from about 150 mg to about 200 mg. In some embodiments, the dose of pembrolizumab ranges from about 200 mg to about 250 mg. In some embodiments, the dose of pembrolizumab ranges from about 250 mg to about 300 mg. In some embodiments, the dose of pembrolizumab ranges from about 350 mg to about 400 mg. In some embodiments, the dose of pembrolizumab ranges from about 400 mg to about 450 mg. In some embodiments, the dose of pembrolizumab ranges from about 450 mg to about 500 mg.

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

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

[0321] In some embodiments, a moderate 3-week dosing cycle is used, and nivolumab is administered in an amount ranging from about 300 mg to about 400 mg. In some embodiments, nivolumab is administered in an amount ranging from about 300 mg to about 310 mg. In some embodiments, nivolumab is administered in an amount ranging from about 310 mg to about 320 mg. In some embodiments, nivolumab is administered in an amount ranging from about 320 mg to about 330 mg. In some embodiments, nivolumab is administered in an amount ranging from about 330 mg to about 340 mg. In some embodiments, nivolumab is administered in an amount ranging from about 340 mg to about 350 mg. In some embodiments, nivolumab is administered in an amount ranging from about 350 mg to about 360 mg. In some embodiments, nivolumab is administered in an amount ranging from about 360 mg to about 370 mg. In some embodiments, nivolumab is administered in an amount ranging from about 370 mg to about 380 mg. In some embodiments, nivolumab is administered in an amount ranging from about 380 mg to about 390 mg. In some embodiments, nivolumab is administered in an amount ranging from about 390 mg to about 400 mg.

[0322] In some embodiments, nivolumab is administered in combination with another agent, such as ipilimumab. In some such embodiments, nivolumab is administered in an amount ranging from about 1 mg / kg to about 5 mg / kg. In some embodiments, nivolumab is administered in an amount ranging from about 1 mg / kg to about 2 mg / kg. In some embodiments, nivolumab is administered in an amount ranging from about 2 mg / kg to about 3 mg / kg. In some embodiments, nivolumab is administered in an amount ranging from about 3 mg / kg to about 4 mg / kg. In some embodiments, nivolumab is administered in an amount ranging from about 4 mg / kg to about 5 mg / kg. In some embodiments, ipilimumab is administered in an amount ranging from about 0.5 mg / kg to about 1.5 mg / kg. In some embodiments, ipilimumab is administered in an amount ranging from about 0.5 mg / kg to about 0.75 mg / kg. In some embodiments, ipilimumab is administered in an amount ranging from about 0.75 mg / kg to about 1.0 mg / kg. In some embodiments, ipilimumab is administered in an amount ranging from about 1.0 mg / kg to about 1.5 mg / kg. In some embodiments, ipilimumab is administered 2, 3, 4, or 5 times on the same day as nivolumab infusion, and then nivolumab is administered alone continuously at the doses listed above.

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

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

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

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

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

[0328] In some embodiments, ipilimumab is administered in combination with nivolumab, as appropriate, as discussed above.

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

[0330] In some embodiments, ramucirumab is administered in combination with an additional agent, as appropriate. For example, in some embodiments, ramucirumab is administered in combination with docetaxel (e.g., at about 50, about 75, or about 100 mg / m on day 1 of a 28-day cycle). 2For example, in some embodiments, ramucirumab is administered in combination with erlotinib (e.g., at a dose of about 100, about 150, or about 150 mg daily). For example, in some embodiments, ramucirumab is administered in combination with paclitaxel (e.g., at a dose of about 60, about 80, or about 100 mg / m on days 1, 8, and 15 of a 28-day cycle). 2 (at a dose of

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

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

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

[0334] In some embodiments, the administered therapeutic agent is doxorubicin. In some embodiments, doxorubicin is administered intravenously (e.g., by bolus injection). In some embodiments, doxorubicin is administered on day 1 of a 21-day administration cycle. In some embodiments, the dose of doxorubicin is about 20 mg / m 2to approximately 100 mg / m 2 In some embodiments, the dose of doxorubicin is about 20 mg / m 2 ~about 30mg / m 2 In some embodiments, the dose of doxorubicin is about 30 mg / m 2 ~about 40mg / m 2 In some embodiments, the dose of doxorubicin is about 40 mg / m 2 ~about 50mg / m 2 In some embodiments, the dose of doxorubicin is about 50 mg / m 2 ~about 60mg / m 2 In some embodiments, the dose of doxorubicin is about 60 mg / m 2 ~about 65mg / m 2 In some embodiments, the dose of doxorubicin is about 65 mg / m 2 ~about 70mg / m 2 In some embodiments, the dose of doxorubicin is about 70 mg / m 2 ~about 75mg / m 2 In some embodiments, the dose of doxorubicin is about 75 mg / m 2 ~about 80mg / m 2 In some embodiments, the dose of doxorubicin is about 80 mg / m 2 ~about 90mg / m 2 In some embodiments, the dose of doxorubicin is about 90 mg / m 2 ~about 100mg / m 2 The range is.

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

[0336] In some embodiments, the administered therapeutic agent is gemcitabine. In some embodiments, gemcitabine is administered intravenously (e.g., by infusion). In some embodiments, the dose of gemcitabine is about 850 mg / m 2 to approximately 1500 mg / m 2 In some embodiments, the dosage of gemcitabine is about 850 mg / m 2 ~about 900mg / m 2 In some embodiments, the dosage of gemcitabine is about 900 mg / m 2 ~about 950mg / m 2 In some embodiments, the dosage of gemcitabine is about 950 mg / m 2 ~about 1000mg / m 2 In some embodiments, the dosage of gemcitabine is about 1000 mg / m 2 ~About 1050mg / m 2 In some embodiments, the dosage of gemcitabine is about 1050 mg / m 2 ~about 1100mg / m 2 In some embodiments, the dosage of gemcitabine is about 1100 mg / m 2 ~about 1150mg / m 2 In some embodiments, the dosage of gemcitabine is about 1150 mg / m 2 ~about 1200mg / m 2 In some embodiments, the dosage of gemcitabine is about 1200 mg / m 2 ~about 1250mg / m 2 In some embodiments, the dosage of gemcitabine is about 1250 mg / m 2 ~About 1300mg / m 2 In some embodiments, the dosage of gemcitabine is about 1300 mg / m 2 ~about 1400mg / m 2 In some embodiments, the dosage of gemcitabine is about 1400 mg / m 2 ~about 1500mg / m2 The range is.

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

[0338] In some embodiments, the administered therapeutic agent is cetuximab. In some embodiments, the cetuximab is administered intravenously (e.g., by infusion). In some embodiments, the cetuximab is given in a weekly dosing format. In some embodiments, the cetuximab is given in a biweekly dosing format. For a weekly format, in some embodiments, the first dose is infused at a higher concentration (e.g., as a loading dose), followed by infusions at a lower concentration for each subsequent dose. In some embodiments, the first dose of cetuximab is about 300 mg / m 2 to approximately 500 mg / m 2 In some embodiments, the first dose of cetuximab is in the range of about 300 mg / m 2 ~about 325mg / m 2 In some embodiments, the first dose of cetuximab is in the range of about 325 mg / m 2 ~about 350mg / m 2 In some embodiments, the first dose of cetuximab is in the range of about 350 mg / m 2 ~about 375mg / m 2 In some embodiments, the first dose of cetuximab is in the range of about 375 mg / m 2 ~about 400mg / m 2 In some embodiments, the first dose of cetuximab is in the range of about 400 mg / m 2 ~about 425mg / m 2In some embodiments, the first dose of cetuximab is in the range of about 425 mg / m 2 ~about 450mg / m 2 In some embodiments, the first dose of cetuximab is in the range of about 450 mg / m 2 ~about 475mg / m 2 In some embodiments, the first dose of cetuximab is in the range of about 475 mg / m 2 ~about 500mg / m 2 In some embodiments, subsequent doses of cetuximab range from about 200 mg / m 2 to approximately 300 mg / m 2 In some embodiments, subsequent doses of cetuximab range from about 200 mg / m 2 to approximately 225 mg / m 2 In some embodiments, subsequent doses of cetuximab range from about 225 mg / m 2 to approximately 250 mg / m 2 In some embodiments, subsequent doses of cetuximab range from about 250 mg / m 2 to approximately 275 mg / m 2 In some embodiments, subsequent doses of cetuximab range from about 275 mg / m 2 to approximately 300 mg / m 2 When administered biweekly, in some embodiments, each dose of cetuximab is in the range of about 400 mg / m 2 to approximately 600 mg / m 2 In some embodiments, each dose of cetuximab is in the range of about 400 mg / m 2 to approximately 425 mg / m 2 In some embodiments, each dose of cetuximab is in the range of about 425 mg / m 2 ~about 450mg / m 2 In some embodiments, each dose of cetuximab is in the range of about 450 mg / m 2 ~about 475mg / m 2 In some embodiments, each dose of cetuximab is in the range of about 475 mg / m 2 ~about 500mg / m2 In some embodiments, each dose of cetuximab is in the range of about 500 mg / m 2 ~about 525mg / m 2 In some embodiments, each dose of cetuximab is in the range of about 525 mg / m 2 ~about 550mg / m 2 In some embodiments, each dose of cetuximab is in the range of about 550 mg / m 2 ~about 575mg / m 2 In some embodiments, each dose of cetuximab is in the range of about 575 mg / m 2 ~about 600mg / m 2 The range is.

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

[0340] In some embodiments, the administered therapeutic agent is irinotecan (e.g., irinotecan hydrochloride). In some embodiments, the irinotecan is administered intravenously (e.g., by infusion). In some embodiments, the dose of irinotecan is about 10 mg / m 2 to approximately 1000 mg / m 2 In some embodiments, the dose of irinotecan is about 20 mg / m 2 to approximately 500 mg / m 2 In some embodiments, the dose of irinotecan is about 50 mg / m 2 to approximately 125 mg / m 2 In some embodiments, the dose of irinotecan is about 20 mg / m 2 In some embodiments, the dose of irinotecan is about 20 mg / m 2In some embodiments, the dose of irinotecan is about 30 mg / m 2 In some embodiments, the dose of irinotecan is about 40 mg / m 2 In some embodiments, the dose of irinotecan is about 50 mg / m 2 In some embodiments, the dose of irinotecan is about 60 mg / m 2 In some embodiments, the dose of irinotecan is about 70 mg / m 2 In some embodiments, the dose of irinotecan is about 75 mg / m 2 In some embodiments, the dose of irinotecan is about 80 mg / m 2 In some embodiments, the dose of irinotecan is about 90 mg / m 2 In some embodiments, the dose of irinotecan is about 100 mg / m 2 In some embodiments, the dose of irinotecan is about 120 mg / m 2 In some embodiments, the dose of irinotecan is about 125 mg / m 2 In some embodiments, the dose of irinotecan is about 150 mg / m 2 In some embodiments, the dose of irinotecan is about 180 mg / m. In some embodiments, the dose of irinotecan is about 200 mg / m. 2 In some embodiments, the dose of irinotecan is about 240 mg / m 2 In some embodiments, the dose of irinotecan is about 250 mg / m 2 In some embodiments, the dose of irinotecan is about 300 mg / m 2 In some embodiments, the dose of irinotecan is about 320 mg / m 2 In some embodiments, the dose of irinotecan is about 350 mg / m 2 In some embodiments, the dose of irinotecan is about 360 mg / m 2 In some embodiments, the dose of irinotecan is about 400 mg / m 2In some embodiments, the dose of irinotecan is about 450 mg / m 2 In some embodiments, the dose of irinotecan is about 480 mg / m 2 In some embodiments, the dose of irinotecan is about 500 mg / m 2 In some embodiments, the dose of irinotecan is about 600 mg / m 2 In some embodiments, the dose of irinotecan is about 700 mg / m 2 In some embodiments, the dose of irinotecan is about 750 mg / m 2 In some embodiments, the dose of irinotecan is about 800 mg / m 2 In some embodiments, the dose of irinotecan is about 900 mg / m 2 In some embodiments, the dose of irinotecan is about 1000 mg / m 2 is.

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

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

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

[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, giving a three-week cycle. In some embodiments, the dose is divided between two administrations. For example, in some embodiments, 2500 mg / m 2 daily dose of 1250 mg / m 2 In some embodiments, the dose is 1250 mg / m divided into two doses. 2 of capecitabine is administered orally twice daily (e.g., morning and evening) for a total daily dose of 2500 mg / m 2 is administered for two weeks, followed by a one-week rest period, in three-week cycles. In some embodiments, the subject is administered a total of four cycles. In some embodiments, the subject is administered a total of eight cycles.

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

[0346] In some embodiments, the administered therapeutic agent is vinorelbine. In some embodiments, the vinorelbine is administered intravenously (e.g., by infusion). In some embodiments, the dose of vinorelbine is about 5 mg / m 2 to approximately 50 mg / m 2 In some embodiments, the dose of vinorelbine is about 10 mg / m 2 to approximately 45 mg / m 2 In some embodiments, the dose of vinorelbine is about 15 mg / m 2 to approximately 40 mg / m 2 In some embodiments, the dose of vinorelbine is about 20 mg / m 2 to approximately 35 mg / m 2 In some embodiments, the dose of vinorelbine is about 25 mg / m 2 to approximately 30 mg / m 2 In some embodiments, the dose of vinorelbine is about 6.25 mg / m 2 In some embodiments, the dose of vinorelbine is about 7.5 mg / m 2 In some embodiments, the dose of vinorelbine is about 10 mg / m 2 In some embodiments, the dose of vinorelbine is about 12.5 mg / m 2 In some embodiments, the dose of vinorelbine is about 15 mg / m 2 In some embodiments, the dose of vinorelbine is about 17.5 mg / m 2 In some embodiments, the dose of vinorelbine is about 18.75 mg / m 2In some embodiments, the dose of vinorelbine is about 20 mg / m 2 In some embodiments, the dose of vinorelbine is about 22.5 mg / m 2 In some embodiments, the dose of vinorelbine is about 25 mg / m 2 In some embodiments, the dose of vinorelbine is about 27.5 mg / m 2 In some embodiments, the dose of vinorelbine is about 30 mg / m 2 is.

[0347] In some embodiments, a dose of vinorelbine is administered once a 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 a week for 4 weeks (e.g., a 28-day cycle).

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

[0349] It is understood that various combinations of additional anti-cancer agents may also be used, for example, cetuximab and doxorubicin may be used with the engineered immune cells provided herein.

[0350] XII. Additional Cancer Targets 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 (BCMA); CD38; DLL3; G protein-coupled receptor class C group 5, member D (GPRC5D); epidermal growth factor receptor (EGFR); CD138; prostate-specific membrane antigen (P). SMA; Fms-like tyrosine kinase 3 (FLT3); KREMEN2 (kringle-containing transmembrane protein 2), ALPPL2, claudin 4, claudin 6, C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 [aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer]; Tn 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 in acute leukemia or lymphoma but not in hematopoietic progenitor cells; glycosylated CD43 epitopes expressed in non-hematopoietic cancers; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCA M); 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); protease serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta);Stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha (FRa or FR1); folate receptor beta (FRb); receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase cytochrome P450 IX (CAIX); proteasome (prosome, macropenetrate) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3 [aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer]; transglutaminase TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); globoH glycoceramide hexasaccharide moiety (GloboH); mammary differentiation antigen (N Y-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, K9 locus (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ES0-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1);ETS translocation mutant gene 6 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 A-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase; reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG [transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene]; N-acetylglucosaminyltransferase 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 (BORIS or Brother of the Regulator of Imprinted Proteins) Sites), squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (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); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR alpha 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialyl Lewis antigen); fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, IL1 1Ra, IL13Ra2, CD179b-IGL11, TCR gamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Timl- / HVCR1, CSF2RA (GM-CSFR alpha), TGF beta R2, Lewis ag, TCR-beta 1 chain, TCR-beta 2 chain, TCR-gamma chain, TCR-delta chain, FITC, leutenizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV Antigens recognized by antibodies to K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), desmoglein 3 autoantibody (Dsg3), desmoglein 1 autoantibody (Dsg1), 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 8.2 (CLD18A2 or CLDN18.2), P-glycoprotein, STEAP1, Livl, nectin-4, Cripto, gpA33, BST1 / CD157, small-conductance chloride channel, and TNT are included.

[0351] definition The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides include receptors and other polypeptides, such as linkers or peptides, provided they contain amino acid residues, including natural and / or non-natural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, polypeptides may contain modifications relative to the original or native sequence, so long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations of the host producing the protein or errors due to PCR amplification.

[0352] As used herein, a "subject" refers to a mammal, such as a human or other animal, typically a human. In some embodiments, the subject, e.g., a patient, to whom an agent(s), cell, cell population, or composition is administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and may be of any appropriate age, including infant, child, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

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

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

[0355] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at the dosage and for the duration of administration necessary, to achieve a desired therapeutic result, such as treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effects of the treatment. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the cell population administered. In some embodiments, the provided methods involve administering cells and / or compositions in an effective amount, e.g., a therapeutically effective amount.

[0356] The term "about" as used herein refers to a normal error range for each value, which is readily known to one of ordinary skill in the art. As used herein, reference to "about" a value or parameter includes (and describes) embodiments that are directed to 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 a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. Terms such as "up to," "at least," "greater than," "less than," "between," and the like include the recited numerical values. Also, numerical values ​​preceded by terms such as "about" or "approximately" include the recited numerical value. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, as well as any other stated or intervening value within that range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also encompassed within the claimed subject matter. This applies regardless of the scope.

[0359] Furthermore, when a sequence is disclosed as "comprising" a nucleotide or amino acid sequence, such reference is also intended to include that the sequence "comprises," "consists," or "consists essentially of" the recited sequence, unless otherwise specified.

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

[0361] Unless otherwise defined, all terms of art, notation, and other technical and scientific or technical terms used herein are intended to have the same meaning as commonly understood to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from the commonly understood meaning.

[0362] All publications, including patent documents, scientific articles, and databases, referenced 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 a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition 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.

[0364] Non-limiting embodiments Among the embodiments provided herein are the following: 1. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: The cancer is a solid tumor, and each dose of engineered NK cells is approximately 1 × 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 administering a therapeutic agent containing genetically engineered NK cells; (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; Prior to administering the first dose of genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy. A method comprising: 2. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1 × 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 administering a therapeutic agent containing genetically engineered NK cells; (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; Prior to administering the first dose of genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy. A method comprising: 3. The method of embodiment 1 or embodiment 2, wherein the therapeutic agent increases expression of an NKG2D ligand in the subject. 4. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: The cancer is a solid tumor, and each dose of engineered NK cells is approximately 1 × 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 administering a therapeutic agent containing genetically engineered NK cells; (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject; A method comprising: 5. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1 × 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 administering a therapeutic agent containing genetically engineered NK cells; (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject; A method comprising: 6. A method for treating cancer in a subject, comprising: administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 days to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1 × 10 8 genetically engineered NK cells ~ approximately 1 x 10 10 comprising genetically engineered NK cells, The subject has 5% or less peripheral blasts. 7. The method of embodiment 6, comprising 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 an NKG2D ligand in the subject, and any combination thereof. 8. The method of any one of embodiments 1-5, wherein the subject has 5% or less peripheral blasts. 9. The method of any one of embodiments 1-8, wherein the subject has less than 5% peripheral blasts. 10. The method of any one of embodiments 6-9, wherein prior to administering the genetically engineered NK cells to the subject, the percentage of peripheral blasts in the subject is determined, and if the subject has 5% or less peripheral blasts, the subject is selected for treatment. 11. Prior to administering the genetically engineered NK cells to the subject, the method comprises: (a) determining the percentage of peripheral blasts in a subject; (b) selecting the subject for treatment if the subject has 5% or less peripheral blasts; 11. The method of any one of embodiments 6 to 10, comprising: 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 the group consisting of 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 prior to administering the first dose of genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy. 15. The first, second, and third doses of genetically engineered NK cells were administered at approximately 1 x 10 9 genetically engineered NK cells or approximately 1.5 x 10 9 15. The method of any one of embodiments 1-14, comprising genetically engineered NK cells. 16. The method of any one of embodiments 1-15, wherein the administration cycle is from about 14 days to about 35 days. 17. The method of any one of embodiments 1-16, wherein the administration cycle is about 21 days. 18. The method of any one of embodiments 1-16, wherein the administration cycle is about 28 days. 19. The method of any one of embodiments 1-18, comprising administering an additional administration cycle. 20. The method of any one of embodiments 1-19, wherein if the subject exhibits a clinical response, optionally a complete response (CR), after a dosing cycle, the method comprises administering an additional dosing cycle. 21. The method of any one of embodiments 1-20, wherein if the subject shows a clinical response after an administration cycle and then shows disease progression, the method comprises administering an additional administration cycle. 22. The method of any one of embodiments 1-21, comprising administration of 1 to 5 administration cycles. 23. The method of any one of embodiments 19-22, wherein the subject is administered lymphodepleting therapy before each cycle. 24. The method of any one of embodiments 1-18, wherein the second dose of genetically engineered cells is administered to the subject about 7 days after administration of the first dose of genetically engineered cells. 25. The method of any one of embodiments 1-24, wherein the third dose of genetically engineered cells is administered to the subject about 7 days after administration of the second dose of genetically engineered cells. 26. A method for treating cancer in a subject, 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 group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells; and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1.5 × 10 9 Contains genetically engineered NK cells; The subject has 5% or less peripheral blasts and no evidence of extramedullary disease. 27. The method of any one of embodiments 1-25, wherein the administration of the therapeutic agent is prior to administering the first dose of engineered NK cells. 28. The method of any one of embodiments 1-26, wherein the administration of the therapeutic agent is concurrent with the administration of a dose of genetically engineered NK cells, optionally concurrent with the administration of the first dose of genetically engineered NK cells. 29. The method of any one of embodiments 1-26, wherein the administration of the therapeutic agent occurs after administration of a dose of genetically engineered NK cells, and optionally after administration of a third dose of genetically engineered NK cells. 30. The method of any one of embodiments 1-3 and 12-29, wherein the lymphodepletion therapy comprises administration of 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. Each dose of Flu is approximately 10 mg / m 2 ~about 60mg / m 2 32. The method of embodiment 31, comprising: 33. The method of any one of embodiments 1-3 and 12-32, wherein the lymphodepletion therapy comprises administration of 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 is each given 5 days before the start of an administration cycle; a second dose of Flu and Cy is each given 4 days before the start of an administration cycle; and a third dose of Flu and Cy is each given 3 days before the start of an administration cycle. Each dose of 36.Cy is approximately 200 mg / m 2 ~about 600mg / m 2 36. The method of embodiment 34 or embodiment 35, comprising: 37. Each dose of Flu is approximately 30 mg / m 2 and each dose of Cy is about 300 mg / m 2 37. The method of any one of embodiments 34 to 36, comprising: 38. Each dose of Flu is approximately 30 mg / m 2 and each dose of Cy is about 500 mg / m 2 37. The method of any one of embodiments 34 to 36, comprising: 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 lymphodepletion therapy comprises administration of Flu and cytosine arabinoside (Ara-C). 41. The method of any one of embodiments 1-3, 12-32, 39, and 40, wherein the lymphodepletion therapy comprises administering five doses of Ara-C. 42. The method of embodiment 41, wherein a first dose of Flu and Ara-C is each given 7 days before the start of an administration cycle; a second dose of Flu and Ara-C is each given 6 days before the start of an administration cycle; a third dose of Flu and Ara-C is each given 5 days before the start of an administration cycle; a fourth dose of Flu and Ara-C is each given 3 days before the start of an administration cycle; and a fifth dose of Flu and Ara-C is each given 4 days before the start of an administration cycle. 43. Each dose of Ara-C is approximately 1 g / m 2 ~approx. 4g / m 2 43. The method of embodiment 41 or embodiment 42, comprising: 44. Each dose of Flu is approximately 30 mg / m 2 Each dose of Ara-C is about 2 g / m 2 44. The method of any one of embodiments 41 to 43, comprising: 45. The method of any one of embodiments 1-44, wherein the therapeutic agent is a chemotherapeutic agent. 46. ​​The method of any one of embodiments 1-45, wherein the therapeutic agent is decitabine. 47. The method of embodiment 46, wherein a dose of decitabine is administered to the subject daily for 5 days prior to the start of an administration cycle. 48. A dose of decitabine is approximately 20 mg / m 248. The method of embodiment 47, comprising: 49. A method for treating cancer in a subject, 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 group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells; and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1.5 × 10 9 Contains genetically engineered NK cells; Subjects have 5% or less peripheral blasts; Subjects were randomly assigned to receive the first dose of engineered NK cells at a dose of approximately 20 mg / m for 5 days prior to receiving the first dose. 2 wherein decitabine is administered daily. 50. The method of any one of embodiments 2, 3, 5-49, wherein the cancer is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), and optionally the cancer is relapsed / refractory (r / r) AML or very high-risk MDS. 51. The method of any one of embodiments 1-3 and 11-45, wherein the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly(ADP-ribose) polymerase (PARP), or any combination thereof. 52. The method of any one of embodiments 1-3 and 11-51, 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. 53. The method of any one of embodiments 1-3 and 11-52, wherein the NK cell engager binds to an activating receptor on the NK cell and an antigen expressed by cells of the cancer, and optionally the activating receptor is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof. 54. The method of any one of embodiments 1-49 and 51-53, wherein the cancer is carcinoma, sarcoma, or melanoma. 55. The method of any one of embodiments 1-49 and 51-54, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colon 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. 56. The method of any one of embodiments 1-49 and 51-55, wherein the cancer comprises liver cancer or colon cancer. 57. The method of any one of embodiments 1-45 and 51-56, wherein the therapeutic agent comprises capecitabine, cisplatin, doxorubicin, regorafenib, or sorafenib. 58. The method of any one of embodiments 1-49 and 51-55, wherein the cancer comprises bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, or uterine cancer. 59. The method of any one of embodiments 1-45, 51-55, and 58, wherein the therapeutic agent comprises doxorubicin or gemcitabine. 60. The method of any one of embodiments 1-49 and 51-55, wherein the cancer comprises esophageal cancer, head and neck cancer, or lung cancer. 61. The method of any one of embodiments 1-45, 51-55, and 60, wherein the therapeutic agent comprises gemcitabine, irinotecan, or vinorelbine. 62. The method of any one of embodiments 1-49 and 51-55, wherein the cancer comprises melanoma. 63. The method of any one of embodiments 1-44, 51-55, and 62, 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. 64. The method of any one of embodiments 1-63, wherein the cancer is a relapsed / refractory (R / R) cancer. 65. The method of any one of embodiments 1-64, wherein the subject has been treated with one prior line of therapy. 66. The method of any one of embodiments 1-65, wherein the subject has been treated with two prior lines of therapy. 67. The method of any one of embodiments 1-66, wherein the subject has been treated with three ...

Claims

1. 1. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: The cancer is a solid tumor, and each dose of engineered NK cells is approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ administering a therapeutic agent containing genetically engineered NK cells; (b) administering a therapeutic agent that increases expression of an NKG2D ligand in the subject; Prior to administering the first dose of genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy. A method comprising:

2. 10. 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. 10. The method of claim 1, wherein the therapeutic agent is a chemotherapeutic agent.

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

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

6. 10. The method of 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 5% or less peripheral blasts, the subject is selected for treatment.

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

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

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

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

11. 10. The method of any one of claims 1 to 9, wherein the administration of the therapeutic agent follows the administration of a dose of the genetically engineered NK cells, and optionally follows the administration of a third dose of the genetically engineered NK cells.

12. 10. The method of claim 1, wherein the lymphodepletion therapy comprises administration of fludarabine (Flu).

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

14. Each dose of Flu is about 10 mg / m ２ ~about 60mg / m ２ 14. The method of claim 13, comprising:

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

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

17. 17. The method of claim 16, wherein a first dose of Flu and Cy is each given 5 days before the start of an administration cycle; a second dose of Flu and Cy is each given 4 days before the start of an administration cycle; and a third dose of Flu and Cy is each given 3 days before the start of an administration cycle.

18. Each dose of Cy was approximately 200 mg / m ２ ~Approx. 600mg / m ２ 18. The method of claim 16 or 17, comprising:

19. Each dose of Flu is about 30 mg / m ２ wherein each dose of Cy is about 300 mg / m ２ 18. The method of claim 16 or 17, comprising:

20. Each dose of Flu is about 30 mg / m ２ wherein each dose of Cy is about 500 mg / m ２ 18. The method of claim 16 or 17, comprising:

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

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

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

24. 24. The method of claim 23, wherein a first dose of Flu and Ara-C is each given 7 days prior to the start of an administration cycle; a second dose of Flu and Ara-C is each given 6 days prior to the start of an administration cycle; a third dose of Flu and Ara-C is each given 5 days prior to the start of an administration cycle; a fourth dose of Flu and Ara-C is each given 3 days prior to the start of an administration cycle; and a fifth dose of Flu and Ara-C is each given 4 days prior to the start of an administration cycle.

25. Each dose of Ara-C is approximately 1 g / m ２ ~ about 4 g / m ２ 25. The method of claim 23 or 24, comprising:

26. Each dose of Flu is about 30 mg / m ２ and each dose of Ara-C is about 2 g / m ２ 25. The method of claim 23 or 24, comprising:

27. 1. 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 of natural killer group 2D (NKG2D), the method comprising: (a) assessing the level or amount of an NKG2D ligand in a biological sample from a subject with 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 NKG2D ligand exceeds 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; (c) administering the genetically engineered NK cells to the subject; Including, The method wherein the biological sample is obtained from the subject prior to administration of the genetically engineered NK cells.

28. Genetically engineered NK cells (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. administered to a subject in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ 28. The method of claim 27, comprising genetically engineered NK cells.

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

30. 29. The method of claim 27 or 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. 29. The method of claim 27 or 28, wherein the NKG2D ligand comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof.

32. 29. The method of claim 27 or 28, wherein the NKG2D ligands include MICA and MICB.

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

34. The first, second, and third doses of genetically engineered NK cells were each approximately 1 x 10 ９ genetically engineered NK cells or approximately 1.5 x 10 ９ 34. The method of any one of claims 1 to 26 or 28 to 33, comprising genetically engineered NK cells.

35. 35. The method of any one of claims 1-26 or 28-34, wherein the administration cycle is from about 14 days to about 35 days.

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

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

38. 38. The method of any one of claims 1 to 26 or 28 to 37, comprising administering an additional cycle of administration.

39. 39. The method of any one of claims 1-26 or 28-38, wherein if the subject exhibits a clinical response after a dosing cycle, as appropriate, a partial response (PR), a complete response with incomplete hematologic recovery (CRi), or a complete response (CR), the method comprises administering an additional dosing cycle as consolidation treatment.

40. 39. The method of any one of claims 1-26 or 28-38, wherein if the subject shows a clinical response after a dosing cycle and then shows disease progression, the method comprises administering an additional dosing cycle as re-treatment.

41. 41. The method of any one of claims 1 to 26 or 28 to 40, comprising the administration of 1 to 5 dosing cycles.

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

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

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

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

47. 27. The method of any one of claims 1 to 26, wherein 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.

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

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

50. 49. The method of any one of claims 1 to 44 or 46 to 48, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colon 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. 49. The method of any one of claims 1 to 44 or 46 to 48, wherein the cancer comprises liver cancer or colon cancer.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

42.

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

68. The method of any one of claims 65 to 67, wherein the intracellular signaling region comprises a costimulatory domain and CD3 zeta.

69. 69. The method of claim 68, wherein the costimulatory domain comprises an OX40 domain.

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

39.

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

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

40.

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

74. 74. The method of any one of claims 1 to 73, wherein the population of engineered NK cells is derived from a subject not afflicted with cancer.

75. 75. The method of any one of claims 1 to 74, wherein a dose of the genetically engineered NK cells is administered to the subject on an exogenous basis, and where appropriate, each dose of the genetically engineered NK cells is administered to the subject on an exogenous basis.

76. 1. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D) to treat cancer in a subject, comprising: (a) Genetically engineered NK cells (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. administered to a subject with cancer in an administration cycle comprising: The cancer is a solid tumor, and each dose of engineered NK cells is approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ comprising genetically engineered NK cells; (b) 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; Use wherein prior to administering the first dose of genetically engineered NK cells to the subject, the subject is subjected to lymphodepletion therapy.

77. 1. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D) to treat cancer in a subject, comprising: (a) Genetically engineered NK cells (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. administered to a subject with cancer in an administration cycle comprising: Each dose of genetically engineered NK cells was approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ comprising genetically engineered NK cells; (b) 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; Use wherein prior to administering the first dose of genetically engineered NK cells to the subject, the subject is subjected to lymphodepletion therapy.

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

79. 1. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D) to treat cancer in a subject, comprising: (a) Genetically engineered NK cells (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. administered to a subject with cancer in an administration cycle comprising: The cancer is a solid tumor, and each dose of engineered NK cells is approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ comprising genetically engineered NK cells; (b) The use, wherein the subject is administered a therapeutic agent that increases expression of an NKG2D ligand in the subject.

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

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

82. 82. The use of claim 81, wherein the population of genetically engineered NK cells is for use in combination 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 a subject, and any combination thereof.

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

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

85. 85. The use of any one of claims 76 to 84, wherein the subject has no evidence of extramedullary disease.

86. 86. The use of any one of claims 79 to 85, 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.

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

88. 1. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D) to treat cancer in a subject, wherein the genetically engineered NK cells are: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells; and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1.5 x 10 ９ comprising genetically engineered NK cells; Subjects have 5% or less peripheral blasts and no evidence of extramedullary disease.

89. 1. Use of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D) to treat cancer in a subject, wherein the genetically engineered NK cells are: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells; and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1.5 x 10 ９ comprising genetically engineered NK cells; Subjects were administered a dose of approximately 20 mg / m for 5 days prior to receiving the first dose of engineered NK cells. ２ Decitabine, which is administered daily, is used.

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

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

92. 92. The use of any one of claims 76 to 91, wherein if the subject exhibits a clinical response, as appropriate, a partial response (PR), a complete response with incomplete hematologic recovery (CRi), or a complete response (CR), after a dosing cycle, additional dosing cycles are administered to the subject as consolidation treatment.

93. 92. The use of any one of claims 76 to 91, wherein if the subject shows a clinical response after a dosing cycle and then shows disease progression, the subject is administered an additional dosing cycle as re-treatment.

94. 94. The use of any one of claims 76 to 93, wherein the subject is administered 1 to 5 administration cycles.

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

96. 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. 97. The use of any one of claims 76 to 96, wherein a dose of the genetically engineered NK cells is administered to the subject on an exogenous basis, where appropriate each dose of the genetically engineered NK cells is administered to the subject on an exogenous basis.

98. 1. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: The cancer is a solid tumor, and each dose of engineered NK cells is approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ administering a therapeutic agent containing genetically engineered NK cells; (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; Prior to administering the first dose of genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy. A method comprising:

99. 1. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ administering a therapeutic agent containing genetically engineered NK cells; (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; Prior to administering the first dose of genetically engineered NK cells to the subject, the subject is administered lymphodepletion therapy. A method comprising:

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

101. 1. A method for treating cancer in a subject, comprising: (a) administering to a subject having cancer a population of natural killer (NK) cells that have been genetically engineered to express a chimeric receptor that binds to a ligand of natural killer group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: The cancer is a solid tumor, and each dose of engineered NK cells is approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ administering a therapeutic agent containing genetically engineered NK cells; (b) administering to the subject a therapeutic agent that increases expression of an NKG2D ligand in the subject; A method comprising:

102. 1. A method for treating cancer in a subject, 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 group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 5 to about 10 days after administration of the second dose of genetically engineered NK cells. and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1 x 10 ８ genetically engineered NK cells to approximately 1 x 10 １０ 100% of said genetically engineered NK cells, and wherein the subject has 5% or less peripheral blasts.

103. 1. A method for treating cancer in a subject, 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 group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells; and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1.5 x 10 ９ comprising genetically engineered NK cells; The subject has less than 5% peripheral blasts and no evidence of extramedullary disease.

104. 1. A method for treating cancer in a subject, 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 group 2D (NKG2D), wherein the genetically engineered NK cells: (i) a first dose of genetically engineered NK cells; (ii) a second dose of genetically engineered NK cells administered to the subject about 7 days after administration of the first dose of genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject about 7 days after administration of the second dose of genetically engineered NK cells; and administered in a dosing cycle comprising: Each dose of genetically engineered NK cells was approximately 1.5 x 10 ９ comprising genetically engineered NK cells; The subject has peripheral blasts less than or equal to 5%; The subject receives a dose of approximately 20 mg / m before receiving the first dose of genetically engineered NK cells. ２ wherein decitabine is administered daily for five days.

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