WO2025006631A1

WO2025006631A1 - Dosing regimens for treatment of cancer

Info

Publication number: WO2025006631A1
Application number: PCT/US2024/035654
Authority: WO
Inventors: David Richard SHOOK; Nishi KOTHARI
Original assignee: Nkarta Inc
Current assignee: Nkarta Inc
Priority date: 2023-06-27
Filing date: 2024-06-26
Publication date: 2025-01-02
Anticipated expiration: 2025-12-27

Abstract

Several embodiments of the methods and compositions disclosed herein relate to immune cells that are engineered to express cytotoxic chimeric receptors and various dosing regimens for administering such cells. In several embodiments, the immune cells express a chimeric receptor that targets ligands of NKG2D on tumor cells. In several embodiments, the cancer is a blood cancer, for example, acute myeloid leukemia (e.g., relapsed/refractory acute myeloid leukemia) or myelodysplastic syndrome. In several embodiments, the tumor is a solid tumor.

Description

DOSING REGIMENS FOR TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent Application Nos. 63/523,604, filed June 27, 2023; 63/535,240 filed August 29, 2023, the entire contents of each of which are incorporated by reference herein.

FIELD

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

BACKGROUND

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

INCORPORATION BY REFERENCE OF MATERIAL IN SEQUENCE LISTING FILE

[0004] This application incorporates by reference the material contained in the Sequence Listing XML file being submitted concurrently herewith: File name: NKT.109WO_ST26.xml; created on June 18, 2024 and is 46,527 bytes in size.

SUMMARY

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

[0006] Provided herein is a method of treating a cancer in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle. In some embodiments, the dosing cycle comprises a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of the genetically engineered NK cells. In some embodiments, all three doses are administered to the subject within between about 4 days and about 10 days. In some embodiments, the second dose is administered to the subject between 2-4 days after administration of the first dose to the subject; the third dose is administered to the subject between 2-4 days after administration of the second dose to the subject.

[0007] In some embodiments, the dosing cycle comprises: (I) a first dose of the genetically engineered NK cells; (ii) a second dose of the genetically engineered NK cells administered to the subject 2-4 days after administration of the first dose of the genetically engineered NK cells; and (Hi) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 1 .5 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 1 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 1 .5 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 2 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 2.5 x 10⁹ genetically engineered NK cells. In some embodiments, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 10 days of conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 4-10 days. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 4 days. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 5 days. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 6 days. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 7 days. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 8 days. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 9 days. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 10 days. [0008] Also provided herein is a method of treating a cancer in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 1 .5 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells. In some embodiments, prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 7-10 days of conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 10 days of conclusion of administration of the lymphodepleting therapy.

[0009] In some embodiments, each dose is separated by between about 24 hours and about 72 hours. In some embodiments, each dose is separated by at least about 24 hours. In some embodiments, each dose is separated by about 24 hours. In some embodiments, each dose is separated by at least about 48 hours. In some embodiments, each dose is separated by about 48 hours. In some embodiments, each dose is separated by at least about 72 hours. In some embodiments, each dose is separated by about 72 hours.

[0010] In some embodiments, the first dose is administered on about Day 0 of the dosing cycle. In some embodiments, the second dose is administered on about Day 2 of the dosing cycle. In some embodiments, the second dose is administered on about Day 3 of the dosing cycle. In some embodiments, the second dose is administered on about Day 4 of the dosing cycle. In some embodiments, the third dose is administered on about Day 4 of the dosing cycle. In some embodiments, the third dose is administered on about Day 5 of the dosing cycle. In some embodiments, the third dose is administered on about Day 6 of the dosing cycle. In some embodiments, the third dose is administered on about Day 7 of the dosing cycle. In some embodiments, the third dose is administered on about Day 8 of the dosing cycle.

[0011] In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 2 of the dosing cycle, and the third dose is administered on about Day 4 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 2 of the dosing cycle, and the third dose is administered on about Day 5 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 2 of the dosing cycle, and the third dose is administered on about Day 6 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 3 of the dosing cycle, and the third dose is administered on about Day 5 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 3 of the dosing cycle, and the third dose is administered on about Day 6 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 3 of the dosing cycle, and the third dose is administered on about Day 7 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 4 of the dosing cycle, and the third dose is administered on about Day 6 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 4 of the dosing cycle, and the third dose is administered on about Day 7 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 4 of the dosing cycle, and the third dose is administered on about Day 8 of the dosing cycle.

[0012] In some embodiments, the first dose of genetically engineered NK cells is administered to the subject about 2 to 5 days after administration of the lymphodepleting therapy has concluded. In some embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 2 days after administration of the lymphodepleting therapy has concluded. In some embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 3 days after administration of the lymphodepleting therapy has concluded. In some embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 4 days after administration of the lymphodepleting therapy has concluded. In some embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 5 days after administration of the lymphodepleting therapy has concluded. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 7-10 days of conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 10 days of conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

[0013] In some embodiments, the second dose of the genetically engineered NK cells is administered to the subject 2-4 days after administration of the first dose of the genetically engineered NK cells; and the third dose of the genetically engineered NK cells is administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells.

[0014] In some embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 3 days after administration of the first dose of the genetically engineered cells. In some embodiments, the third dose of genetically engineered NK cells is administered to the subject about 4 days after administration of the second dose of the genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises between about 1 x 10⁹ and about 5 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 1 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 1 .5 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises between about 2 x 10⁹ and about 5x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 2 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 2.5 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises about 3 x 10⁹ genetically engineered NK cells. In some embodiments, all three doses of the genetically engineered NK cells are administered to the subject within about 10 days of conclusion of administration of the lymphodepleting therapy.

[0015] In some embodiments, the lymphodepleting therapy comprises administration of fludarabine (Flu). In some embodiments, each dose of Flu comprises between about 10 mg/m2 and about 40 mg/m2. In some embodiments, the lymphodepleting therapy comprises administration of five doses of Flu. In some embodiments, the lymphodepleting therapy comprises administration cytosine arabinoside (Ara-C). In some embodiments, each dose of Ara-C comprises between about 1 g/m2 and about 4 g/m2. In some embodiments, the lymphodepleting therapy comprises administration of five doses of Ara-C. In some embodiments, the first doses of Flu and Ara-C are each given 7 days prior to initiation of the dosing cycle; the second doses of Flu and Ara-C are each given 6 days prior to initiation of the dosing cycle; the third doses of Flu and Ara-C are each given 5 days prior to initiation of the dosing cycle; the fourth doses of Flu and Ara-C are each given 4 days prior to initiation of the dosing cycle; and the fifth doses of Flu and Ara-C are each given 3 days prior to initiation of the dosing cycle. In some embodiments, each dose of Flu comprises about 30 mg/m² and each dose of Ara-C comprises about 2 g/m².

[0016] In some embodiments, the cancer is a relapsed/refractory acute myeloid leukemia (r/r AML).

[0017] Also provided herein is a method of treating a relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle. In some embodiments, the dosing cycle comprises: (I) a first dose of the genetically engineered NK cells; (II) a second dose of the genetically engineered NK cells administered to the subject 2-4 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 1 .5 x 10⁹ genetically engineered NK cells. In some embodiments, each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells. In some embodiments, the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m² fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

[0018] In some embodiments, the dosing cycle is between about 21 days and about 35 days. In some embodiments, the dosing cycle is about 28 days.

[0019] In some embodiments, if the subject exhibits a clinical response following the dosing cycle, the method comprises administering an additional dosing cycle as a consolidation treatment. In some embodiments, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the method comprises administering an additional dosing cycle as retreatment. In some embodiments, the clinical response is a partial response (PR). In some embodiments, the clinical response is a complete response with incomplete hematologic recovery (CRi). In some embodiments, the clinical response is a complete response (CR).

[0020] In some embodiments, the method comprises administration of between one dosing cycle and five dosing cycles. In some embodiments, the subject is administered the lymphodepleting therapy prior to each dosing cycle.

[0021] In some embodiments, the subject has less than or equal to 5% peripheral blasts. In some embodiments, the subject does not have evidence of extramedullary disease.

[0022] In some embodiments, the method further comprises: (a) assessing the level or amount of a NKG2D ligand in a biological sample from the subject, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or a polynucleotide encoded by the NKG2D ligand gene; and (b) selecting the subject for treatment with the genetically engineered NK cells if the level or amount of the NKG2D ligand is above a reference value, wherein the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells. In some embodiments, the reference value is within 25%, within 20%, within 15%, within 10%, or within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the genetically engineered NK cells who did not exhibit a clinical response following administration of the genetically engineered NK cell. In some embodiments, the reference value is the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the genetically engineered NK cells who did not exhibit a clinical response following administration of the genetically engineered NK cell. 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 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.

[0023] In some embodiments, the method further comprises administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, a therapeutic agent that increases expression of a NKG2D ligand in the subject, and any combination thereof. In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, and a NK cell engager. In some embodiments, administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells.

[0024] In some embodiments, the cancer is a NKG2D ligand-expressing cancer. In some embodiments, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is relapsed/refractory (R/R). In some embodiments, the cancer is relapsed after hematopoetic cell transplantation (HOT).

[0025] In some embodiments, subject has been treated with at least one prior line of therapy. In some embodiments, the subject has been treated with one, two, or three prior lines of therapy. In some embodiments, the subject has been treated with one, two, three, or four prior lines of therapy. In some embodiments, the subject has been treated with one prior line of therapy. In some embodiments, the subject has been treated with two prior lines of therapy. In some embodiments, the subject has been treated with three prior lines of therapy. In some embodiments, the subject has been treated with four prior lines of therapy. In some embodiments, if the subject has a FLT3-mutated cancer or a IHD 1/2-mutated cancer, the subject has been treated with four prior lines of therapy. In some embodiments, the subject has a FLT3- mutated cancer. In some embodiments, the subject has a IHD 1/2-mutated cancer. In some embodiments, the subject has an ECOG of 0-2. In some embodiments, the subject has an ECOG of 0 or 1 . In some embodiments, the subject has an ECOG of 0. In some embodiments, the subject has an ECOG of 1 . In some embodiments, the subject has an ECOG of 2. In some embodiments, the subject is 18 years of age or older.

[0026] 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 SEO ID NO: 42. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEO ID NO: 42. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the intracellular signaling region comprises a co-stimulatory domain and a CD3zeta. In some embodiments, the co-stimulatory domain comprises an 0X40 domain. In some embodiments, the chimeric receptor has at least 95% sequence identity to the amino acid sequence set forth in SEO ID NO: 39. In some embodiments, the chimeric receptor comprises the amino acid sequence set forth in SEO ID NO: 39. In some embodiments, the genetically engineered NK cells express a membrane-bound interleukin 15 (mblL15). In some embodiments, the mblL15 has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40.

[0027] In some embodiments, the population of engineered NK cells are allogeneic to the subject. In some embodiments, the population of engineered NK cells are derived from a donor not having a cancer. [0028] In some embodiments, a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis. In some embodiments, each dose of the engineered NK cells is administered to the subject on an outpatient basis.

[0029] Also provided herein is use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) administration of a first dose of the genetically engineered NK cells; (ii) administration of a second dose of the genetically engineered NK cells 2-4 days after administration of the first dose of the genetically engineered cells; and (iii) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0030] Also provided herein is use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) administration of a first dose of the genetically engineered NK cells; (ii) administration of a second dose of the genetically engineered NK cells 2-4 days after administration of the first dose of the genetically engineered cells; and (iii) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

[0031] Also provided herein is use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

[0032] Also provided herein is use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

[0033] Also provided herein is use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed/refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) administration of a first dose of the genetically engineered NK cells; (ii) administration of a second dose of the genetically engineered NK cells 2-4 days after administration of the first dose of the genetically engineered cells; and (Hi) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m² fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

[0034] Also provided herein is use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed/refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are administered in a dosing cycle comprising: (i) administration of a first dose of the genetically engineered NK cells; (ii) administration of a second dose of the genetically engineered NK cells 2-4 days after administration of the first dose of the genetically engineered cells; and (Hi) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m² fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

[0035] In some embodiments, all three doses of the genetically engineered NK cells are administered within about 4-10 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 4 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 5 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 6 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 7 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 8 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 9 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 10 days. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 10 days of conclusion of administration of the lymphodepleting therapy.

DETAILED DESCRIPTION

[0036] Some embodiments of the methods and compositions provided herein relate to engineered immune cells and combinations of the same for use in immunotherapy. In several embodiments, the engineered cells are engineered in multiple ways, for example, to express a cytotoxicity-inducing receptor complex. As used herein, the term “cytotoxic receptor complexes” shall be given its ordinary meaning and shall also refer to (unless otherwise indicated), Chimeric Antigen Receptors (CAR), chimeric receptors (also called activating chimeric receptors in the case of NKG2D chimeric receptors). In several embodiments, the cells are further engineered to achieve a modification of the reactivity of the cells against non-tumor tissue and/or other therapeutic cells.

[0037] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0038] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. Cell Types

[0039] Some embodiments of the methods and compositions provided herein relate to a cell such as an immune cell. For example, an immune cell, such as an NK cell or a T cell, may be engineered to include a chimeric receptor such as a NKG2D-ligand-directed chimeric receptor, or engineered to include a nucleic acid encoding said chimeric receptor as described herein. Additional embodiments relate to engineering a second set of cells to express another cytotoxic receptor complex, such as an NKG2D chimeric receptor complex as disclosed herein.

[0040] Traditional anti-cancer therapies relied on a surgical approach, radiation therapy, chemotherapy, or combinations of these methods. As research led to a greater understanding of some of the mechanisms of certain cancers, this knowledge was leveraged to develop targeted cancer therapies. Targeted therapy is a cancer treatment that employs certain drugs that target specific genes or proteins found in cancer cells or cells supporting cancer growth, (like blood vessel cells) to reduce or arrest cancer cell growth. More recently, genetic engineering has enabled approaches to be developed that harness certain aspects of the immune system to fight cancers. In some cases, a patient’s own immune cells are modified to specifically eradicate that patient’s type of cancer. Various types of immune cells can be used, such as T cells, Natural Killer (NK cells), or combinations thereof, as described in more detail below.

[0041] To facilitate cancer immunotherapies, there are also provided for herein polynucleotides, polypeptides, and vectors that encode chimeric receptors that comprise a target binding moiety (e.g., an extracellular binder of a ligand expressed by a cancer cell) and a cytotoxic signaling complex. For example, some embodiments include a polynucleotide, polypeptide, or vector that encodes, for example an activating chimeric receptor comprising an NKG2D extracellular domain that is directed against a tumor marker, for example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others, to facilitate targeting of an immune cell to a cancer and exerting cytotoxic effects on the cancer cell. Also provided are engineered immune cells (e.g., NK cells and/or T cells) expressing such chimeric receptors. There are also provided herein, in several embodiments, polynucleotides, polypeptides, and vectors that encode a construct comprising an extracellular domain comprising two or more subdomains, e.g., first and second ligand binding receptor and a cytotoxic signaling complex. Also provided are engineered immune cells (e.g., NK cells and/or T cells) expressing such bi-specific constructs (in some embodiments the first and second ligand binding domain target the same ligand). Methods of treating cancer and other uses of such cells for cancer immunotherapy are also provided for herein.

Engineered Cells for Immunotherapy

[0042] In several embodiments, cells of the immune system are engineered to have enhanced cytotoxic effects against target cells, such as tumor cells. For example, a cell of the immune system may be engineered to include a tumor-directed chimeric receptor and/or a tumor-directed CAR as described herein. In several embodiments, white blood cells or leukocytes, are used, since their native function is to defend the body against growth of abnormal cells and infectious disease. There are a variety of types of white bloods cells that serve specific roles in the human immune system, and are therefore a preferred starting point for the engineering of cells disclosed herein. White blood cells include granulocytes and agranulocytes (presence or absence of granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells such as those that follow or are otherwise described herein may be engineered to include a chimeric antigen receptor, such as a NKG2D ligand-directed chimeric receptor, or a nucleic acid encoding the chimeric receptor. In several embodiments, the cells are optionally engineered to co-express a membranebound interleukin 15 (mbll_15) domain. Thus, in several embodiments, the immune cells engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membranebound interleukin 15 (mblL15) domain. Monocytes for Immunotherapy

[0043] Monocytes are a subtype of leukocyte. Monocytes can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes are associated with the adaptive immune system and serve the main functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake cellular material, or entire cells, followed by digestion and destruction of the engulfed cellular material. In several embodiments, monocytes are used in connection with one or more additional engineered cells as disclosed herein. Several embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others) and optionally a membrane-bound interleukin 15 (mblL15) domain. In some embodiments, the monocytes engineered to express a chimeric receptor are also engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mblL15) domain. In several embodiments, the monocytes are autologous cells. In some embodiments, the monocytes are allogeneic cells.

Lymphocytes for Immunotherapy

[0044] Lymphocytes, the other primary sub-type of leukocyte include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity). While B cells are engineered according to several embodiments, disclosed herein, several embodiments also relate to engineered T cells or engineered NK cells (mixtures of T cells and NK cells are used in some embodiments, either from the same donor, or different donors). Several embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, the lymphocytes engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mblL15) domain. In several embodiments, the lymphocytes are autologous cells. In some embodiments, the lymphocytes are allogeneic cells.

T Cells for Immunotherapy

[0045] T cells are distinguishable from other lymphocytes sub-types (e.g., B cells or NK cells) based on the presence of a T-cell receptor on the cell surface. T cells can be divided into various different subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cell, mucosal associated invariant T cells and gamma delta T cells. In some embodiments, a specific subtype of T cell is engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. In some embodiments, CD4+ T cells are engineered. In some embodiments, CD8+ T cells are engineered. In some embodiments, regulatory T cells are engineered. In some embodiments, gamma delta T cells are engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. For example, in some embodiments, CD4+ and CD8+ T cells are engineered. In some embodiments, there is no specific selection of a type of T cells to be engineered to express the cytotoxic receptor complexes disclosed herein. In several embodiments, specific techniques, such as use of cytokine stimulation are used to enhance expansion/collection of T cells with a specific marker profile. For example, in several embodiments, activation of certain human T cells, e.g. CD4+ T cells, CD8+ T cells is achieved through use of CD3 and/or CD28 as stimulatory molecules. In several embodiments, there is provided a method of treating or preventing cancer or an infectious disease, comprising administering a therapeutically effective amount of T cells expressing the cytotoxic receptor complex and/or a homing moiety as described herein. In several embodiments, the engineered T cells are autologous cells. In some embodiments, the T cells are allogeneic cells. Several embodiments of the methods and compositions disclosed herein relate to T cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, T cells engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbll_15) co-stimulatory domain.

NK Cells for Immunotherapy

[0046] In several embodiments, there is provided a method of treating or preventing cancer or an infectious disease, comprising administering a therapeutically effective amount of natural killer (NK) cells expressing the cytotoxic receptor complex and/or a homing moiety as described herein. In several embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In several embodiments, the engineered NK cells are autologous cells. In several embodiments, the NK cells are allogeneic cells. In several embodiments, NK cells are preferred because the natural cytotoxic potential of NK cells is relatively high. In several embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, leading to an even more effective activity against target cells (e.g., tumor or other diseased cells). Several embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, NK cells engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membranebound interleukin 15 (mblL15) domain. Thus, in some embodiments, NK cells are engineered to express (e.g., bicistronically express) a chimeric receptor (e.g., a NKG2D ACR) and mbll_15.

[0047] In several embodiments, immortalized NK cells are used and are subject to engineering, as disclosed herein. In some embodiments, the NK cells are derived from cell line NK-92. NK-92 cells are derived from NK cells, but lack major inhibitory receptors displayed by normal NK cells, while retaining the majority of activating receptors. Some embodiments of NK-92 cells described herein related to NK-92 cell engineered to silence certain additional inhibitory receptors, for example, SMAD3, allowing for upregulation of interferon-y (IFNy), granzyme B, and/or perforin production. Additional information relating to the NK-92 cell line is disclosed in WO 1998/49268 and U.S. Patent Application Publication No. 2002-0068044 and incorporated in their entireties herein by reference. NK-92 cells are used, in several embodiments, in combination with one or more of the other cell types disclosed herein. For example, in one embodiment, NK-92 cells are used in combination with NK cells as disclosed herein. In an additional embodiment, NK- 92 cells are used in combination with T cells as disclosed herein.

Hematopoietic Stem Cells for Cancer Immunotherapy

[0048] In some embodiments, hematopoietic stem cells (HSCs) are used in the methods of immunotherapy disclosed herein. In several embodiments, the cells are engineered to express a homing moiety and/or a cytotoxic receptor complex. HSCs are used, in several embodiments, to leverage their ability to engraft for long-term blood cell production, which could result in a sustained source of targeted anti-cancer effector cells, for example to combat cancer remissions. In several embodiments, this ongoing production helps to offset anergy or exhaustion of other cell types, for example due to the tumor microenvironment. In several embodiments allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In several embodiments, the HSCs are autologous cells. In some embodiments, the HSCs are allogeneic cells. In several embodiments, HSCs are used in combination with one or more additional engineered cell type disclosed herein. Several embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, the HSCs engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbll_15) domain.

Induced Pluripotent Stem Cells

[0049] In some embodiments, NK, T, or other immune cells derived from pluripotent stem cells (IPSCs) are used in the method of immunotherapy disclosed herein. In some embodiments, induced pluripotent stem cells (IPSCs) are used in the method of immunotherapy disclosed herein. iPSCs are used, in several embodiments, to leverage their ability to differentiate and derive into non-pluripotent cells, including, but not limited to, CD34 cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells comprising one or several genetic modifications at selected sites through differentiating iPSCs or less differentiated cells comprising the same genetic modifications at the same selected sites. In several embodiments, the iPSCs are used to generate iPSC-derived NK or T cells. In several embodiments, the cells are engineered to express a homing moiety and/or a cytotoxic receptor complex. In several embodiments, iPSCs are used in combination with one or more additional engineered cell type disclosed herein. Several embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express an activating chimeric receptor that targets a ligand on a tumor cell, for example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others). In some embodiments, the iPSCs engineered to express a chimeric receptor are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbll_15) co-stimulatory domain. In several embodiments, the engineered iPSCs are differentiated into NK, T, or other immune cells, such as for use in a composition or method provided herein. In several embodiments, the iPSCs are autologous cells. In some embodiments, the iPSCs are allogeneic cells.

II. Cytotoxic Receptor Constructs

Extracellular domains (Tumor binder)

[0050] Some embodiments of the compositions and methods described herein relate to a chimeric receptor that includes an extracellular domain that comprises a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain) as described herein. Several embodiments of the compositions and methods described herein relate to a chimeric receptor that includes an extracellular domain that comprises a ligand binding domain that binds a ligand expressed by a tumor cell (also referred to as an activating chimeric receptor) as described herein. In some embodiments, the ligand binding domain binds to a ligand of NKG2D. The ligand binding domain, depending on the embodiment, targets for example MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others).

[0051] In some embodiments, the antigen-binding domain is derived from or comprises wild-type or non-wild-type sequence of an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab')2, a single domain antibody (sdAb), a vH or vL domain, a camelid VHH domain, or a non-immunoglobulin scaffold such as a DARPIN, an affibody, an affilin, an adnectin, an affitin, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a kunitz domain, an Armadillo repeat protein, an autoantigen, a receptor or a ligand. In some embodiments, the tumor-binding domain contains more than one antigen binding domain. a. Antigen-Binding Proteins

[0052] There are provided, in several embodiments, antigen-binding proteins. As used herein, the term “antigen-binding protein" shall be given its ordinary meaning, and shall also refer to a protein comprising an antigen-binding fragment that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding fragment to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds to the antigen or from the light chain of an antibody that binds to the antigen. In still some embodiments, the antigen-binding fragment comprises all six CDRs from an antibody that binds to the antigen (three from the heavy chain and three from the light chain). In several embodiments, the antigen-binding fragment comprises one, two, three, four, five, or six CDRs from an antibody that binds to the antigen, and in several embodiments, the CDRs can be any combination of heavy and/or light chain CDRs. The antigen-binding fragment in some embodiments is an antibody fragment.

[0053] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., an antigen-binding fragment of an antibody), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, a single-chain variable fragment (scFv), a nanobody (e.g. VH domain of camelid heavy chain antibodies; VHH fragment,), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a Fd fragment, and a complementarity determining region (CDR) fragment. These molecules can be derived from any mammalian source, such as human, mouse, rat, rabbit, or pig, dog, or camelid. Antibody fragments may compete for binding of a target antigen with an intact (e.g., native) antibody and the fragments may be produced by the modification of intact antibodies (e.g. enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA technologies or peptide synthesis. The antigen-binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigenbinding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. In addition, peptide antibody mimetics (“PAMs") can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold.

[0054] In some embodiments, the antigen-binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or into multiple polypeptide chains. For instance, antigen-binding proteins can include, but are not limited to, a diabody; an intrabody; a domain antibody (single VL or VH domain or two or more VH domains joined by a peptide linker;); a maxibody (2 scFvs fused to Fc region); a triabody; a tetrabody; a minibody (scFv fused to CH3 domain); a peptibody (one or more peptides attached to an Fc region); a linear antibody (a pair of tandem Fd segments (VH-0H1 -VH-CH1 ) which, together with complementary light chain polypeptides, form a pair of antigen binding regions); a small modular immunopharmaceutical; and immunoglobulin fusion proteins (e.g. IgG-scFv, IgG-Fab, 2scFv-lgG, 4scFv-lgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0055] In some embodiments, the antigen-binding protein has the structure of an immunoglobulin. As used herein, the term “immunoglobulin” shall be given its ordinary meaning, and shall also refer to a tetrameric molecule, with each tetramer comprising two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0056] Within light and heavy chains, the variable (V) and constant regions (C) are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. The variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites. [0057] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.

[0058] Human light chains are classified as kappa and lambda light chains. An antibody “light chain”, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (A) light chains refer to the two major antibody light chain isotypes. A light chain may include a polypeptide comprising, from amino terminus to carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL).

[0059] Heavy chains are classified as mu (p), delta (A), gamma (y), alpha (a), and epsilon (E), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. An antibody “heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs. A heavy chain may include a polypeptide comprising, from amino terminus to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1 ), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3), and optionally an immunoglobulin heavy chain constant domain 4 (CH4).

[0060] The IgG-class is further divided into subclasses, namely, IgG 1 , lgG2, lgG3, and lgG4. The IgA-class is further divided into subclasses, namely lgA1 and lgA2. The IgM has subclasses including, but not limited to, lgM1 and lgM2. The heavy chains in IgG, IgA, and IgD antibodies have three domains (CH1 , CH2, and CH3), whereas the heavy chains in IgM and IgE antibodies have four domains (CH1 , CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain (e.g., between the light and heavy chain) and between the hinge regions of the antibody heavy chains.

[0061] In some embodiments, the antigen-binding protein is an antibody. The term “antibody”, as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be monoclonal, or polyclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. The antibody may be “humanized”, “chimeric” or nonhuman. An antibody may include an intact immunoglobulin of any isotype, and includes, for instance, chimeric, humanized, human, and bispecific antibodies. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains. Antibody sequences can be derived solely from a single species, or can be “chimeric,” that is, different portions of the antibody can be derived from two different species as described further below. Unless otherwise indicated, the term “antibody” also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains provided the antibodies retain the same or similar binding and/or function as the antibody comprised of two full length light and heavy chains. For example, antibodies having 1 , 2, 3, 4, or 5 amino acid residue substitutions, insertions or deletions at the N-terminus and/or C-terminus of the heavy and/ or light chains are included in the definition provided that the antibodies retain the same or similar binding and/or function as the antibodies comprising two full length heavy chains and two full length light chains. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies. There is provided, in some embodiments, monoclonal and polyclonal antibodies. As used herein, the term “polyclonal antibody” shall be given its ordinary meaning, and shall also refer to a population of antibodies that are typically widely varied in composition and binding specificity. As used herein, the term “monoclonal antibody” (“mAb”) shall be given its ordinary meaning, and shall also refer to one or more of a population of antibodies having identical sequences. Monoclonal antibodies bind to the antigen at a particular epitope on the antigen.

[0062] In some embodiments, the antigen-binding protein is a fragment or antigen-binding fragment of an antibody. The term “antibody fragment" refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either vL or vH), camelid vHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1 126-1 136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide mini bodies). An antibody fragment may include a Fab, Fab’, F(ab’)2, and/or Fv fragment that contains at least one CDR of an immunoglobulin that is sufficient to confer specific antigen binding to a cancer antigen (e.g., CD19). Antibody fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0063] In some embodiments, Fab fragments are provided. A Fab fragment is a monovalent fragment having the VL, VH, CL and CH1 domains; a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VH and CH1 domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain. In some embodiments, these antibody fragments can be incorporated into single domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. In some embodiments, the antibodies comprise at least one CDR as described herein. [0064] There is also provided for herein, in several embodiments, single-chain variable fragments. As used herein, the term “single-chain variable fragment” (“scFv”) shall be given its ordinary meaning, and shall also refer to a fusion protein in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site). For the sake of clarity, unless otherwise indicated as such, a “single-chain variable fragment” is not an antibody or an antibody fragment as defined herein. Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is configured to reduce or not allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain. According to several embodiments, if the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

[0065] In several embodiments, the antigen-binding protein comprises one or more CDRs. As used herein, the term “CDR" shall be given its ordinary meaning, and shall also refer to the complementarity determining region (also termed “minimal recognition units" or “hypervariable region”) within antibody variable sequences. The CDRs permit the antigen-binding protein to specifically bind to a particular antigen of interest. There are three heavy chain variable region CDRs (CDR-H1 , CDR-H2 and CDR-H3) and three light chain variable region CDRs (CDR-L1 , CDR-L2 and CDR-L3). The CDRs in each of the two chains typically are aligned by the framework regions to form a structure that binds specifically to a specific epitope or domain on the target protein. From N-terminus to C-terminus, naturally-occurring light and heavy chain variable regions both typically conform to the following order of these elements: FW1 , CDR1 , FW2, CDR2, FW3, CDR3, FW4. For heavy chain variable regions, the order is typically: FW-H1 , CDR-H1 , FW-H2, CDR- H2, FW-H3, CDR-H3, and FW-H4 from N-terminus to C-terminus. For light chain variable regions, the order is typically: FW-L1 , CDR-L1 , FW-L2, CDR-L2, FW-L3, CDR-L3, FW-L4 from N-terminus to C-terminus. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991 , NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901 -917; Chothia et al., 1989, Nature 342:878-883. Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody may be identified using this system. Other numbering systems for the amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657- 670; 2001 ). The binding domains disclosed herein may utilize CDRs defined according to any of these systems. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, IMGT, Paratome, AbM, and/or conformational definitions, or a combination of any of the foregoing. Any of the CDRs, either separately or within the context of variable domains, can be interpreted by one of skill in the art under any of these numbering systems as appropriate. One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen-binding protein.

[0066] In some embodiments, the antigen-binding proteins provided herein comprise one or more CDR(s) as part of a larger polypeptide chain. In some embodiments, the antigen-binding proteins covalently link the one or more CDR(s) to another polypeptide chain. In some embodiments, the antigen-binding proteins incorporate the one or more CDR(s) noncovalently. In some embodiments, the antigen-binding proteins may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure comprises a polypeptide or portion thereof that is sufficient to form a conformationally stable structural support, or framework, or scaffold, which is able to display one or more sequences of amino acids that bind to an antigen (e.g., CDRs, a variable region, etc.) in a localized surface region. Such structures can be a naturally occurring polypeptide or polypeptide “fold" (a structural motif), or can have one or more modifications, such as additions, deletions and/or substitutions of amino acids, relative to a naturally occurring polypeptide or fold. Depending on the embodiment, the scaffolds can be derived from a polypeptide of a variety of different species (or of more than one species), such as a human, a non-human primate or other mammal, other vertebrate, invertebrate, plant, bacteria or virus.

[0067] Depending on the embodiment, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. In some such embodiments, those framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST 1 , coiled coil, LACI-D1 , Z domain and/or tendamistat domains.

[0068] There is also provided, in some embodiments, antigen-binding proteins with more than one binding site. In several embodiments, the binding sites are identical to one another while in some embodiments the binding sites are different from one another. For example, an antibody typically has two identical binding sites, while a “bispecific” or “bifunctional” antibody has two different binding sites. The two binding sites of a bispecific antigen-binding protein or antibody will bind to two different epitopes, which can reside on the same or different protein targets. In several embodiments, this is particularly advantageous, as a bispecific chimeric antigen receptor can impart to an engineered cell the ability to target multiple tumor markers. For example, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others, and an additional tumor marker, such as CD70, CD123, CD19, Her2, mesothelin, Claudin 6, BCMA, EGFR, or any other marker disclosed herein or appreciated in the art as a tumor specific antigen or tumor associated antigen can be bound by a bispecific antibody. b. Natural Killer Group Domains that Bind Tumor Ligands

[0069] In several embodiments, engineered immune cells such as NK cells are leveraged for their ability to recognize and destroy tumor cells. NK cells express both inhibitory and activating receptors on the cell surface. Inhibitory receptors bind self-molecules expressed on the surface of healthy cells (thus preventing immune responses against “self” cells), while the activating receptors bind ligands expressed on abnormal cells, such as tumor cells. When the balance between inhibitory and activating receptor activation is in favor of activating receptors, NK cell activation occurs and target (e.g., tumor) cells are lysed.

[0070] Natural killer Group 2 member D (NKG2D) is an NK cell activating receptor that recognizes a variety of ligands expressed on cells. The surface expression of various NKG2D ligands is generally low in healthy cells but is upregulated upon, for example, malignant transformation. Non-limiting examples of ligands recognized by NKG2D include, but are not limited to, MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, as well as other molecules expressed on target cells that control the cytolytic or cytotoxic function of NK cells. In several embodiments, T cells are engineered to express an extracellular domain to binds to one or more tumor ligands and activate the T cell. For example, in several embodiments, T cells are engineered to express an NKG2D receptor as the binder/activation moiety. In several embodiments, engineered cells as disclosed herein are engineered to express another member of the NKG2 family, e.g., NKG2A, NKG2C, and/or NKG2E. Combinations of such receptors are engineered in some embodiments. Moreover, in several embodiments, other receptors are expressed, such as the Killercell immunoglobulin-like receptors (KIRs).

[0071] In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a full length NKG2D as an extracellular component to recognize ligands on the surface of tumor cells (e.g., liver cells). In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a functional fragment of NKG2D (e.g., human NKG2D) as an extracellular component to recognize ligands on the surface of tumor cells (e.g., liver cells). In one embodiment, full length NKG2D (e.g., full length human NKG2D) is encoded by the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, full length NKG2D (e.g., full length human NKG2D) has the amino acid sequence of SEQ ID NO: 43. In several embodiments, the full length NKG2D, or functional fragment thereof is human NKG2D. Additional information about chimeric receptors for use in the presently disclosed methods and compositions can be found in PCT Patent Publication No. WO/2018/183385, which is incorporated in its entirety by reference herein.

[0072] In several embodiments, cells are engineered to express a cytotoxic receptor complex comprising a functional fragment of NKG2D as an extracellular component to recognize ligands on the surface of tumor cells or other diseased cells. In one embodiment, the functional fragment of NKG2D is encoded by the nucleic acid sequence of SEQ ID NO: 25. In several embodiments, the fragment of NKG2D has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with full-length wild-type NKG2D. In several embodiments, the fragment is encoded by a polynucleotide that has one or more additional mutations from SEQ ID NO: 25, but retains, or in some embodiments, has enhanced, ligand-binding function. In several embodiments, the functional fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26. In several embodiments, the NKG2D fragment is provided as a dimer, trimer, or other concatemeric format, such embodiments providing enhanced ligand-binding activity. In several embodiments, the sequence encoding the NKG2D fragment is optionally fully or partially codon optimized. In one embodiment, a polynucleotide sequence encoding a codon optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28. Advantageously, according to several embodiments, the functional fragment lacks its native transmembrane or intracellular domains but retains its ability to bind ligands of NKG2D as well as transduce activation signals upon ligand binding. A further advantage of such fragments is that expression of DAP10 to localize NKG2D to the cell membrane is not required. Thus, in several embodiments, the cytotoxic receptor complex encoded by the polypeptides disclosed herein does not comprise DAP10. In several embodiments, immune cells, such as NK or T cells, are engineered to express one or more chimeric receptors that target, for example CD70, CD19, CD123, Her2, mesothelin, Claudin 6, BCMA, EGFR, and an NKG2D ligand, such as MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6. Such cells, in several embodiments, also express (e.g., bicistronically express) mblL15.

[0073] In several embodiments, the cytotoxic receptor complexes are configured to dimerize. Dimerization may comprise homodimers or heterodimers, depending on the embodiment. In several embodiments, dimerization results in improved ligand recognition by the cytotoxic receptor complexes (and hence the NK cells expressing the receptor), resulting in a reduction in (or lack) of adverse toxic effects. In several embodiments, the cytotoxic receptor complexes employ internal dimers, or repeats of one or more component subunits. For example, in several embodiments, the cytotoxic receptor complexes may optionally comprise a first NKG2D extracellular domain coupled to a second NKG2D extracellular domain, and a transmembrane/signaling region (or a separate transmembrane region along with a separate signaling region).

[0074] In several embodiments, the various domains/subdomains are separated by a linker such as, a GS3 linker (SEQ ID NOs: 15 and 16, nucleotide and protein, respectively) is used (or a GSn linker). Other linkers used according to various embodiments disclosed herein include, but are not limited to those encoded by SEQ ID NOs: 17, 19, 21 or 23. In several embodiments, other linkers comprise the peptide sequence of one of SEQ ID NOs: 18, 20, 22, 24. This provides the potential to separate the various component parts of the receptor complex along the polynucleotide, which can enhance expression, stability, and/or functionality of the receptor complex.

Cytotoxic Signaling Complex

[0075] Some embodiments of the compositions and methods described herein relate to a chimeric receptor, such as a chimeric receptor directed against an NKG2D ligand, such as MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6) that includes a cytotoxic signaling complex. As disclosed herein, according to several embodiments, the provided chimeric receptors comprise one or more transmembrane and/or intracellular domains that initiate cytotoxic signaling cascades upon the extracellular domain(s) binding to ligands on the surface of target cells. Thus, in some embodiments, the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain (e.g., CD3zeta) and a co-stimulatory signaling domain.

[0076] In several embodiments, the chimeric receptor comprises at least one transmembrane domain, at least one co-stimulatory domain, and/or at least one signaling domain. In some embodiments, more than one component part makes up a given domain - e.g., a co-stimulatory domain may comprise two subdomains. Moreover, in some embodiments, a domain may serve multiple functions, for example, a transmembrane domain may also serve to provide signaling function.

Transmembrane Domains

[0077] Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and/or ligand-directed chimeric receptors) that comprise a transmembrane domain. Some embodiments include a transmembrane domain from NKG2D or another transmembrane protein. In several embodiments in which a transmembrane domain is employed, the portion of the transmembrane protein employed retains at least a portion of its normal transmembrane domain.

[0078] several embodiments, however, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells. In several embodiments, the transmembrane domain comprises CD8a. In several embodiments, the transmembrane domain comprises a CD8 (e.g., CD8 a) hinge and a CD8 (e.g., CD8 a) transmembrane region.

[0079] In several embodiments, the transmembrane domain comprises a “hinge,” e.g., a CD8a hinge. In several embodiments, the hinge of CD8a is encoded by the nucleic acid sequence of SEQ ID NO: 1. In several embodiments, the CD8a hinge is truncated or modified and is encoded by a polynucleotide that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD8a hinge having the sequence of SEQ ID NO: 1 . In several embodiments, the hinge of CD8a comprises the amino acid sequence of SEQ ID NO: 2. In several embodiments, the CD8a hinge can be truncated or modified, such that it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 2.

[0080] In several embodiments, the transmembrane domain comprises a CD8a transmembrane region. In several embodiments, the CD8a transmembrane region is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 3. In several embodiments, the CD8a transmembrane region is truncated or modified and is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 3. In several embodiments, the CD8a transmembrane region comprises the amino acid sequence of SEQ ID NO: 4. In several embodiments, the CD8a transmembrane region is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD8a having the sequence of SEQ ID NO: 4. [0081] Taken together in several embodiments, the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 13. In several embodiments, the CD8 transmembrane domain is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD8 transmembrane domain sequence of SEQ ID NO: 13. In several embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 14. In several embodiments, the CD8 transmembrane domain is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 14.

[0082] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof. In several embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 30. In several embodiments, the CD28 transmembrane domain is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 30.

Signaling Domains

[0083] Some embodiments of the compositions and methods described herein relate to a chimeric receptor (e.g., tumor antigen-directed CARs and/or tumor ligand-directed chimeric receptors) that includes an intracellular signaling domain. For example, immune cells engineered according to several embodiments disclosed herein may comprise at least one subunit of the CD3 T cell receptor complex (or a fragment thereof). In several embodiments, the intracellular signaling domain comprises the CD3zeta subunit. In several embodiments, the CD3zeta is encoded by the nucleic acid sequence of SEQ ID NO: 7. In several embodiments, the CD3zeta can be truncated or modified, such that it is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 7. In several embodiments, the CD3zeta domain comprises the amino acid sequence of SEQ ID NO: 8. In several embodiments, the CD3zeta domain is truncated or modified. In some embodiments, the CD3zeta domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD3zeta domain having the sequence of SEQ ID NO: 8.

[0084] In several embodiments, unexpectedly enhanced signaling is achieved through the use of multiple signaling domains whose activities act synergistically. For example, in several embodiments, the intracellular signaling domain further comprises a co-stimulatory domain. For example, in several embodiments, the intracellular signaling domain further comprises an 0X40 domain. In several embodiments, the 0X40 domain is an intracellular signaling domain. In several embodiments, the 0X40 intracellular signaling domain is encoded by a nucleic acid that comprises the sequence of SEQ ID NO: 5. In several embodiments, the 0X40 intracellular signaling domain can be truncated or modified, such that it is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 5. In several embodiments, the 0X40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 6. In several embodiments, the 0X40 intracellular signaling domain is truncated or modified. In some embodiments, the 0X40 intracellular signaling domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 6. In several embodiments, 0X40 is used as the sole signaling domain in the chimeric receptor construct, however, in several embodiments, 0X40 can be used with one or more other domains. For example, combinations of 0X40 and CD3zeta are used in some embodiments. For example, in some embodiments, the intracellular signaling domain comprises CD3zeta and 0X40, or a signaling portion thereof. By way of further example, combinations of CD28, 0X40, 4-1 BB, and/or CD3zeta are used in some embodiments.

[0085] In several embodiments, the signaling domain comprises a 4-1 BB domain. In several embodiments, the 4-1 BB domain is an intracellular signaling domain. In several embodiments, the 4-1 BB domain is encoded by the nucleic acid sequence of SEQ ID NO: 29. In several embodiments, the 4-1 BB domain can be truncated or modified, such that it is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 29. In several embodiments, the 4-1 BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 30. In several embodiments, the 4-1 BB intracellular signaling domain is truncated or modified. In several embodiments, the 4-1 BB intracellular signaling domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the 4-1 BB intracellular signaling domain having the sequence of SEQ ID NO: 30. In several embodiments, 4-1 BB is used as the sole signaling domain in the chimeric receptor construct, however, in several embodiments, 4-1 BB can be used with one or more other domains. For example, combinations of 4-1 BB andCD3zeta are used in some embodiments. For example, in some embodiments, the intracellular signaling domain comprises CD3zeta and 4-1 BB, or a signaling portion thereof. By way of further example, combinations of CD28, 0X40, 4-1 BB, and/or CD3zeta are used in some embodiments.

[0086] In several embodiments, the signaling domain comprises a CD28 domain. In several embodiments the CD28 domain is an intracellular signaling domain. In several embodiments, the CD28 intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 31 . In several embodiments, the CD28 intracellular signaling domain can be truncated or modified, such that it is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 32. In several embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 32. In several embodiments, the CD28 intracellular signaling domain is truncated or modified. In several embodiments, the CD28 intracellular signaling domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 32. In several embodiments, CD28 is used as the sole signaling domain in the construct, however, in several embodiments, CD28 can be used with one or more other domains. For example, combinations of CD28 andCD3zeta are used in some embodiments. For example, in some embodiments, the intracellular signaling domain comprises CDSzeta and CD28, or a signaling portion thereof. By way of further example, combinations of CD28, 0X40, 4-1 BB, and/or CD3zeta are used in some embodiments.

Exemplary Cytotoxic Receptor Constructs

[0087] Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as an activating chimeric receptor (ACR) that targets ligands of NKG2D. The expression of these cytotoxic receptor complexes in immune cells, such as NK cells, allows the targeting and destruction of particular target cells, such as cancerous cells. Non-limiting examples of such cytotoxic receptor complexes are discussed in more detail below.

[0088] In several embodiments, there is provided a polynucleotide encoding (e.g., from N- to C- terminus) antigen-binding domain/CD8hinge-CD8TM/QX40/CD3zeta chimeric receptor complex. In several embodiments, there is provided a polypeptide comprises (e.g., from N- to C-terminus) antigen-binding domain/CD8hinge-CD8TM/OX40/CD3zeta chimeric receptor complex. The polynucleotide or polypeptide comprises or is composed of a NKG2D ligand binding moiety, a CD8a hinge, a CD8a transmembrane domain, an 0X40 domain, a CD3zeta domain. In several embodiments, the polynucleotide further encodes a 2A cleavage site, and an mblL-15 domain as described herein. In several embodiments, this receptor complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of sequences disclosed herein or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In several embodiments, the encoding nucleic acid sequence, or the amino acid sequence, comprises a sequence in accordance with one or more SEQ ID NOS as described herein, such as those included herein as examples of constituent parts. In several embodiments, the encoding nucleic acid sequence, or the amino acid sequence, comprises a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, sequence identity, homology and/or functional equivalence with a sequence resulting from the combination one or more SEQ ID NOS as described herein. It shall be appreciated that certain sequence variability, extensions, and/or truncations of the disclosed sequences may result when combining sequences, as a result of, for example, ease or efficiency in cloning (e.g., for creation of a restriction site). In several embodiments, the chimeric receptor comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or a range defined by any two of the aforementioned percentages, identical to the sequence of one or more of the SEQ IDs provided for herein, or a portion thereof (e.g. a portion excluding the mbll_15 sequence and/or self-cleaving peptide sequence)

[0089] In several embodiments, there is provided a polynucleotide encoding (e.g., from N- to C- terminus) an NKG2D/CD8a hinge/CD8a transmembrane domain/QX40/CD3zeta activating chimeric receptor complex. In several embodiments, there is provided a polypeptide comprising (e.g., from N- to C- terminus) an NKG2D/CD8a hinge/CD8a transmembrane domain/QX40/CD3zeta activating chimeric receptor complex. The polynucleotide or polypeptide comprises or is composed of a fragment of the NKG2D receptor capable of binding a ligand of the NKG2D receptor (e.g., an extracellular domain of NKG2D), a CD8alpha hinge, a CD8a transmembrane domain, an 0X40 domain, and a CD3zeta domain as described herein. In several embodiments, this receptor complex is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 33. In yet another embodiment, this chimeric receptor comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the sequence of the chimeric receptor may be encoded by a nucleic acid sequence that varies from SEQ ID NO: 33, but remains expressed as a amino acid sequence, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 40. In several embodiments, while the chimeric receptor amino acid sequence may vary from SEQ ID NO: 40, the chimeric receptor retains, or in some embodiments, has enhanced, NK cell activating and/or cytotoxic function. Additionally, in several embodiments, this construct can optionally be co-expressed with mblL15, such as the mblL15 encoded by SEQ ID NO: 35 or 37. In several embodiments, the mblL15 comprises the amino acid sequence of SEQ ID NO: 36, 38, or 40. In several embodiments, the mblL15 is comprises the amino acid sequence of SEQ ID NO: 36. In several embodiments, the mbll_15 is comprises the amino acid sequence of SEQ ID NO: 38. In several embodiments, the mblL15 is comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the amino acid sequence of the mblL15 may vary from SEQ ID NO: 36, 38, or 40, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 36, 38, or 40. In some embodiments, the amino acid sequence of the mblL15 may vary from SEQ ID NO: 36, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 36. In some embodiments, the amino acid sequence of the mblL15 may vary from SEQ ID NO: 38, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 38. In some embodiments, the amino acid sequence of the mblL15 may vary from SEQ ID NO: 40, but remains, depending on the embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 40.

[0090] Additional information about chimeric receptors for use in the presently disclosed methods and compositions can be found in PCT Patent Publication No. WO 2018/183385, filed March 27, 2018, which is incorporated in its entirety by reference herein.

III. Stimulatory Molecules

[0091] In several embodiments, in addition to the various transmembrane domains and signaling domains (and the combination transmembrane/signaling domains), constructs encoding a chimeric receptor can also include a stimulatory molecule as described herein. These can be certain molecules that, for example, further enhance activity of the immune cells. Cytokines may be used in some embodiments. For example, certain interleukins, such as IL-2 and/or IL-15 as non-limiting examples, are used. In some embodiments, the immune cells for therapy are engineered to express such molecules as a secreted form. In additional embodiments, such stimulatory molecules are engineered to be membrane bound, acting as autocrine stimulatory molecules (or even as paracrine stimulators to neighboring cells).

[0092] In several embodiments, the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15). In some embodiments, the IL15 is expressed from a separate cassette on the construct comprising any one of the CARs disclosed herein. In some embodiments, the IL15 is expressed from the same cassette as any one of the CARs disclosed herein. In some embodiments, the chimeric receptor and IL15 are separated by a nucleic acid sequence encoding a cleavage site, for example, a proteolytic cleavage site or a T2A, P2A, E2A, or F2A self-cleaving peptide cleavage site. In some embodiments, the chimeric receptor and IL15 are separated by a T2A sequence.

[0093] I n some embodiments, the IL15 is membrane-bound IL15 (mblL15). In some embodiments, the mblL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain. In some embodiments, the native IL15 sequence is encoded by a sequence having at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 1 1 . In some embodiments, the native IL15 sequence comprises a peptide sequence having at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the native IL15 sequence comprises SEQ ID NO: 12.

[0094] In some embodiments, IL15 is membrane-bound by virtue of its coupling to at least one transmembrane domain. In some embodiments, the at least one transmembrane domain comprises a CD8 transmembrane domain (e.g., SEQ ID NO: 4). In some embodiments, the mblL15 may comprise additional components, such as a leader sequence and/or a hinge sequence. In some embodiments, the leader sequence is a CD8 leader sequence. In some embodiments, the hinge sequence is a CD8 hinge sequence (e.g., SEQ ID NO: 14). In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain (e.g., SEQ ID NO: 4) and a CD8 hinge (e.g., SEQ ID NO: 2). In some embodiments, the mblL15 comprises an amino acid sequence having at least about 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 40. In some embodiments, the mblL15 comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0095] In some embodiments, the tumor antigen-directed CARs and/or tumor ligand-directed chimeric receptors are encoded by a polynucleotide that encodes for one or more cytosolic protease cleavage sites. Such sites are recognized and cleaved by a cytosolic protease, which can result in separation (and separate expression) of the various component parts of the receptor encoded by the polynucleotide. In some embodiments, the tumor antigen-directed CARs and/or tumor ligand-directed chimeric receptor are encoded by a polynucleotide that encodes for one or more self-cleaving peptides, for example a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and/or an F2A cleavage site. As a result, depending on the embodiment, the various constituent parts of an engineered cytotoxic receptor complex can be delivered to an NK cell or T cell in a single vector or by multiple vectors. Thus, a construct can be encoded by a single polynucleotide, but also include a cleavage site, such that downstream elements of the constructs are expressed by the cells as a separate protein (as is the case in some embodiments with IL-15). In several embodiments, a T2A cleavage site is used. In several embodiments, a T2A cleavage site is encoded by the nucleic acid sequence of SEQ ID NO: 9. In several embodiments, T2A cleavage site can be truncated or modified, such that it is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 9. In several embodiments, the T2A cleavage site comprises the amino acid sequence of SEQ ID NO: 10. In several embodiments, the T2A cleavage site is truncated or modified. In some embodiments, the T2A cleavage site has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 10.

[0096] In several embodiments, NK cells are engineered to express membrane-bound interleukin 15 (mblL15). In such embodiments, mblL15 expression on the NK enhances the cytotoxic effects of the engineered NK cell by enhancing the proliferation and/or longevity of the NK cells. In several embodiments, the mblL15 is encoded by the same polynucleotide as the CAR. In some embodiments, mblL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1 1 . In some embodiments, mblL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1 1 and a sequence that encodes for a transmembrane domain. In some embodiments, mblL15 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, mblL15 comprises the amino acid sequence of SEQ ID NO: 12 functionally coupled to an amino acid sequence of a transmembrane domain. In several embodiments, mblL15 is encoded by the nucleic acid sequence of SEQ ID NO: 35. In several embodiments, the nucleic acid encoding mblL15 can be truncated or modified. In some embodiments, mblL15 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 35. In several embodiments, the mblL15 comprises the amino acid sequence of SEQ ID NO: 40. In several embodiments, the mblL15 is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the mblL15 having the sequence of SEQ ID NO: 40. Membrane-bound IL15 sequences are explored in PCT publications WO 2018/183385 and WO 2020/056045, each of which is hereby expressly incorporated by reference in its entirety and pertaining to membrane-bound IL15 sequences.

IV. Methods of Treatment

[0097] Some embodiments relate to a method of treating, ameliorating, inhibiting, or preventing cancer with a cell or immune cell comprising a chimeric antigen receptor and/or an activating chimeric receptor, as disclosed herein. Some embodiments relate to a method of treating, ameliorating, inhibiting, or preventing cancer with a cell or immune cell comprising a chimeric receptor provided herein, in combination with a therapeutic agent. In some embodiments, the method includes treating or preventing cancer. In some embodiments, the method includes administering a therapeutically effective amount of immune cells expressing a tumor-directed chimeric antigen receptor and/or tumor-directed chimeric receptor as described herein. Examples of types of cancer that may be treated as such are described herein. [0098] Disclosed herein are methods of treating cancer in a subject. In some embodiments, the methods comprise administering to the subject any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, or any one of the cells disclosed herein, or any combination thereof, and a therapeutic agent.

[0099] Also disclosed herein are uses of any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, any one of the cells disclosed herein, and a therapeutic agent for the treatment of cancer. Also disclosed herein are uses of any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, any one of the cells disclosed herein, and a therapeutic agent in the manufacture of a medicament for the treatment of cancer. In some embodiments, 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, or any combination thereof, is for use in combination with a therapeutic agent.

[00100] In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, or a combination thereof. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a monoclonal antibody. In some embodiments, the therapeutic agent is a NK cell engager. In some embodiments, the therapeutic agent increases expression of a NKG2D ligand in a subject.

[00101] In some embodiments, the therapeutic agent is administered prior to, concurrent with, and/or after administration of genetically engineered cells.

[00102] In some embodiments, the therapeutic agent is administered prior to administration of genetically engineered cells. In some embodiments, the therapeutic agent is administered prior to treatment with a lymphodepleting therapy. In some embodiments, the therapeutic agent is administered after treatment with a lymphodepleting therapy and prior to administration of the genetically engineered cells. In some embodiments, the therapeutic agent is administered concurrently with administration of genetically engineered cells. In some embodiments, the therapeutic agent is administered after administration of genetically engineered cells.

[00103] In certain embodiments, treatment of a subject with genetically engineered cell(s) and a therapeutic agent as described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; and (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy. [00104] In particular, methods of treating subjects with dosing regimens as provided herein and described in the Working Examples (e.g., with NK cells engineered to express a NKG2D ligand-directed chimeric receptor) may result in unexpected efficacy and safety, including an efficacy and safety profile allowing for outpatient administration. It is contemplated that such unexpected effects are due, at least in part, to providing an increased number of genetically engineered NK cells during a time period in which a subject’s immune response has not yet fully recovered following lymphodepleting therapy, thereby allowing increased engraftment of adoptively transferred immune cells (e.g., NK cells). Such a time period may include, for example, within about 7 days of administration of the genetically engineered NK cells and/or within about 14 days of administration of a first dose of lymphodepleting therapy. For example, by providing higher doses of genetically engineered NK cells, an increased number of doses, or both, within this time period, the efficacy of genetically engineered NK cells may be improved. Further, the opportunity to provide higher and/or an increased number of doses of genetically engineered NK cells is afforded by the lack of graft vs. host disease and toxicity associated with NK cells. This is in contrast with, for example, cytokine release syndrome (CRS) and neurotoxicity frequently observed with adoptive transfer of engineered T cells, such as CAR T cells.

[00105] Administration of the engineered cells can be by a variety of routes, including, without limitation, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and/or local delivery to an affected tissue. Administration of the therapeutic agent can be by a variety of routes, including, without limitation, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and/or local delivery to an affected tissue.

V. Selection of Subjects

[00106] Provided herein are methods for selecting subjects for treatment. In some aspects, the methods for selecting subjects are used to identify subjects who are likely to exhibit a clinical response (e.g., a partial response (PR), a complete response with incomplete hematologic recovery (CRi) or a complete response (CR)) to the treatment. In some embodiments, identified subjects are selected for treatment and/or administered the treatment.

NKG2D Ligand(s)

[00107] In some embodiments, the methods comprise selecting a subject having a level or amount of a NKG2D ligand above a reference value. For example, in some cases, a subject having a level or amount of a NKG2D ligand above the reference value is predicted to exhibit a clinical response to treatment. Conversely, a subject having a level or amount of expression of a NKG2D ligand below the reference value would not be predicted to exhibit a clinical response to treatment. In some embodiments, the subject has acute myeloid leukemia (AML), optionally relapsed/refractory AML (r/r AML). In some embodiments, the treatment comprises administration of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D. [00108] In some embodiments, the comparison of a level or amount of a NKG2D ligand to a reference value of the NKG2D ligand allows for the assessment, measurement, and/or determination of the probability and/or likelihood of a clinical outcome (e.g. CR, PR, or RD) following administration of and/or associated with a treatment. In some embodiments, the level or amount of a NKG2D ligand in a biological sample is compared to a reference value, e.g., a NKG2D ligand reference value. In some embodiments, the reference value is a value of a level, amount, or concentration of the NKG2D ligand. In some embodiments, the reference value is or is derived from an amount or level of an RNA gene product or a protein gene product. In particular embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, that indicate a likelihood of a clinical response (e.g. CR or PR) and/or an increased, elevated, or high probability of a clinical response(e.g. CR or PR), following administration of a treatment and values or measurements of the NKG2D ligand gene or protein product that indicate an absent or low likelihood and/or a decreased, reduced, or low probability of a clinical response (e.g. CR or PR), following administration of a treatment. In some embodiments, the reference value is a boundary, divide, and/or threshold value between the amounts or levels of the NKG2D ligand gene or protein product where a majority of one or more clinical responses take place or have previously taken place and amounts or levels of the NKG2D ligand gene or protein product where a minority of one or more clinical responses take place or previously taken place.

[00109] In certain embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, associated with a particular type of clinical response from amounts or levels associated with one or more other types of clinical response. In particular embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, associated with clinical response (e.g., CR and/or PR) from the amounts or levels that are associated with no clinical response (e.g., PD). In particular embodiments, the reference value is an amount or level of the NKG2D ligand gene or protein product, or a transformation thereof, that is a boundary between or a threshold value that separates the amounts or levels of the NKG2D ligand gene or protein product, or transformations thereof, associated with CR or PR from the amounts or levels that are associated with other clinical responses, e.g., NR/SD or PD.

[00110] In some embodiments, the reference value is a predetermined value. In particular embodiments, the reference value has been calculated and/or derived from data from a study. In some embodiments, the study is a clinical study. In particular embodiments, the clinical study is a completed clinical study. In certain embodiments, the data from the study included NKG2D ligand expression, e.g., expression of a NKG2D ligand gene or protein product, in samples taken or obtained from subjects in the study. In particular embodiments, the data from the study includes the number and types of clinical responses experienced by subjects during the study. In certain embodiments, the subjects in the clinical study had or have a clinical response, such as CR or PR. In some embodiments, the clinical response is CR. In certain embodiments, the data from the study includes the number and types of diseases or conditions, such as cancer (e.g. AML). In particular embodiments, the data from the study includes the number and types of treatment experienced by subjects during the study. In certain embodiments, the subjects are or were treated with NK cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D.

[00111] In some embodiments, the expression of a NKG2D ligand gene or protein product is compared to a reference value and an elevated, increased and/or high probability and/or likelihood of a clinical response (e.g., CR or PR) is indicated. In particular embodiments, the expression of a NKG2D ligand gene or protein product is compared to a reference value and a reduced, decreased and/or low probability and/or likelihood of a clinical response (e.g., CR or PR) is indicated.

[00112] In some embodiments, the reference value is within 25%, within 20%, within 15%, within 10%, or within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 25% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 20% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 15% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 15% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 10% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment. In some embodiments, the reference value is the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the treatment who did not exhibit a clinical response following administration of the treatment.

[00113] In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is above the reference value, the subject is identified as likely to exhibit a clinical response to the treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is above the reference value, the subject is selected for treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is above the reference value, the subject is administered the treatment.

[00114] In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is below the reference value, the subject is identified as not likely to exhibit a clinical response to the treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is below the reference value, the subject is not selected for treatment. In some embodiments, if the amount or level of the NKG2D ligand in the biological sample obtained from a subject is below the reference value, the subject is not administered the treatment.

[00115] In some embodiments, the level of amount of a NKG2D ligand is assessed in a biological sample from the subject. In some embodiments, the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells. In some embodiments, the biological sample comprises a blood sample. In some embodiments, the biological sample is a peripheral blood sample. In some embodiments, the biological sample comprises a bone marrow sample. In some embodiments, the biological sample is a bone marrow biopsy.

[00116] In some embodiments, the biological sample is obtained from the subject within about 14 days, within about 10 days, within about 7 days, within about 6 days, within about 5 days, within about 4 days, within about 3 days, within about 2 days, or within about 1 day prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 14 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 10 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 7 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 6 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 5 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 4 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 3 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 2 days prior to administration of the genetically engineered NK cells to the subject. In some embodiments, the biological sample is obtained from the subject within about 1 day prior to administration of the genetically engineered NK cells to the subject.

[00117] In some embodiments, the NKG2D ligand comprises MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof. In some embodiments, the NKG2D ligand comprises MICA. In some embodiments, the NKG2D ligand comprises MICA and MICB. In some embodiments, the NKG2D ligand comprises MICA and MICB. In some embodiments, the NKG2D ligand comprises ULBP1. In some embodiments, the NKG2D ligand comprises ULBP2. In some embodiments, the NKG2D ligand comprises ULBP3. In some embodiments, the NKG2D ligand comprises ULBP4. In some embodiments, the NKG2D ligand comprises ULBP5. In some embodiments, the NKG2D ligand comprises ULBP6. In some embodiments, the NKG2D ligand comprises ULBP1 and ULBP3. In some embodiments, the NKG2D ligand comprises ULBP2, ULBP5, and ULBP6. In some embodiments, the NKG2D ligand comprises MICA, MIB, and ULBP1 . In some embodiments, the NKG2D ligand comprises MICA, MIB, and ULBP3. In some embodiments, the NKG2D ligand comprises MICA, MIB, ULBP1 , and ULBP3.

[00118] Methods for determining the amount or level of a NKG2D ligand protein product are known in the art. Suitable methods for assessing, measuring, determining, and/or quantifying the level, amount, or concentration of one or more protein products include, but are not limited to detection with immunoassays, nucleic acid-based or protein-based aptamer techniques, high precision liquid chromatography (HPLC), peptide sequencing, and microarray adaptations of any of the foregoing (including nucleic acid, antibody or protein-protein (i.e., non-antibody) arrays). In some embodiments, the immunoassay is or includes methods or assays that detect proteins based on an immunological reaction, e.g., by detecting the binding of an antibody or antigen binding antibody fragment to a gene product. Immunoassays include, but are not limited to, quantitative immunocytochemistry or immunohistochemistry, ELISA (including direct, indirect, sandwich, competitive, multiple and portable ELISAs), Western blotting (including one, two or higher dimensional blotting or other chromatographic means, optionally including peptide sequencing), enzyme immunoassay, radioimmunoassay, and surface plasmon resonance. In some embodiments, the level or amount of a NKG2D ligand protein product is determined by the number or percentage of cells positive for the NKG2D ligand protein product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand protein product is determined by the intensity of expression of the NKG2D ligand protein product among cells positive for the NKG2D ligand protein product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand protein product is determined by the number or percentage of cells positive for the NKG2D ligand protein product in a biological sample and the intensity of expression of the NKG2D ligand protein product among cells positive for the NKG2D ligand protein product in a biological sample.

[00119] Methods for determining the amount or level of a NKG2D ligand gene product are also known in the art. Suitable methods for assessing, measuring, determining, and/or quantifying the level, amount, or concentration of one or more gene products include, but are not limited to polymerase chain reaction (PCR), including reverse transcriptase (rt) PCR, droplet digital PCR, real-time and quantitative PCR (qPCR) methods, Northern blotting; Southern blotting, e.g., of reverse transcription products and derivatives; array based methods, including blotted arrays, microarrays, or in situ-synthesized arrays; and sequencing, e.g., sequencing by synthesis, pyrosequencing, dideoxy sequencing, or sequencing by ligation, or any other methods known in the art. In some embodiments, the level or amount of a NKG2D ligand gene product is determined by the number or percentage of cells positive for the NKG2D ligand gene product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand gene product is determined by the intensity of expression of the NKG2D ligand gene product among cells positive for the NKG2D ligand gene product in a biological sample. In some embodiments, the level or amount of a NKG2D ligand gene product is determined by the number or percentage of cells positive for the NKG2D ligand gene product in a biological sample and the intensity of expression of the NKG2D ligand gene product among cells positive for the NKG2D ligand gene product in a biological sample.

Peripheral Blasts

[00120] In some embodiments, the methods comprise selecting a subject having marrow limited disease (also known as marrow localized disease) and/or less than or equal to a particular percentage of peripheral blasts (blasts in peripheral blood) for treatment. For example, in some cases, a subject having less than or equal to 5% peripheral blasts (e.g., without evidence of extramedullary disease) is predicted to exhibit a clinical response to treatment. Conversely, a subject having greater than 5% peripheral blasts would not be predicted to exhibit a clinical response to treatment. In some embodiments, the subject has acute myeloid leukemia (AML), optionally relapsed/refractory AML (r/r AML). In some embodiments, the subject has relapsed following HCT. In some embodiments, the treatment comprises administration of a population of natural killer (NK) cells genetically engineered to express a chimeric receptor that binds ligands of NKG2D.

[00121] Methods for assessing the percentage of blasts in peripheral blood of a subject are known in the art.

[00122] In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining whether a subject has marrow limited disease and/or selecting a subject for treatment that has marrow limited disease. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining what a subject has marrow limited disease. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises selecting a subject for treatment that has marrow limited disease. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining whether the subject has marrow limited disease and selecting the subject for treatment if the subject has marrow limited disease.

[00123] In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining the percentage of peripheral blasts in a subject and/or selecting a subject for treatment that has less than or equal to 5% peripheral blasts. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining the percentage of peripheral blasts in a subject. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises selecting a subject for treatment that has less than or equal to 5% peripheral blasts. In some embodiments, prior to administering to a subject a population of NK cells, the method comprises determining the percentage of peripheral blasts in a subject and selecting the subject for treatment if the subject that has less than or equal to 5% peripheral blasts.

[00124] In some embodiments, the subject has marrow limited disease (also known as marrow localized disease). In some embodiments, marrow limited disease is defined as less than or equal to 5% peripheral blasts with no evidence of extramedullary disease. Thus, in some embodiments, the subject has less than or equal to 5% peripheral blasts. Thus, in some embodiments, the subject has less than or equal to 5% peripheral blasts with no evidence of extramedullary disease. In some embodiments, the subject has less than 5% peripheral blasts. In some embodiments, the subject has less than 5% peripheral blasts with no evidence of extramedullary disease. In some embodiments, at the time of administration of the lymphodepleting therapy, the subject has less than or equal to 5% peripheral blasts. In some embodiments, at the time of administration of the first dose of the dosing cycle, the subject has less than or equal to 5% peripheral blasts. In some embodiments, at the time of administration of the lymphodepleting therapy, the subject has less than 5% peripheral blasts. In some embodiments, at the time of administration of the first dose of the dosing cycle, the subject has less than 5% peripheral blasts.

Other

[00125] In some embodiments, the subject has a r/r AML as determined by standard European and LeukemiaNet (ELN) criteria (Dohner et al., Blood (2022) 140(12) : 1345-77) . In some embodiments, the subject has minimum residual disease. In some embodiments, the subject has r/r AML with MRD. In some embodiments, the subject is in complete response (CR) with MRD.

[00126] In some embodiments, the subject has r/r MDS as determined by standard International Working Group (IWG) criteria (Cheson et al., Blood (2006) 108(2):419-25). In some embodiments, the subject has a high-risk genetic mutation (e.g., TP53 or monosomy 7).

[00127] In some embodiments, the subject has received a prior line of therapy for the AML. In some embodiments, the subject has received one, two, three, or four prior lines of therapy for the AML. In some embodiments, the subject has received one prior line of therapy for the AML. In some embodiments, the subject has received two prior lines of therapy for the AML. In some embodiments, the subject has received three prior lines of therapy for the AML. In some embodiments, the subject has received no more than three prior lines of therapy for the AML.

[00128] In some embodiments, the subject has a FLT3-mutated cancer or a IHD 1/2-mutated cancer. In some embodiments, the subject has a FLT3-mutated cancer. In some embodiments, the subject has a IHD 1/2-mutated cancer. In some embodiments, the subject has a FLT3-mutated cancer or a IHD 1/2-mutated cancer, the subject has received four prior lines of therapy. In some embodiments, if the subject has a FLT3-mutated cancer or a IHD 1/2-mutated cancer, the subject has received a prior line of therapy that targets the mutation. In some embodiments, the subject has received four prior lines of therapy for the AML.

[00129] In some embodiments, the subject is at least 18 years of age. In some embodiments, the subject is less than 75 years of age. In some embodiments, the subject is between about 18 years of age and about 75 years of age.

[00130] In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. In some embodiments, the subject has an ECOG performance status of 0. In some embodiments, the subject has an ECOG performance status of 1 . In some embodiments, the subject has an ECOG performance status of 2. VI. Administration and Dosing

[00131] Further provided herein are methods of treating a subject having cancer, comprising administering to the subject a composition comprising immune cells (e.g., natural killer cells) engineered to express a cytotoxic receptor complex as disclosed herein. In some embodiments, a therapeutic agent is also administered to the subject. For example, some embodiments of the compositions and methods described herein relate to use of (a) a tumor-directed chimeric antigen receptor and/or tumor-directed chimeric receptor, or use of cells expressing a tumor-directed chimeric antigen receptor and/or tumor- directed chimeric receptor; and (b) a therapeutic agent, for treating a cancer patient. Uses of such engineered immune cells and a therapeutic agent for treating cancer are also provided.

[00132] In certain embodiments, treatment of a subject with genetically engineered cell(s) (and a optionally a therapeutic agent as described herein) achieves one, two, three, four, or more of the following effects, including, for example: (I) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; and (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy. Each of these comparisons are versus, for example, a different therapy for a disease, which includes a cell-based immunotherapy for a disease using cells that do not express the constructs disclosed herein.

[00133] In particular, methods of treating subjects with dosing regimens as provided herein and described in the Working Examples (e.g., with NK cells engineered to express a NKG2D ligand-directed chimeric receptor) may result in unexpected efficacy and safety, including an efficacy and safety profile allowing for outpatient administration. It is contemplated that such unexpected effects are due, at least in part, to providing an increased number of genetically engineered NK cells during a time period in which a subject’s immune response has not yet fully recovered following lymphodepleting therapy, thereby allowing increased engraftment of adoptively transferred immune cells (e.g., NK cells). Such a time period may include, for example, within about 7 days of administration of the genetically engineered NK cells and/or within about 14 days of administration of a first dose of lymphodepleting therapy. For example, by providing higher doses of genetically engineered NK cells, an increased number of doses, or both, within this time period, the efficacy of genetically engineered NK cells may be improved. Further, the opportunity to provide higher and/or an increased number of doses of genetically engineered NK cells is afforded by the lack of graft vs. host disease and toxicity associated with NK cells. This is in contrast with, for example, cytokine release syndrome (CRS) and neurotoxicity frequently observed with adoptive transfer of engineered T cells, such as CAR T cells.

[00134] Administration can be by a variety of routes, including, without limitation, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and/or local delivery to an affected tissue. The cells (in particular, NK cells) engineered to express a chimeric receptor complex described herein can be formulated for parenteral administration by injection, e.g., by bolus injection or infusion.,

[00135] In several embodiments, multiple doses are used, for example, two, three, four, or more doses within a dosing cycle. In some embodiments, a dosing cycle comprises administration of two doses of NK cells. In some embodiments, a dosing cycle consists of two doses of NK cells. In several embodiments, a dosing cycle comprises administration of three doses of NK cells. In several embodiments, a dosing cycle consists of three doses of NK cells. In several embodiments, a dosing cycle comprises administration of four doses of NK cells. In several embodiments, a dosing cycle consists of four doses of NK cells. In several embodiments, a dosing cycle comprises administration of five doses of NK cells. In several embodiments, a dosing cycle consists of five doses of NK cells. Such multi-dose cycles can be repeated one or more times, as needed to treat a cancer or disease progression.

[00136] Doses of immune cells such as NK cells can be readily determined for a given subject based on their body mass, disease type and state, and desired aggressiveness of treatment, but range, depending on the embodiments, from about 10⁵ cells per kg to about 10¹² cells per kg (e.g., 10⁵-10⁷, 10⁷- 10¹⁰, 10¹⁰-10¹² and overlapping ranges therein). In one embodiment, a dose escalation regimen is used. In several embodiments, a range of immune cells such as NK and/or T cells is administered, for example between about 1 x 10⁶ cells/kg to about 1 x 10⁸ cells/kg. In several embodiments, a range of immune cells such as NK cells is administered, for example between about 1 x 10⁹ genetically engineered immune cells to about 3 x 10⁹ genetically engineered immune cells. In several embodiments, 1 x 10⁸ NK cells are administered (2 x 10⁶/kg for subject under 50kg) three times over a 28-day cycle. In several embodiments, 1 .5 x 10⁸ NK cells are administered (3 x 10⁶/kg for a subject under 50kg) three times over a 28-day cycle. In several embodiments, 3 x 10⁸ NK cells are administered three times over a 28-day cycle. In several embodiments, 4.5 x 10⁸ NK cells are administered three times over a 28-day cycle. In several embodiments, 1 x 10⁹ NK cells are administered three times over a 28-day cycle. In several embodiments, 1 .5 x 10⁹ NK cells are administered three times over a 28-day cycle. In several embodiments, 2 x 10⁹ NK cells are administered three times over a 28-day cycle. In several embodiments, 2.5 x 10⁹ NK cells are administered three times over a 28-day cycle. In several embodiments, 3 x 10⁹ NK cells are administered three times over a 28-day cycle.

[00137] In several embodiments, 1 .5 x 10⁸ NK cells are administered (3 x 10⁶/kg for a subject under 50kg) two times over a 28-day cycle. In several embodiments, 4.5 x 10⁸ NK cells are administered two times over a 28-day cycle. In several embodiments, 1 .5 x 10⁹ NK cells are administered two times over a 28-day cycle. [00138] In several embodiments, 1 .5 x 10⁹ NK cells are administered (3 x 10⁷/kg for a subject under 50kg) three times over a 28-day cycle. In several embodiments, 3 x 10⁹ NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 x 1 o¹⁰ NK cells are administered three times over a 28-day cycle. In several embodiments, at least 4.5 x 10⁹ NK cells are administered over the cycle. In several embodiments, 2 x 1 O¹⁰ NK cells are administered three times over a 28-day cycle. In several embodiments, at least 6 x 10⁹ NK cells are administered over the cycle. In several embodiments, 2.5 x 10¹⁰ NK cells are administered three times over a 28-day cycle. In several embodiments, at least 7.5 x 10⁹ NK cells are administered over the cycle. In several embodiments, 3 x 1 O¹⁰ NK cells are administered three times over a 28-day cycle. In several embodiments, at least 9 x 10⁹ NK cells are administered over the cycle.

[00139] In several embodiments, 1 .5 x 10⁹ NK cells are administered (3 x 10⁷/kg for a subject under 50kg) three times over a 28-day cycle. In several embodiments, 3 x 10⁹ NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 x 10¹⁰ NK cells are administered three times over a 28-day cycle. In several embodiments, at least 4.5 x 10⁹ NK cells are administered over the cycle.

[00140] In several embodiments, three doses of the genetically engineered NK cells are administered within about 4-10 days. In several embodiments, three doses of the genetically engineered NK cells are administered within about 6 days, within about 7 days, within about 8 days, within about 9 days, or within about 10 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 4 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 5 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 6 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 7 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 8 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 9 days. In some embodiments, three doses of the genetically engineered NK cells are administered within about 10 days.

[00141] In several embodiments, all three doses of the genetically engineered NK cells are administered within about 7 days, within about 8 days, within about 9 days, within about 10 days, within about 1 1 days, within about 12 days, within about 13 days, or within about 14 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 7 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 8 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 9 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 10 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 1 1 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 12 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 1 13 days of the conclusion of administration of the lymphodepleting therapy. In some embodiments, all three doses of the genetically engineered NK cells are administered within about 14 days of the conclusion of administration of the lymphodepleting therapy.

[00142] In some embodiments, a lymphodepleting therapy is administered to the subject prior to administration of the first dose of genetically engineered NK cells. In some embodiments, a lymphodepleting therapy is administered to the subject on each of 5 days, 4 days, and 3 days prior to administration of the first dose of genetically engineered NK cells. In some embodiments, a lymphodepleting therapy is administered to the subject on each of 7 days, 6 days, 5 days, 4 days, and 3 days prior to administration of the first dose of genetically engineered NK cells. In several embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 3 days after administration of a lymphodepleting therapy to the subject has concluded.

[00143] In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 2-4 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 2 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 3 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 4 days after the first dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the third dose of the genetically engineered NK cells is administered to the subject about 2-4 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the third dose of the genetically engineered NK cells is administered to the subject about 2 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the third dose of the genetically engineered NK cells is administered to the subject about 3 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the third dose of the genetically engineered NK cells is administered to the subject about 4 days after the second dose of the genetically engineered NK cells is administered to the subject. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered between days 2-4 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered between days 4-8 of the dosing cycle, wherein each dose is administered between about 2-4 days after the preceding dose.

[00144] In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 2 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 2 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 2 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 3 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 2 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 4 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 3 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 2 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 3 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 3 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 3 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 4 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 4 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 2 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 4 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 3 days after the second dose of the genetically engineered NK cells is administered to the subject. In several embodiments, the second dose of the genetically engineered NK cells is administered to the subject about 4 days after the first dose of the genetically engineered NK cells is administered to the subject, and the third dose of the genetically engineered NK cells is administered to the subject about 4 days after the second dose of the genetically engineered NK cells is administered to the subject.

[00145] In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 2 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 4 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 2 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 5 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 2 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 6 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 3 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 5 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 3 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 6 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 3 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 7 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 4 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 6 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 4 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 7 of the dosing cycle. In some embodiments, a first dose of the genetically engineered NK cells is administered on day 0 of the dosing cycle, the second dose of the genetically engineered NK cells is administered on day 4 of the dosing cycle, and the third dose of the genetically engineered NK cells is administered on day 8 of the dosing cycle.

[00146] In several embodiments, each dose comprises between about 1 x 10⁹ genetically engineered NK cells and about 5 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises between about 1 .5 x 10⁹ genetically engineered NK cells and about 5 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises between about 2 x 10⁹ genetically engineered NK cells and about 5 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises between about 2 x 10⁹ genetically engineered NK cells and about 3 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 1 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 1 .5 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 2 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 2.5 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 3 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 3.5 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 4 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 4.5 x 10⁹ genetically engineered NK cells. In several embodiments, each dose comprises about 5 x 10⁹ genetically engineered NK cells.

[00147] In several embodiments, 1 x 10⁹ genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 1 .5 x 10⁹ genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, 2 x 1 o⁹ genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, at least 6 x 1 o⁹ genetically engineered NK cells are administered over the cycle. In several embodiments, 2.5 x 10⁹ genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, at least 7.5 x 10⁹ genetically engineered NK cells are administered over the cycle. In several embodiments, 3 x 10⁹ genetically engineered NK cells are administered three times over a 28-day cycle. In several embodiments, at least 9 x 10⁹ genetically engineered NK cells are administered over the cycle.

[00148] In several embodiments, a dosing cycle comprises administration of three doses of genetically engineered NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises at least about 1.5 x 10⁹ NK cells. In several embodiments, a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises at least about 1 .5 x 10⁹ NK cells.

[00149] In several embodiments, a dosing cycle comprises administration of three doses of genetically engineered NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 2 x 10⁹ NK cells. In several embodiments, a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 2 x 10⁹ NK cells.

[00150] In several embodiments, a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 2.5 x 10⁹ NK cells. In several embodiments, a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 2.5 x 10⁹ NK cells.

[00151] In several embodiments, a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 3 x 10⁹ NK cells. In several embodiments, a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 3 x 10⁹ NK cells.

[00152] Methods of treating subjects with dosing regimens as provided herein may result in unexpected efficacy and safety, including an efficacy and safety profile allowing for outpatient administration. It is contemplated that such unexpected effects are due, at least in part, to providing an increased number of genetically engineered NK cells during a time period in which a subject's immune response has not yet fully recovered following lymphodepleting therapy, thereby allowing increased engraftment of adoptively transferred immune cells (e.g., NK cells). Such a time period may include, for example, within about 7 days of administration of the genetically engineered NK cells and/or within about 14 days of administration of a first dose of lymphodepleting therapy. For example, by providing higher doses of genetically engineered NK cells, an increased number of doses, or both, within this time period, the efficacy of genetically engineered NK cells may be improved. Further, the opportunity to provide higher and/or an increased number of doses of genetically engineered NK cells is afforded by the lack of graft vs. host disease and toxicity associated with NK cells. This is in contrast with, for example, the cytokine release syndrome (CRS) and neurotoxicity frequently observed with adoptive transfer of engineered T cells, such as CAR T cells.

[00153] In several embodiments, subjects exhibiting at least a partial response will receive at least one additional dosing cycle. Dosing cycles may continue, depending on the embodiment as long as the subject is exhibiting an anti-tumor response and tolerating the engineering NK cells. In several embodiments, a subject will not receive an additional dosing cycle if they are not responding (e.g., no tumor response) and/or if the therapy is not tolerated. However, as discussed herein, in several embodiments the disclosed dosing regimens have limited, or no, adverse impacts or toxicities. In several embodiments, a determination about receiving/administering an additional dosing cycle is made at an evaluation 30 days after the inception of a dosing cycle (whether that be the first dosing cycle, or a subsequent cycle). In several embodiments, not more than 5 additional cycles are given to a subject.

[00154] In some embodiments, a dose of NK cells of the dosing cycle is administered on an outpatient basis. In some embodiments, two doses of NK cells of the dosing cycle are administered on an outpatient basis. In some embodiments, each dose of NK cells of the dosing cycle is administered on an outpatient basis. In some embodiments, a dosing cycle is administered to the subject on an outpatient basis. In some embodiments, at least one dosing cycle is administered to the subject on an outpatient basis. In some embodiments, each dosing cycle is administered to the subject on an outpatient basis.

[00155] In several embodiments, the administration of engineered NK cells is preceded by one or more preparatory treatments. In several embodiments, the administration of engineered NK cells is preceded by a lymphodepleting therapy (also referred to as “lymphodepletion”). In several embodiments, each dosing cycle is preceded by lymphodepletion. In several embodiments, a combination of chemotherapeutic agents is used for lymphodepletion. In several embodiments, a single chemotherapeutic agent is used for lymphodepletion. In several embodiments, wherein a combination of chemotherapeutic agents is used, agents with different mechanisms of actions are optionally used. In several embodiments, different classes of agents are optionally used. In several embodiments, an antimetabolic agent is used. In several embodiments, the antimetabolic agent inhibits and/or prevents cell replication. In several embodiments, the antimetabolic agent is an altered nucleotide that disrupts DNA replication, making it effective in targeting rapidly dividing tumor cells.

[00156] In several embodiments, cytosine arabinoside (Ara-C) is used. In several embodiments, a dose of between about 0.2 - about 10 g/m² Ara-C is administered, including doses of about 0.2 g/ m², about 0.5 g/ m², about 1 .0 g/ m², about 1 .5 g/ m², about 2.0 g/ m², about 2.5 g/ m², about 3.0 g/ m², about 3.5 g/ m², about 4.0 g/ m², about 5.0 g/ m², about 6.0 g/m², about 7.0 g/ m², about 8.0 g/ m², about 9.0 g/ m², about 10.0 g/m², about 10.5 g/ m², or any dose between those listed. In several embodiments, a dose of about 2 g/ m² of Ara-C is administered. In several embodiments, the dose of Ara-C is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In several embodiments, the dose of Ara-C is given daily for about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily. In several embodiments, an additional agent is used in combination with the Ara-C. In several embodiments, the additional agent is also an antimetabolite. In several embodiments, the additional agent inhibits one or more of DNA polymerase alpha, ribonucleotide reductase and/or DNA primase, thus inhibiting DNA synthesis.

[00157] In several embodiments, the additional agent is fludarabine. In several embodiments, a dose of between about 5.0 mg/m² - about 200 mg/m² fludarabine is administered, including doses of about 5.0 mg/m², about 10.0 mg/m², about 15.0 mg/m², about 20.0 mg/m², about 25.0 mg/m², about 30.0 mg/m², about 35.0 mg/m², about 40.0 mg/m², about 45.0 mg/m², about 50.0 mg/m², about 60.0 mg/m², about 70.0 mg/m², about 80.0 mg/m², about 90.0 mg/m², about 100.0 mg/m², about 125.0 mg/m², about 150.0 mg/m², about 175.0 mg/m², about 200.0 mg/m², or any dose between those listed. In several embodiments, a dose of about 30 mg/m² of fludarabine is administered. In several embodiments, the dose of fludarabine is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In several embodiments, the dose of fludarabine is given daily for at about 3 days. In several embodiments, the dose of fludarabine is given daily for at about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[00158] In several embodiments, a combination of fludarabine and Ara-C is used with a daily dose of fludarabine of between about 20 mg/m² and 40 mg/m² and a daily dose of Ara-C of between about 1 .5 g/m² and 2.5 g/m². In several embodiments, a combination of fludarabine and Ara-C is used with a daily dose of fludarabine of about 30 mg/m² and a daily dose of Ara-C of about 2 g/m². In several embodiments, the combination of fludarabine and Ara-C (or any other agent or agents as disclosed herein) is administered for about 5 days, with the administration started about 7 days prior to the first administration of the engineered NK cells (for example day -7 to day -3). In several embodiments, lymphodepletion is started at day -5 prior to administration of engineered NK cells. In several embodiments, this combination advantageously functions not only as a lymphodepletion regimen, but as an anti-cancer agent as well (in addition to the engineered NK cells). In several embodiments, the lymphodepletion regimen works synergistically with the engineered NK cells to provide effect reduction and/or elimination of cancerous cells.

[00159] In several embodiments, the additional agent is cyclophosphamide. In several embodiments, a dose of between about 100 mg/m² - about 100 mg/m² fludarabine is administered, including doses of about 100.0 mg/m², about 200 mg/m², about 300 mg/m², about 400 mg/m², about 500 mg/m², about 600 mg/m², about 700 mg/m², about 800 mg/m², about 900 mg/m², about 1000 mg/m², or any dose between those listed. In several embodiments, a dose of about 300 mg/m² of cyclophosphamide is administered. In several embodiments, a dose of about 500 mg/m² of cyclophosphamide is administered. In several embodiments, the dose of cyclophosphamide is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In several embodiments, the dose of cyclophosphamide is given daily for at about 3 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.

[00160] In several embodiments, a combination of fludarabine and cyclophosphamide is used. In several embodiments, cyclophosphamide (300 mg/m²) and fludarabine (30 mg/m²) are administered daily for 3 days. In several embodiments, cyclophosphamide (500 mg/m²) and fludarabine (30 mg/m²) are administered daily for 3 days. In some embodiments, fludarabine and cyclophosphamide are each administered daily 5 days, 4 days, and 3 days prior to administration of the engineered NK cells.

[00161] In certain embodiments, a dose of a genetically engineered cell(s) described herein or composition thereof is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks. In other embodiments, two, three or four doses of a genetically engineered cell(s) described herein or composition thereof is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks. In some embodiments, a dose(s) of a genetically engineered cell(s) described herein or composition thereof is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, a dose of a genetically engineered cell(s) described herein or composition thereof is administered for 1 month, 1 .5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.

[00162] In several embodiments, a subject is subject to lymphodepletion at least one time prior to administration of genetically engineered cells as disclosed herein. In several embodiments, lymphodepletion is performed before one or more additional doses of engineered cells are administered. In several embodiments, lymphodepletion is performed before each dosing cycle. In several embodiments, lymphodepletion is not performed only before a first dosing cycle. In several embodiments, a dosing cycle is used that comprises lymphodepletion followed by at least two doses of engineered cells as disclosed herein, with the two doses separated by a time interval. In several embodiments, the time interval is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or more days (including intervals falling between the time marking a price interval since the last administration, e.g., 84 hours, or 3.5 days). In several embodiments, the dosing cycle itself is approximately 14, 21 , 28, 35, 42 or more days, several embodiments, three doses are each administered about 2-4 days apart from each other. In several embodiments, a subject receives a first dose on day 0 of the cycle, a second dose on day 3 of the cycle and a third dose on day 7 of the cycle. In several such embodiments, a 28 day cycle is used with primary outcome measures evaluated at about day 28.

[00163] In some embodiments, if a subject exhibits a clinical response following a dosing cycle, an additional dosing cycle is administered to the subject as consolidation treatment. Clinical responses may include complete response (CR; e.g., complete remission), complete response with incomplete hematologic recovery (CRi; e.g., CR with residual thrombocytopenia), morphologic leukemia-free state (MLFS), and partial response (PR; e.g., partial remission). Methods for assessing clinical response are known in the art (Dohner et al., Blood (2017) 129(4):424-47).

[00164] For example, in some embodiments, if a subject exhibits a complete response (CR) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject exhibits a complete response with incomplete hematologic recovery (CRi) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject exhibits morphologic leukemia-free state (MLFS) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment. In some embodiments, if a subject exhibits a partial response (PR) following a dosing cycle, a subsequent dosing cycle is administered as consolidation treatment.

[00165] In some embodiments, in a subject does not exhibit a clinical response from a dosing cycle, an additional dosing cycle is administered to the subject. In some embodiments, if a subject does not exhibit a CR following a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if a subject does not exhibit a CRi following a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if a subject does not exhibit MLFS following a dosing cycle, a subsequent dosing cycle is administered. In some embodiments, if a subject does not exhibit a PR following a dosing cycle, a subsequent dosing cycle is administered.

[00166] In some embodiments, if a subject exhibits a clinical response from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject exhibits a CR from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject exhibits a CRi from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject exhibits MLFS from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. In some embodiments, if a subject exhibits a PR from a dosing cycle but subsequently exhibits disease progression, the subject is administered an additional cycle as retreatment. [00167] In some embodiments, the dosing regimen comprises between one dosing cycle and five dosing cycles. In some embodiments, the dosing regimen consists of between one dosing cycle and five dosing cycles. In some embodiments, the dosing regimen consists of between one dosing cycle and five dosing cycles. In some embodiments, the dosing regimen consists of one dosing cycle. In some embodiments, the dosing regimen consists of two dosing cycles. In some embodiments, the dosing regimen consists of three dosing cycles. In some embodiments, the dosing regimen consists of four dosing cycles. In some embodiments, the dosing regimen consists of five dosing cycles. In some embodiments, the subject is administered a lymphodepleting therapy prior to each dosing cycle.

[00168] In some embodiments, among subjects treated according to the method, the overall response rate (ORR) is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65%. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of subjects treated according to the method exhibit a CR or a CRi. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or 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 a CRi. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of subjects treated according to the method exhibit MLFS. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65% of subjects treated according to the method exhibit a PR.

[00169] In several embodiments, lymphodepletion is performed prior to the inception of each dosing cycle, if subsequent dosing cycles are required (e.g., the subject requires further treatment). For example, in several embodiments, a subject undergoes lymphodepletion, receives a plurality of doses of engineered cells according to a cycle, is evaluated at the end of the cycle time and, if deemed necessary undergoes a second lymphodepletion followed by a second dosing cycle. In such embodiments where multiple dosing cycles are used, a first and a second dosing cycle need not be the same (e.g., a first cycle may have 2 doses, while a second uses three doses). Depending on the subject 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dosing cycles are performed. In some embodiments, no more than five dosing cycles are administered.

[00170] Depending on the embodiment, various types of cancer can be treated. In several embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a leukemia or a lymphoma. In several embodiments, the cancer being treated is acute myeloid leukemia (AML). In several embodiments, the cancer being treated is relapsed/refractory acute myeloid leukemia (r/r AML). In some embodiments, the cancer is relapsed to HCT. In several embodiments, the cancer being treated is myelodysplastic syndrome (MDS).

[00171] In several embodiments, the cancer is a solid cancer.

[00172] Additional embodiments provided for herein include treatment or prevention of the following non-limiting examples of cancers including, but not limited to, acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi sarcoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including but not limited to astrocytomas, spinal cord tumors, brain stem glioma, glioblastoma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell leukemia, renal cell cancer, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to, non-small cell lung cancer, (NSCLC) and small cell lung cancer), pancreatic cancer, bowel cancer, melanoma, ocular cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is 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.

[00173] In some embodiments, also provided herein are nucleic acid and amino acid sequences that have sequence identity and/or homology of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges therein) as compared with the respective nucleic acid or amino acid sequences of SEQ ID NOS. 1 - 44 (or combinations of two or more of SEQ ID NOS: 1 -44) and that also exhibit one or more of the functions as compared with the respective SEQ ID NOS. 1 -44 (or combinations of two or more of SEQ ID NOS: 1 - 44) including but not limited to, (i) enhanced proliferation, (ii) enhanced activation, (iii) enhanced cytotoxic activity against cells presenting ligands to which NK cells harboring receptors encoded by the nucleic acid and amino acid sequences bind, (iv) enhanced homing to tumor or infected sites, (v) reduced off target cytotoxic effects, (vi) enhanced secretion of immunostimulatory cytokines and chemokines (including, but not limited to IFNy, TNFa, IL-22, CCL3, CCL4, and CCL5), and (vii) enhanced ability to stimulate further innate and adaptive immune responses, and (viii) combinations thereof.

[00174] Additionally, in several embodiments, there are provided amino acid sequences that correspond to any of the nucleic acids disclosed herein, while accounting for degeneracy of the nucleic acid code. Furthermore, those sequences (whether nucleic acid or amino acid) that vary from those expressly disclosed herein, but have functional similarity or equivalency are also contemplated within the scope of the present disclosure. The foregoing includes mutants, truncations, substitutions, or other types of modifications.

[00175] In several embodiments, polynucleotides encoding the disclosed cytotoxic receptor complexes are mRNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is operably linked to at least one regulatory element for the expression of the cytotoxic receptor complex.

[00176] Additionally provided, according to several embodiments, is a vector comprising the polynucleotide encoding any of the polynucleotides provided for herein, wherein the polynucleotides are optionally operatively linked to at least one regulatory element for expression of a cytotoxic receptor complex. In several embodiments, the vector is a retrovirus.

[00177] Further provided herein are engineered immune cells (such as NK and/or T cells) comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein. Further provided herein are compositions comprising a mixture of engineered immune cells (such as NK cells and/or engineered T cells), each population comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein.

VII. Cancer Types

[00178] Some embodiments of the compositions and methods described herein relate to administering immune cells comprising a tumor-directed chimeric antigen receptor and/or tumor-directed chimeric receptor to a subject with cancer. Several embodiments provided for herein relate to combination therapies wherein immune cells comprising a tumor-directed chimeric antigen receptor and/or tumor- directed chimeric receptor to a subject with cancer in conjunction with a therapeutic agent (an additional anti-cancer agent). Several embodiments provided for herein relate to combination therapies wherein immune cells comprising a tumor-directed chimeric antigen receptor and/or tumor-directed chimeric receptor to a subject with a solid tumor in conjunction with a therapeutic agent (an additional anti-cancer agent.

[00179] Various embodiments provided for herein include treatment or prevention of the following non-limiting examples of cancers. In several embodiments, the cancer is a NKG2D ligand-expressing cancer. In some embodiments, the NKG2D ligand comprises MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5 and/or ULBP6. In some embodiments, the cancer is a solid tumor. In several embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a leukemia or a lymphoma. Examples of cancer include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi sarcoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including but not limited to astrocytomas, spinal cord tumors, brain stem glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell leukemia, renal cell cancer, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to, non-small cell lung cancer, (NSCLC) and small cell lung cancer), pancreatic cancer, bowel cancer, melanoma, ocular cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the 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.

[00180] In several embodiments, acute myeloid leukemia (AML) is treated using genetically engineered immune cells in combination with a therapeutic agent (as described more fully below). In several embodiments, the cancer is AML. In several embodiments, the cancer is r/r AML. In some embodiments, the cancer relapsed following HCT.

[00181] In myelodysplastic syndrome (MDS) is treated using genetically engineered immune cells in combination with a therapeutic agent (as described more fully below). In several embodiments, the cancer is MDS. In several embodiments, the cancer is very high-risk MDS.

[00182] In several embodiments, a solid tumor is treated using genetically engineered immune cells in combination with a therapeutic agent (as described more fully below).

[00183] In several embodiments, breast cancer is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00184] In several embodiments, cervical cancer (e.g., cervical squamous cell carcinoma and/or endocervical adenocarcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00185] In several embodiments, uterine corpus endometrial carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00186] In several embodiments, ovarian serous carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00187] In several embodiments, bladder urothelial carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00188] In several embodiments, colorectal cancer (e.g., colorectal carcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00189] In several embodiments, rectal adenocarcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent. [00190] In several embodiments, gastric cancer is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00191] In several embodiments, a head and neck cancer (e.g., head and neck squamous cell carcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00192] In several embodiments, esophageal carcinoma is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00193] In several embodiments, hepatocellular carcinoma (HCC) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00194] In several embodiments, lung cancer (e.g., lung squamous cell carcinoma and/or lung adenocarcinoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00195] In several embodiments, melanoma (e.g., skin cutaneous melanoma) is treated using genetically engineered immune cells in combination with a therapeutic agent.

[00196] In particular embodiments, the cancer to be treated is one that exhibits an upregulation of NKG2D ligands. In several embodiments, the cancer is associated with a current or previous viral infection. For example, in several embodiments, the cancer to be treated is selected from one or more of head and neck squamous cell carcinoma (HNSCC), cervical carcinoma, esophageal carcinoma, and lung squamous cell carcinoma.

[00197] In some embodiments, the additional anti-cancer agent is a therapeutic agent that upregulates a NKG2D ligand in the subject; or is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, or any combination thereof, or both. Examples of additional anti-cancer agents are described in further detail in the section below.

VIII. Combination Therapies: Therapeutic Agents

[00198] In several embodiments, subjects who are administered the genetically engineered immune cells are also administered a therapeutic agent (an additional anti-cancer agent). In several embodiments, the therapeutic agent comprises a chemotherapeutic agent. In several embodiments, the chemotherapeutic agent comprises an antimetabolite, an alkylating agent, a topoisomerase inhibitor, a mitotic inhibitor, an antibiotic, a protein kinase inhibitor, a proteasome inhibitor, an inhibitor of poly (ADP- ribose) polymerase (PARP), or a combination thereof.

[00199] 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 anti metabolite 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 oxaliplatin (CAPOX).

[00200] 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).

[00201] In some embodiments, the therapeutic agent comprises a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor comprises irinotecan, topotecan, etoposide, or any combination thereof. In some embodiments, the topoisomerase inhibitor comprises irinotecan. In some embodiments, the topoisomerase inhibitor comprises topotecan. In some embodiments, the therapeutic agent comprises etoposide.

[00202] In some embodiments, the therapeutic agent comprises a mitotic inhibitor. In some embodiments, the mitotic inhibitor comprises vincristine, vinblastine, vinorelbine, docetaxel, paclitaxel, eribulin, izabepilone, epothilone, or any combination thereof. In some embodiments, the therapeutic agent comprises vincristine. In some embodiments, the therapeutic agent comprises vinblastine. In some embodiments, the mitotic inhibitor comprises vinorelbine. In some embodiments, the therapeutic agent comprises paclitaxel. In some embodiments, the therapeutic agent comprises docetaxel. In some embodiments, the mitotic inhibitor comprises eribulin. In some embodiments, the mitotic inhibitor comprises izabepilone. In some embodiments, the mitotic inhibitor comprises epothilone.

[00203] In some embodiments, the therapeutic agent comprises an antibiotic. In some embodiments, the antibiotic comprises bleomycin; actinomycin D; an anthracycline, optionally doxorubicin, daunorubicin, or idarubicin, mitomycin, or any combination thereof. In some embodiments, the therapeutic agent comprises bleomycin. In some embodiments, the therapeutic agent comprises actinomycin D. In some embodiments, the therapeutic agent comprises an anthracycline. In some embodiments, the therapeutic agent comprises doxorubicin. In some embodiments, the therapeutic agent comprises daunorubicin. In some embodiments, the therapeutic agent comprises idarubicin. In some embodiments, the therapeutic agent comprises mytomycin.

[00204] 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.

[00205] 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. 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.

[00206] 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.

[00207] In several embodiments, the therapeutic agent comprises a HDAC inhibitor. In some embodiments, the HDAC inhibitor comprises tricostatin A (TSA), valproic acid, sodium butyrate, sodium valproate (VPA), FR901228, MS-275, phenylbutyrate, PDX101 , suberoylanilide hydroxamic acid, or any combination thereof. In some embodiments, the HDAC inhibitor comprises TSA. In some embodiments, the HDAC inhibitor comprises valproic acid. In some embodiments, the HDAC inhibitor comprises sodium buryrate. In some embodiments, the HDAC inhibitor comprises VPA. In some embodiments, the HDAC inhibitor comprises FR901228. In some embodiments, the HDAC inhibitor comprises MS-275. In some embodiments, the HDAC inhibitor comprises phenylbutyrate. In some embodiments, the HDAC inhibitor comprises PDX101 . In some embodiments, the HDAC inhibitor comprises suberoylanilide hydroxamic acid, [00208] In several embodiments, the therapeutic agent comprises a small molecule.

[00209] In several embodiments, the therapeutic agent comprises a monoclonal antibody. In several embodiments, the monoclonal antibody comprises an anti-CD20 antibody, an anti-CTLA4 antibody, an anti-EGFR antibody, an anti-HER2/neu antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti- VEGF antibody, or any combination thereof. In several embodiments, the monoclonal antibody comprises an anti-CD20 antibody (e.g., rituximab). In several embodiments, the monoclonal antibody comprises an anti-CTLA4 antibody. In several embodiments, the monoclonal antibody comprises an anti-EGFR antibody (e.g., cetuximab). In several embodiments, the monoclonal antibody comprises an anti-HER2/neu antibody. In several embodiments, the monoclonal antibody comprises an anti-PD1 antibody. In several embodiments, the monoclonal antibody comprises an anti-PD-L1 antibody. In several embodiments, the monoclonal antibody comprises anti-VEGF antibody, or any combination thereof.

[00210] In several embodiments, the therapeutic agent is a hormone therapy or a therapeutic agent that disrupts or modifies a hormone pathway. In some embodiments, the therapeutic agent comprises tamoxifen. In several embodiments, the therapeutic agent comprises hyperthermia. In several embodiments, the therapeutic agent is an additional form of immunotherapy. In several embodiments, the therapeutic agent comprises photodynamic therapy (PDT). In several embodiments, the therapeutic agent comprises radiation therapy. In several embodiments, the therapeutic agent comprises transplanted stem cells.

[00211] In several embodiments, the therapeutic agent increases a ligand of an NKG2D receptor. In some embodiments, the ligand comprises MICA, MICB, ULBP1 , 2, 3, 4, 5 and/or 6.

[00212] In some embodiments, the therapeutic agent comprises a hypomethylating agent (HMA). In some embodiments, the therapeutic agent comprises azacitadine. In some embodiments, a dose of azacitadine is administered to the subject daily for between about 1 day and about 10 days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of azacitadine is administered to the subject daily for five days, six days, or seven days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of azacitadine is administered to the subject daily for five days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of azacitadine is administered to the subject daily for six days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of azacitadine is administered to the subject daily for sevendays prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of azacitadine is between about 50 mg/m2 and about 100 mg/m2. In some embodiments, a dose of azacitadine is about 75 mg/m2. In some embodiments, azacitadine is administered intravenously (IV) or subcutaneously (SC). In some embodiments, azacitadine is administered IV. In some embodiments, azacitadine is administered SC. In some embodiments, the therapeutic agent comprises decitabine. In some embodiments, a dose of decitabine is administered to the subject daily for between about 1 day and about 10 days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of decitabine is administered to the subject daily for five days prior to administration of the first dose of engineered NK cells. In some embodiments, a dose of decitabine is between about 10 mg/m2 and about 30 mg/m2. In some embodiments, a dose of decitabine is about 20 mg/m2. In some embodiments, decitabine is administered intravenously (IV).

[00213] 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.

[00214] In several embodiments, the therapeutic agent is an NK cell engager (e.g., a molecule that binds both an antigen expressed by cells of the cancer and an antigen expressed by NK cells). In several embodiments, the NK cell engager binds to an activating receptor on an NK cell and an antigen expressed by cells of the cancer. In some embodiments, the activating receptor on the NK cell is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof

[00215] In several embodiments, the therapeutic agent administered is cisplatin. In several embodiments, cisplatin is administered as an IV bolus, or as a series of infusions. In several embodiments, the dose of cisplatin ranges from about 20 mg/m² to about 150 mg/m². In several embodiments, the dose of cisplatin ranges from about 20 mg/m² to about 30 mg/m². In several embodiments, the dose of cisplatin ranges from about 30 mg/m² to about 40 mg/m². In several embodiments, the dose of cisplatin ranges from about 40 mg/m² to about 50 mg/m². In several embodiments, the dose of cisplatin ranges from about 50 mg/m² to about 60 mg/m². In several embodiments, the dose of cisplatin ranges from about 60 mg/m² to about 70 mg/m². In several embodiments, the dose of cisplatin ranges from about 70 mg/m² to about 80 mg/m². In several embodiments, the dose of cisplatin ranges from about 80 mg/m² to about 90 mg/m². In several embodiments, the dose of cisplatin ranges from about 90 mg/m² to about 100 mg/m². In several embodiments, the dose of cisplatin ranges from about 100 mg/m² to about 1 10 mg/m². In several embodiments, the dose of cisplatin ranges from about 110 mg/m² to about 120 mg/m².

several embodiments, the dose of cisplatin ranges from about 120 mg/m² to about 130 mg/m² In several embodiments, the dose of cisplatin ranges from about 130 mg/m² to about 140 mg/m². In several embodiments, the dose of cisplatin ranges from about 140 mg/m² to about 150 mg/m².

[00216] Depending on the embodiment, cisplatin is administered IV on a weekly basis for 3, 4, 5, or 6 total administrations. In several embodiments, cisplatin is administered every 3 to weeks for a total of 3 doses. In several embodiments, cisplatin is administered daily for 5 days. In several embodiments, a series of 3 to 4 doses comprises a cycle, and optionally more than one cycle is administered.

[00217] In several embodiments, cisplatin is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, cisplatin is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, cisplatin is administered concurrently with engineered immune cells, In several embodiments, cisplatin is administered after administration of engineered immune cells. [00218] In several embodiments, the therapeutic agent administered is sorafenib. In several embodiments, sorafenib is administered orally (e.g., in a tableted form). In several embodiments, the dose of sorafenib ranges from about 200 mg to about 800 mg per day. In several embodiments, the dose of sorafenib ranges from about 200 mg to about 250 mg per day. In several embodiments, the dose of sorafenib ranges from about 250 mg to about 300 mg per day. In several embodiments, the dose of sorafenib ranges from about 300 mg to about 350 mg per day. In several embodiments, the dose of sorafenib ranges from about 350 mg to about 400 mg per day. In several embodiments, the dose of sorafenib ranges from about 400 mg to about 450 mg per day. In several embodiments, the dose of sorafenib ranges from about 450 mg to about 500 mg per day. In several embodiments, the dose of sorafenib ranges from about 500 mg to about 550 mg per day. In several embodiments, the dose of sorafenib ranges from about 550 mg to about 600 mg per day.

several embodiments, the dose of sorafenib ranges from about 600 mg to about 650 mg per day.

several embodiments,

dose of sorafenib ranges from about 650 mg to about 700 mg per day.

several embodiments, the dose of sorafenib ranges from about 700 mg to about 750 mg per day. In several embodiments, the dose of sorafenib ranges from about 750 mg to about 800 mg per day. In several embodiments, the total dose is broken into two administrations per day, for example separated by 8 to 12 hours. For example, for a total daily dose of 400 mg, a 200 mg administration (e.g., tablet) is ingested by a subject in the morning and a second 200 mg administration is ingested in the evening.

[00219] In several embodiments, sorafenib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, sorafenib is administered between lymphodepletion and administration of engineered immune cells, In several embodiments, sorafenib is administered concurrently with engineered immune cells, In several embodiments, sorafenib is administered after administration of engineered immune cells.

[00220] In several embodiments, the therapeutic agent administered is regorafenib. In several embodiments, regorafenib is administered orally (e.g., in a tableted form). In several embodiments, the dose of regorafenib ranges from about 100 mg to about 200 mg per day. In several embodiments, the dose of regorafenib ranges from about 100 mg to about 110 mg per day. In several embodiments, the dose of regorafenib ranges from about 1 10 mg to about 120 mg per day. In several embodiments, the dose of regorafenib ranges from about 120 mg to about 130 mg per day.

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

[00221] In several embodiments, regorafenib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, regorafenib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, regorafenib is administered concurrently with engineered immune cells. In several embodiments, regorafenib is administered after administration of engineered immune cells.

[00222] In several embodiments, the therapeutic agent administered is atezolizumab. In several embodiments, the atezolizumab is administered as an IV infusion for a duration of 60 minutes. If 60 minutes is tolerated by a subject a shorter, 30-minute, infusion is optionally used. Depending on the embodiment, the dose of atezolizumab varies with the amount of elapsed time between administrations. For example, in several embodiments two weeks are allowed to elapse between administrations. A lower dose is used in such embodiments based on the higher frequency of administration. In several embodiments, three weeks are allowed to elapse between administrations. An intermediate dose is used in such embodiments based on the intermediate frequency of administration. In several embodiments, four weeks are allowed to lapse between administrations. A higher dose is used in such embodiments based on the lower frequency of administration.

[00223] In several embodiments employing administration every two weeks, the atezolizumab is administered in a dose ranging from about 800 to about 900 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 800 to about 810 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 810 to about 820 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 820 to about 830 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 830 to about 840 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 840 to about 850 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 850 to about 860 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 860 to about 870 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 870 to about 880 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 880 to about 890 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 890 to about 900 mg.

[00224] In several embodiments employing administration every three weeks, the atezolizumab is administered in a dose ranging from about 1000 to about 1400 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1000 to about 1050 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1050 to about 1 100 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1100 to about 1 150 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1150 to about 1200 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1200 to about 1250 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1250 to about 1300 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1300 to about 1350 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1350 to about 1400 mg.

[00225] In several embodiments employing administration every four weeks, the atezolizumab is administered in a dose ranging from about 1500 to about 1800 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1500 to about 1550 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1550 to about 1600 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1600 to about 1640 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1640 to about 1680 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1680 to about 1700 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1700 to about 1750 mg. In several embodiments, the atezolizumab is administered in a dose ranging from about 1750 to about 1800 mg.

[00226] In several embodiments, atezolizumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, atezolizumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, atezolizumab is administered concurrently with engineered immune cells. In several embodiments, atezolizumab is administered after administration of engineered immune cells.

[00227] In several embodiments, the therapeutic agent administered is bevacizumab. In several embodiments, the bevacizumab is administered as an IV infusion. Depending on the embodiment, bevacizumab is infused every two weeks or every three weeks. In several embodiments, the bevacizumab is administered in a dose ranging from about 2.5 mg/kg to about 20 mg/kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 2.5 mg/kg to about 5 mg/kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 5 mg/kg to about 7.5 mg/kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 7.5 mg/kg to about 10 mg/kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 10 mg/kg to about 12.5 mg/kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 12.5 mg/kg to about 15 mg/kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 15 mg/kg to about 17.5 mg/kg. In several embodiments, the bevacizumab is administered in a dose ranging from about 17.5 mg/kg to about 20 mg/kg.

[00228] According to several embodiments, the bevacizumab is optionally combined with one or more of cisplatin, paclitaxel, topotecan, interferon (e.g., interferon alpha), paclitaxel and carboplatin, atezolizumab, pegylated liposomal doxorubicin, carboplatin, gemcitabine, fluoropyrimidine-based chemotherapy, 5-fl uorouracil (5-FU)/leucovorin (LV)Zirinotecan (IFL), and 5-FU/LV/oxaliplatin (FOLFOX4). [00229] In several embodiments, bevacizumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, bevacizumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, bevacizumab is administered concurrently with engineered immune cells. In several embodiments, bevacizumab is administered after administration of engineered immune cells.

[00230] In several embodiments, the therapeutic agent administered is levantinib. In several embodiments, levantinib is administered orally (e.g., in a tableted form). In several embodiments, the dose of levantinib ranges from about 1 mg to about 12 mg per day. In several embodiments, the dose of levantinib ranges from about 1 mg to about 2 mg per day. In several embodiments, the dose of levantinib ranges from about 2 mg to about 3 mg per day. In several embodiments, the dose of levantinib ranges from about 3 mg to about 4 mg per day. In several embodiments, the dose of levantinib ranges from about

4 mg to about 5 mg per day. In several embodiments, the dose of levantinib ranges from about 5 mg to about 6 mg per day. In several embodiments, the dose of levantinib ranges from about 6 mg to about 7 mg per day. In several embodiments, the dose of levantinib ranges from about 7 mg to about 8 mg per day. In several embodiments, the dose of levantinib ranges from about 8 mg to about 9 mg per day. In several embodiments, the dose of levantinib ranges from about 9 mg to about 10 mg per day. In several embodiments, the dose of levantinib ranges from about 10 mg to about 1 1 mg per day. In several embodiments, the dose of levantinib ranges from about 1 1 mg to about 12 mg per day. In several embodiments, the total dose is broken into two administrations per day, for example separated by 8 to 12 hours. For example, for a total daily dose of 10 mg, a 5mg administration (e.g., a 5 mg tablet) is ingested by a subject in the morning and a second 5 mg administration is ingested in the evening (e.g., an additional

5 mg tablet).

[00231] In several embodiments, the levantinib is optionally administered in conjunction with a dose of between about 150 mg and about 300 mg of pembrolizumab (on a 3 week cycle) or a dose of between about 350 mg and about 500 mg of pembrolizumab (on a 6 week cycle). In several embodiments, the dose of pembrolizumab ranges from about 150 mg to about 200 mg. In several embodiments, the dose of pembrolizumab ranges from about 200 mg to about 250 mg. In several embodiments, the dose of pembrolizumab ranges from about 250 mg to about 300 mg. In several embodiments, the dose of pembrolizumab ranges from about 350 mg to about 400 mg. In several embodiments, the dose of pembrolizumab ranges from about 400 mg to about 450 mg. In several embodiments, the dose of pembrolizumab ranges from about 450 mg to about 500 mg.

[00232] In several embodiments, levantinib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, levantinib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, levantinib is administered concurrently with engineered immune cells, In several embodiments, levantinib is administered after administration of engineered immune cells. [00233] In several embodiments, the therapeutic agent administered is nivolumab. In several embodiments, the nivolumab is administered as an IV infusion. Depending on the embodiment, nivolumab is infused every two weeks or every four weeks. With the higher frequency dosing schedule, nivolumab is administered in an amount ranging from about 200 to about 300 mg. In several embodiments, the nivolumab is administered in an amount ranging from about 200 mg to about 210 mg. In several embodiments, the nivolumab is administered an amount ranging from about 210 mg to about 220 mg. In several embodiments, the nivolumab is administered an amount ranging from about 220 mg to about 230 mg. In several embodiments, the nivolumab is administered an amount ranging from about 230 mg to about 240 mg. In several embodiments, the nivolumab is administered an amount ranging from about 240 mg to about 250 mg. In several embodiments, the nivolumab is administered an amount ranging from about 250 mg to about 260 mg. In several embodiments, the nivolumab is administered an amount ranging from about 260 mg to about 270 mg. In several embodiments, the nivolumab is administered an amount ranging from about 270 mg to about 280 mg. In several embodiments, the nivolumab is administered an amount ranging from about 280 mg to about 290 mg. In several embodiments, the nivolumab is administered an amount ranging from about 290 mg to about 300 mg. With the lower frequency dosing schedule, the nivolumab is administered an amount ranging from about 400 mg to about 500 mg. In several embodiments, the nivolumab is administered an amount ranging from about 400 mg to about 410 mg. In several embodiments, the nivolumab is administered an amount ranging from about 410 mg to about 420 mg. In several embodiments, the nivolumab is administered an amount ranging from about 420 mg to about 430 mg. In several embodiments, the nivolumab is administered in a dose between about 430 mg to about 440 mg. In several embodiments, the nivolumab is administered an amount ranging from about 440 mg to about 450 mg. In several embodiments, the nivolumab is administered an amount ranging from about 450 mg to about 460 mg. In several embodiments, the nivolumab is administered an amount ranging from about 460 mg to about 470 mg. In several embodiments, the nivolumab is administered in a dose between about 470 mg to about 480 mg. In several embodiments, the nivolumab is administered an amount ranging from about 480 mg to about 490 mg. In several embodiments, the nivolumab is administered an amount ranging from about 490 mg to about 500 mg.

[00234] In several embodiments, an intermediate 3-week administration cycle is used, with the nivolumab administered in an amount ranging from about 300 mg and about 400 mg. In several embodiments, the nivolumab is administered an amount ranging from about 300 mg to about 310 mg. In several embodiments, the nivolumab is administered an amount ranging from about 310 mg to about 320 mg. In several embodiments, the nivolumab is administered an amount ranging from about 320 mg to about 330 mg. In several embodiments, the nivolumab is administered an amount ranging from about 330 mg to about 340 mg. In several embodiments, the nivolumab is administered an amount ranging from about 340 mg to about 350 mg. In several embodiments, the nivolumab is administered an amount ranging from about 350 mg to about 360 mg. In several embodiments, the nivolumab is administered an amount ranging from about 360 mg to about 370 mg. In several embodiments, the nivolumab is administered an amount ranging from about 370 mg to about 380 mg. In several embodiments, the nivolumab is administered an amount ranging from about 380 mg to about 390 mg. In several embodiments, the nivolumab is administered an amount ranging from about 390 mg to about 400 mg.

[00235] In several embodiments, the nivolumab is administered in conjunction with another agent, such as ipilimumab. In several such embodiments, the nivolumab is administered an amount ranging from about 1 mg/kg to about 5 mg/kg. In several embodiments, the nivolumab is administered an amount ranging from about 1 mg/kg to about 2 mg/kg. In several embodiments, the nivolumab is administered an amount ranging from about 2 mg/kg to about 3 mg/kg. In several embodiments, the nivolumab is administered an amount ranging from about 3 mg/kg to about 4 mg/kg. In several embodiments, the nivolumab is administered an amount ranging from about 4 mg/kg to about 5 mg/kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 0.5 mg/kg to about 1 .5 mg/kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 0.5 mg/kg to about .75 mg/kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 0.75 mg/kg to about 1 .0 mg/kg. In several embodiments, the ipilimumab is administered in an amount ranging from about 1 .0 mg/kg to about 1 .5 mg/kg. In several embodiments, the ipilimumab is administered on the same day as a nivolumab infusion for 2, 3, 4, or 5 doses, and then nivolumab is administered alone on an ongoing basis at a dose as described above.

[00236] In several embodiments, nivolumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, nivolumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, nivolumab is administered concurrently with engineered immune cells. In several embodiments, nivolumab is administered after administration of engineered immune cells.

[00237] In several embodiments, the therapeutic agent administered is pembrolizumab. In several embodiments, the pembrolizumab is administered as an IV infusion. Depending on the embodiment, nivolumab is infused every two weeks or every four weeks. With the higher frequency dosing schedule, pembrolizumab is administered in an amount ranging from about 150 to about 250 mg. In several embodiments, the pembrolizumab is administered in an amount ranging from about 150 mg to about 160 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 160 mg to about 170 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 170 mg to about 180 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 180 mg to about 190 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 190 mg to about 200 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 200 mg to about 210 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 210 mg to about 220 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 220 mg to about 230 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 230 mg to about 240 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 240 mg to about 250 mg. With the lower frequency dosing schedule, the pembrolizumab is administered an amount ranging from about 350 mg to about 400 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 350 mg to about 360 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 360 mg to about 370 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 370 mg to about 380 mg. In several embodiments, the pembrolizumab is administered in a dose between about 380 mg to about 390 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 390 mg to about 400 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 400 mg to about 410 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 410 mg to about 420 mg. In several embodiments, the pembrolizumab is administered in a dose between about 420 mg to about 430 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 430 mg to about 440 mg. In several embodiments, the pembrolizumab is administered an amount ranging from about 440 mg to about 450 mg.

[00238] In several embodiments, pembrolizumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, pembrolizumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, pembrolizumab is administered concurrently with engineered immune cells. In several embodiments, pembrolizumab is administered after administration of engineered immune cells.

[00239] In several embodiments, the therapeutic agent administered is ipilimumab. In several embodiments, the ipilimumab is administered as an IV infusion. In several embodiments, the ipilimumab is infused every three weeks. In some embodiments, ipilimumab is administered on a ten-week cycle (for example after several three-week cycles). In several embodiments, ipilimumab is administered in an amount ranging from about 1 mg/kg to about 10 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 1 mg/kg to about 2 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 2 mg/kg to about 3 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 3 mg/kg to about 4 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 4 mg/kg to about 5 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 5 mg/kg to about 6 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 6 mg/kg to about 7 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 7 mg/kg to about 8 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 8 mg/kg to about 9 mg/kg. In several embodiments, ipilimumab is administered in an amount ranging from about 9 mg/kg to about 10 mg/kg.

[00240] In several embodiments, ipilimumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, ipilimumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, ipilimumab is administered concurrently with engineered immune cells. In several embodiments, ipilimumab is administered after administration of engineered immune cells.

[00241] In several embodiments, ipilimumab is optionally administered in conjunction with nivolumab, as discussed above.

[00242] In several embodiments, the therapeutic agent administered is ramucirumab. In several embodiments, the ramucirumab is administered as an IV infusion. In several embodiments, the ramucirumab is infused every two weeks. In some embodiments, ipilimumab is administered on a 21 -day cycle (e.g., on day 1 ). In several embodiments, ramucirumab is administered on a 28-day cycle (e.g., on days 1 and 15). In several embodiments, ramucirumab is administered in an amount ranging from about 5 mg/kg to about 15 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 5 mg/kg to about 6 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 6 mg/kg to about 7 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 7 mg/kg to about 8 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 8 mg/kg to about 9 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 9 mg/kg to about 10 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 10 mg/kg to about 1 1 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 1 1 mg/kg to about 12 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 12 mg/kg to about 13 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 13 mg/kg to about 14 mg/kg. In several embodiments, ramucirumab is administered in an amount ranging from about 14 mg/kg to about 15 mg/kg.

[00243] In several embodiments, ramucirumab is optionally administered in conjunction additional agents. For example, in several embodiments, ramucirumab is administered in conjunction with docetaxel (e.g., at a dose of about 50, about 75, or about 100 mg/m2 on day 1 of a 28-day cycle). For example, in several embodiments, ramucirumab is administered in conjunction with eroltinib (e.g., at a dose of about 100, about 150, or about 150 mg daily). For example, in several embodiments, ramucirumab is administered in conjunction with paclitaxel (e.g., at a dose of about 60, about 80, or about 100 mg/m2 on days 1 , 8, and 15 of a 28-day cycle).

[00244] In several embodiments, ramucirumab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, ramucirumab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, ramucirumab is administered concurrently with engineered immune cells. In several embodiments, ramucirumab is administered after administration of engineered immune cells.

[00245] In several embodiments, the therapeutic agent administered is cabozantinib. In several embodiments, cabozantinib is administered orally (e.g., in a tableted form). In several embodiments, the dose of cabozantinib ranges from about 10 mg to about 100 mg per day. In several embodiments, the dose of cabozantinib ranges from about 10 mg to about 20 mg per day. In several embodiments, the dose of cabozantinib ranges from about 20 mg to about 30 mg per day. In several embodiments, the dose of cabozantinib ranges from about 30 mg to about 40 mg per day. In several embodiments, the dose of cabozantinib ranges from about 40 mg to about 50 mg per day. In several embodiments, the dose of cabozantinib ranges from about 50 mg to about 60 mg per day. In several embodiments, the dose of cabozantinib ranges from about 60 mg to about 70 mg per day. In several embodiments, the dose of cabozantinib ranges from about 70 mg to about 80 mg per day. In several embodiments, the dose of cabozantinib ranges from about 80 mg to about 90 mg per day. In several embodiments, the dose of cabozantinib ranges from about 90 mg to about 100 mg per day.

[00246] In several embodiments, cabozantinib is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, cabozantinib is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, cabozantinib is administered concurrently with engineered immune cells. In several embodiments, cabozantinib is administered after administration of engineered immune cells.

[00247] In several embodiments, the therapeutic agent administered is doxorubicin. In several embodiments, doxorubicin is administered intravenously (e.g., by bolus injection). In several embodiments, doxorubicin is administered on day 1 of a 21 -day dosing cycle. In several embodiments, the dose of doxorubicin ranges from about 20 mg/m² to about 100 mg/m². In several embodiments, the dose of doxorubicin ranges from about 20 mg/m² to about 30 mg/m². In several embodiments, the dose of doxorubicin ranges from about 30 mg/m² to about 40 mg/m².

several embodiments, the dose of doxorubicin ranges from about 40 mg/m² to about 50 mg/m².

several embodiments, the dose of doxorubicin ranges from about 50 mg/m² to about 60 mg/m².

several embodiments, the dose of doxorubicin ranges from about 60 mg/m² to about 65 mg/m².

several embodiments, the dose of doxorubicin ranges from about 65 mg/m² to about 70 mg/m². In several embodiments, the dose of doxorubicin ranges from about 70 mg/m² to about 75 mg/m². In several embodiments, the dose of doxorubicin ranges from about 75 mg/m² to about 80 mg/m². In several embodiments, the dose of doxorubicin ranges from about 80 mg/m² to about 90 mg/m². In several embodiments, the dose of doxorubicin ranges from about 90 mg/m² to about 100 mg/m².

[00248] In several embodiments, doxorubicin is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, doxorubicin is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, doxorubicin is administered concurrently with engineered immune cells. In several embodiments, doxorubicin is administered after administration of engineered immune cells.

[00249] In several embodiments, the therapeutic agent administered is gemcitabine. In several embodiments, gemcitabine is administered intravenously (e.g., by infusion). In several embodiments, the dose of gemcitabine ranges from about 850 mg/m² to about 1500 mg/m². In several embodiments, the dose of gemcitabine ranges from about 850 mg/m² to about 900 mg/m². In several embodiments, the dose of gemcitabine ranges from about 900 mg/m² to about 950 mg/m². In several embodiments, the dose of gemcitabine ranges from about 950 mg/m² to about 1000 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1000 mg/m² to about 1050 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1050 mg/m² to about 1 100 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1 100 mg/m² to about 1 150 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1 150 mg/m² to about 1200 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1200 mg/m² to about 1250 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1250 mg/m² to about 1300 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1300 mg/m² to about 1400 mg/m². In several embodiments, the dose of gemcitabine ranges from about 1400 mg/m² to about 1500 mg/m².

[00250] In several embodiments, gemcitabine is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, gemcitabine is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, gemcitabine is administered concurrently with engineered immune cells. In several embodiments, gemcitabine is administered after administration of engineered immune cells.

[00251] In several embodiments, the therapeutic agent administered is cetuximab. In several embodiments, cetuximab is administered intravenously (e.g., by infusion). In several embodiments, cetuximab is given in a weekly dose format. In several embodiments, cetuximab is given in a bi-weekly format. For the weekly format, in several embodiments, an initial dose is infused at an elevated concentration (e.g., as a loading dose) followed by a lower concentration for each subsequent dose, In several embodiments, the first dose of cetuximab ranges from about 300 mg/m² to about 500 mg/m². In several embodiments, the first dose of cetuximab ranges from about 300 mg/m² to about 325 mg/m². In several embodiments, the first dose of cetuximab ranges from about 325 mg/m² to about 350 mg/m². In several embodiments, the first dose of cetuximab ranges from about 350 mg/m² to about 375 mg/m². In several embodiments, the first dose of cetuximab ranges from about 375 mg/m² to about 400 mg/m². In several embodiments, the first dose of cetuximab ranges from about 400 mg/m² to about 425 mg/m². In several embodiments, the first dose of cetuximab ranges from about 425 mg/m² to about 450 mg/m². In several embodiments, the first dose of cetuximab ranges from about 450 mg/m² to about 475 mg/m². In several embodiments, the first dose of cetuximab ranges from about 475 mg/m² to about 500 mg/m². In several embodiments, the subsequent doses of cetuximab range from about 200 mg/m² to about 300 mg/m². In several embodiments, the subsequent doses of cetuximab range from about 200 mg/m² to about 225 mg/m². In several embodiments, the subsequent doses of cetuximab range from about 225 mg/m² to about 250 mg/m². In several embodiments, the subsequent doses of cetuximab range from about 250 mg/m² to about 275 mg/m². In several embodiments, the subsequent doses of cetuximab range from about 275 mg/m² to about 300 mg/m². For the bi-weekly format, in several embodiments, each dose of cetuximab ranges from about 400 mg/m² to about 600 mg/m². In several embodiments, each dose of cetuximab ranges from about 400 mg/m² to about 425 mg/m². In several embodiments, each dose of cetuximab ranges from about 425 mg/m² to about 450 mg/m². several embodiments, each dose of cetuximab ranges from about 450 mg/m² to about 475 mg/m². In several embodiments, each dose of cetuximab ranges from about 475 mg/m² to about 500 mg/m². In several embodiments, each dose of cetuximab ranges from about 500 mg/m² to about 525 mg/m². In several embodiments, each dose of cetuximab ranges from about 525 mg/m² to about 550 mg/m². In several embodiments, each dose of cetuximab ranges from about 550 mg/m² to about 575 mg/m². In several embodiments, each dose of cetuximab ranges from about 575 mg/m² to about 600 mg/m².

[00252] In several embodiments, cetuximab is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, cetuximab is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, cetuximab is administered concurrently with engineered immune cells. In several embodiments, cetuximab is administered after administration of engineered immune cells.

[00253] In several embodiments, the therapeutic agent administered is irinotecan (e.g., irinotecan hydrochloride). In several embodiments, irinotecan is administered intravenously (e.g., by infusion). In several embodiments, the dose of irinotecan ranges from about 10 mg/m² to about 1000 mg/m². In several embodiments, the dose of irinotecan ranges from about 20 mg/m² to about 500 mg/m². In several embodiments, the dose of irinotecan ranges from about 50 mg/m² to about 125 mg/m². In several embodiments, the dose of irinotecan is about 20 mg/m². In several embodiments, the dose of irinotecan is about 20 mg/m². In several embodiments, the dose of irinotecan is about 30 mg/m². In several embodiments, the dose of irinotecan is about 40 mg/m². In several embodiments, the dose of irinotecan is about 50 mg/m². In several embodiments, the dose of irinotecan is about 60 mg/m². In several embodiments, the dose of irinotecan is about 70 mg/m². In several embodiments, the dose of irinotecan is about 75 mg/m². In several embodiments, the dose of irinotecan is about 80 mg/m². In several embodiments, the dose of irinotecan is about 90 mg/m². In several embodiments, the dose of irinotecan is about 100 mg/m². In several embodiments, the dose of irinotecan is about 120 mg/m². In several embodiments, the dose of irinotecan is about 125 mg/m². In several embodiments, the dose of irinotecan is about 150 mg/m². In several embodiments, the dose of irinotecan is about 180 mg/m². In several embodiments, the dose of irinotecan is about 200 mg/m². In several embodiments, the dose of irinotecan is about 240 mg/m². In several embodiments, the dose of irinotecan is about 250 mg/m². In several embodiments, the dose of irinotecan is about 300 mg/m². In several embodiments, the dose of irinotecan is about 320 mg/m². In several embodiments, the dose of irinotecan is about 350 mg/m². In several embodiments, the dose of irinotecan is about 360 mg/m². In several embodiments, the dose of irinotecan is about 400 mg/m². In several embodiments, the dose of irinotecan is about 450 mg/m². In several embodiments, the dose of irinotecan is about 480 mg/m². In several embodiments, the dose of irinotecan is about 500 mg/m². In several embodiments, the dose of irinotecan is about 600 mg/m². In several embodiments, the dose of irinotecan is about 700 mg/m². In several embodiments, the dose of irinotecan is about 750 mg/m². In several embodiments, the dose of irinotecan is about 800 mg/m². In several embodiments, the dose of irinotecan is about 900 mg/m². In several embodiments, the dose of irinotecan is about 1000 mg/m².

[00254] In several embodiments, one dose of irinotecan is administered each week (e.g., every 7 days). In several embodiments, one dose of irinotecan is administered each two-week period (e.g., every 14 days). In several embodiments, one dose of irinotecan is administered each three-week period (e.g., every 21 days). In several embodiments, two doses of irinotecan are administered each week. In several embodiments, two doses of irinotecan are administered each two-week period (e.g., each 14 days). In several embodiments, two doses of irinotecan are administered each three-week period (e.g., each 21 days).

[00255] In several embodiments, irinotecan is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, irinotecan is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, irinotecan is administered concurrently with engineered immune cells. In several embodiments, irinotecan is administered after administration of engineered immune cells.

[00256] In several embodiments, the therapeutic agent administered is capecitabine. In several embodiments, capecitabine is administered orally. In several embodiments, the dose of capecitabine ranges from about 1000 mg/m² to about 5000 mg/m². In several embodiments, the dose of capecitabine ranges from about 1250 mg/m² to about 2500 mg/m². In several embodiments, the dose of capecitabine is about 1250 mg/m². In several embodiments, the dose of capecitabine is about 2500 mg/m². In several embodiments, the dose of capecitabine is about 3750 mg/m². In several embodiments, the dose of capecitabine is about 5000 mg/m².

[00257] 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, following by a one-week rest period, given as a three-week cycle. In some embodiments, the dose is divided between two administrations. For example, in some embodiments, a daily dose of 2500 mg/m² is divided between two administrations of 1250 mg/m². In some embodiments, 1250 mg/m² capecitabine is administered orally two times a day (e.g., morning and evening) for a 2500 mg/m² total daily dose, for two weeks followed by a one-week rest period, given as three-week cycle. In some embodiments, a subject is administered a total of 4 cycles. In some embodiments, a subject is administered a total of 8 cycles.

[00258] In several embodiments, capecitabine is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, capecitabine is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, capecitabine is administered concurrently with engineered immune cells. In several embodiments, capecitabine is administered after administration of engineered immune cells.

[00259] In several embodiments, the therapeutic agent administered is vinorelbine. In several embodiments, vinorelbine is administered intravenously (e.g., by infusion). In several embodiments, the dose of vinorelbine ranges from about 5 mg/m² to about 50 mg/m². In several embodiments, the dose of vinorelbine ranges from about 10 mg/m² to about 45 mg/m². In several embodiments, the dose of vinorelbine ranges from about 15 mg/m² to about 40 mg/m². In several embodiments, the dose of vinorelbine ranges from about 20 mg/m² to about 35 mg/m². In several embodiments, the dose of vinorelbine ranges from about 25 mg/m² to about 30 mg/m². In several embodiments, the dose of vinorelbine is about 6.25 mg/m². In several embodiments, the dose of vinorelbine is about 7.5 mg/m². In several embodiments, the dose of vinorelbine is about 10 mg/m². In several embodiments, the dose of vinorelbine is about 12.5 mg/m². In several embodiments, the dose of vinorelbine is about 15 mg/m². In several embodiments, the dose of vinorelbine is about 17.5 mg/m². In several embodiments, the dose of vinorelbine is about 18.75 mg/m². In several embodiments, the dose of vinorelbine is about 20 mg/m². In several embodiments, the dose of vinorelbine is about 22.5 mg/m². In several embodiments, the dose of vinorelbine is about 25 mg/m². In several embodiments, the dose of vinorelbine is about 27.5 mg/m². In several embodiments, the dose of vinorelbine is about 30 mg/m².

[00260] In some embodiments, a dose of vinorelbine is administered once each week (e.g., every 7 days). In some embodiments, vinorelbine is administered in a 28-day cycle. In some embodiments, a dose of vinorelbine is administered once each week for four weeks (e.g., in a 28-day cycle).

[00261] In several embodiments, vinorelbine is administered prior to lymphodepletion as described herein (e.g., also prior to administration of engineered immune cells). In several embodiments, vinorelbine is administered between lymphodepletion and administration of engineered immune cells. In several embodiments, vinorelbine is administered concurrently with engineered immune cells. In several embodiments, vinorelbine is administered after administration of engineered immune cells.

IV. Additional Cancer Targets

[00262] Some embodiments of the compositions and methods described herein relate to immune cells comprising a chimeric receptor that targets a cancer antigen, such as MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, and/or ULBP6. Additional non-limiting examples of target antigens include: CD70, CD5, CD19; CD123; CD22; CD30; CD171 ; CS1 (also referred to as CD2 subset 1 , CRACC, SLAMF7, CD319, and 19A24); TNF receptor family member B cell maturation (BCMA) ; CD38; DLL3; G protein coupled receptor class C group 5, member D (GPRC5D); epidermal growth factor receptor (EGFR) CD138; prostate-specific membrane antigen (PSMA); Fms Like Tyrosine Kinase 3 (FLT3); KREMEN2 (Kringle Containing Transmembrane Protein 2), ALPPL2, Claudin 4, Claudin 6, C-type lectin-like molecule-1 (CLL- 1 or CLECL1 ); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4 )bDGIcp(l-l)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; a glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors, a glycosylated CD43 epitope expressed on non-hematopoietic cancers, Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD1 17); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 1 1 receptor alpha (IL-IIRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21 ); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR- beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha (FRa or FR1 ); Folate receptor beta (FRb); Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1 , cell surface associated (MUC1 ); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (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)bDCIalp(l-4)bDGIcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); chromosome X open reading frame 61 (CXORF61 ); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1 ); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1 ); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1 ); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51 E2 (OR51 E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1 ); Cancer/testis antigen 1 (NY-ES0-1 ); Cancer/testis antigen 2 (LAGE-la); Melanoma- associated antigen 1 (MAGE-A1 ); ETS translocation-variant gene 6, located on chromosome 12p (ETV6- AML); sperm protein 17 (SPA17); X Antigen Family, Member 1 A (XAGE1 ); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1 ); melanoma cancer testis antigen-2 (MAD- CT-2); Fos-related antigen 1 ; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCT A-l or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase; reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 IB 1 (CYPIB 1 ); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator oflmprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1 ); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Gly cation Endproducts (RAGE-1 ); renal ubiquitous 1 (RU1 ); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1 ); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLLI), MPL, Biotin, c-MYC epitope Tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1 , CDH19, CD200R, Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GMI, PTK7, gpNMB, CDH1 -CD324, DLL3, CD276/B7H3, ILI IRa, IL13Ra2, CD179b-IGLII, TCRgamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Timl-/HVCR1 , CSF2RA (GM-CSFR-alpha), TGFbetaR2, Lews Ag, TCR-betal chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, Leutenizing hormone receptor (LHR), Follicle stimulating hormone receptor (FSHR), Gonadotropin Hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLVI-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1 , KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1 , Guanylyl cyclase C (GCC), auto antibody to desmoglein 3 (Dsg3), auto antibody to desmoglein 1 (Dsgl), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA- DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1 ), AFP, GPRC5D, Claudinl 8.2 (CLD18A2 or CLDN18A.2)), P-glycoprotein, STEAP1 , Livl, Nectin-4, Cripto, gpA33, BST1/CD157, low conductance chloride channel, and the antigen recognized by TNT antibody.

DEFINITIONS

[00263] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood.

[00264] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PGR amplification. [00265] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[00266] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[00267] “Preventing” (and grammatical variations thereof such as “prevent” or “prevention”) as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.

[00268] A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.

[00269] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[00270] 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.”

[00271] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[00272] In addition, when a sequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a reference shall also include, unless otherwise indicated, that the sequence “comprises”, “consists of” or “consists essentially of” the recited sequence.

[00273] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, nonaqueous or any combination thereof.

NON-LIMITING EMBODIMENTS

[00274] Among the embodiments provided herein are:

1 . A method of treating a cancer in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) a first dose of the genetically engineered NK cells;

(II) a second dose of the genetically engineered NK cells administered to the subject 2-4 days after administration of the first dose of the genetically engineered cells; and

(ill) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

2. A method of treating a cancer in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) a first dose of the genetically engineered NK cells;

(ii) a second dose of the genetically engineered NK cells administered to the subject 2-4 days after administration of the first dose of the genetically engineered cells; and (iii) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

3. The method of embodiment 1 or embodiment 2, wherein all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

4. A method of treating a cancer in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

5. A method of treating a cancer in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

6. The method of embodiment 4 or embodiment 5, wherein the second dose of the genetically engineered NK cells is administered to the subject 2-4 days after administration of the first dose of the genetically engineered cells; and the third dose of the genetically engineered NK cells is administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells.

7. The method of any one of embodiments 1 -6, wherein the second dose of the genetically engineered NK cells is administered to the subject about 3 days after administration of the first dose of the genetically engineered cells.

8. The method of any one of embodiments 1 -7, wherein the third dose of genetically engineered NK cells is administered to the subject about 4 days after administration of the second dose of the genetically engineered NK cells.

9. The method of any one of embodiments 1 -8, wherein each dose of the genetically engineered NK cells comprises between about 1 x 10⁹ and about 5x 10⁹ genetically engineered NK cells.

10. The method of any one of embodiments 1 -9, wherein each dose of the genetically engineered NK cells comprises between about 2 x 10⁹ and about 5x 10⁹ genetically engineered NK cells.

1 1 . The method of any one of embodiments 1 -9, wherein each dose of the genetically engineered NK cells comprises about 1 .5 x 10⁹ genetically engineered NK cells.

12. The method of any one of embodiments 1 -10, wherein each dose of the genetically engineered NK cells comprises about 2 x 10⁹ genetically engineered NK cells.

13. The method of any one of embodiments 1 -10, wherein each dose of the genetically engineered NK cells comprises about 2.5 x 10⁹ genetically engineered NK cells.

14. The method of any one of embodiments 1 -13, wherein all three doses of the genetically engineered NK cells are administered to the subject within about 10 days of conclusion of administration of the lymphodepleting therapy.

15. The method of any one of embodiments 1 -14, wherein the lymphodepleting therapy comprises administration of fludarabine (Flu).

16. The method of embodiment 15, wherein each dose of Flu comprises between about 10 mg/m² and about 40 mg/m².

17. The method of any one of embodiments 1 -16, wherein the lymphodepleting therapy comprises administration of five doses of Flu.

18. The method of any one of embodiments 1 -17, wherein the lymphodepleting therapy comprises administration cytosine arabinoside (Ara-C).

19. The method of embodiment 18, wherein each dose of Ara-C comprises between about 1 g/m² and about 4 g/m².

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

21 . The method of embodiment 20, wherein the first doses of Flu and Ara-C are each given 7 days prior to initiation of the dosing cycle; the second doses of Flu and Ara-C are each given 6 days prior to initiation of the dosing cycle; the third doses of Flu and Ara-C are each given 5 days prior to initiation of the dosing cycle; the fourth doses of Flu and Ara-C are each given 4 days prior to initiation of the dosing cycle; and the fifth doses of Flu and Ara-C are each given 3 days prior to initiation of the dosing cycle.

22. The method of any one of embodiments 18-21 , wherein each dose of Flu comprises about 30 mg/m² and each dose of Ara-C comprises about 2 g/m².

23. The method of any one of embodiments 1 -22, wherein the cancer is a relapsed/refractory acute myeloid leukemia (r/r AML).

24. A method of treating a relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) a first dose of the genetically engineered NK cells;

(ill) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m²fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

25. A method of treating a relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) a first dose of the genetically engineered NK cells;

(Hi) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m²fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

26. The method of any one of embodiments 1 -25, wherein the dosing cycle is between about 21 days and about 35 days.

27. The method of any one of embodiments 1 -26, wherein the dosing cycle is about 28 days. 28. The method of any one of embodiments 1 -27, wherein, if the subject exhibits a clinical response, optionally a partial response (PR), a complete response with incomplete hematologic recovery (CRi), or a complete response (CR), following the dosing cycle, the method comprises administering an additional dosing cycle as a consolidation treatment.

29. The method of any one of embodiments 1 -28, wherein, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the method comprises administering an additional dosing cycle as retreatment.

30. The method of any one of embodiments 1 -29, wherein the method comprises administration of between one dosing cycle and five dosing cycles.

31 . The method of any one of embodiments 28-30, wherein the subject is administered the lymphodepleting therapy prior to each dosing cycle.

32. The method of any one of embodiments 1 -31 , wherein the subject has less than or equal to 5% peripheral blasts, optionally wherein the subject does not have evidence of extramedullary disease.

33. The method of any one of embodiments 1 -32, further comprising:

(a) assessing the level or amount of a NKG2D ligand in a biological sample from the subject, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or a polynucleotide encoded by the NKG2D ligand gene; and

(b) selecting the subject for treatment with the genetically engineered NK cells if the level or amount of the NKG2D ligand is above a reference value, wherein the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells.

34. The method of embodiment 33, wherein the reference value is within 25%, within 20%, within 15%, within 10%, or within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the genetically engineered NK cells who did not exhibit a clinical response following administration of the genetically engineered NK cell.

35. The method of embodiment 33 or embodiment 34, wherein the NKG2D ligand comprises MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof.

36. The method of any one of embodiments 1 -35, wherein the method further comprises administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, a therapeutic agent that increases expression of a NKG2D ligand in the subject, and any combination thereof, optionally wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, and a NK cell engager.

37. The method of embodiment 36, wherein administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells.

38. The method of embodiment any one of embodiments 1 -22 and 26-37, wherein the cancer is acute myeloid leukemia (AML). 39. The method of any one of embodiments 1 -38, wherein the cancer is relapsed/refractory

(R/R).

40. The method of any one of embodiments 1 -39, wherein the subject has been treated with at least one prior line of therapy.

41 . The method of any one of embodiments 1 -40, wherein the subject has been treated with one, two, three, or four prior lines of therapy.

42. The method of any one of embodiments 1 -41 , wherein the subject has been treated with one, two, or three prior lines of therapy.

43. The method of any one of embodiments 1 -43, wherein the subject has an ECOG of 0-2, optionally 0 or 1 .

44. The method of any one of embodiments 1 -43, wherein the subject is 18 years of age or older.

45. The method of any one of embodiments 1 -44, wherein the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling region.

46. The method of embodiment 45, wherein the extracellular binding domain has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42

47. The method of embodiment 45 or embodiment 46, wherein the transmembrane domain comprises a CD8 transmembrane domain.

48. The method of any one of embodiments 45-47, wherein the intracellular signaling region comprises a co-stimulatory domain and a CD3zeta.

49. The method of embodiment 48, wherein the co-stimulatory domain comprises an 0X40 domain.

50. The method of any one of embodiments 1 -49, wherein the chimeric receptor has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39.

51 . The method of any one of embodiments 1 -50, wherein the genetically engineered NK cells express a membrane-bound interleukin 15 (mbll_15).

52. The method of embodiment 51 , wherein the mbll_15 has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40.

53. The method of any one of embodiments 1 -52, wherein the population of engineered NK cells are allogeneic to the subject, optionally wherein the population of engineered NK cells are derived from a donor not having a cancer.

54. The method of any one of embodiments 1 -53, wherein a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis, optionally wherein each dose of the engineered NK cells is administered to the subject on an outpatient basis.

55. The method of any one of embodiments 1 -54, wherein all three doses of the genetically engineered NK cells are administered within about 4-10 days. 56. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) administration of a first dose of the genetically engineered NK cells;

(II) administration of a second dose of the genetically engineered NK cells 2-4 days after administration of the first dose of the genetically engineered cells; and

(ill) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

57. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) administration of a first dose of the genetically engineered NK cells;

(ill) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

58. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy. 59. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

60. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed/refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(I) administration of a first dose of the genetically engineered NK cells;

(ill) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m²fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

61 . Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed/refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) administration of a first dose of the genetically engineered NK cells;

(iii) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m²fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

62. The use of any one of embodiments 58-61 , wherein all three doses of the genetically engineered NK cells are administered within about 4-10 days.

63. The use of any one of embodiments 58-62, wherein all three doses of the genetically engineered NK cells are administered within about 10 days of conclusion of administration of the lymphodepleting therapy

EXAMPLES

[00275] The following are non-limiting descriptions of experimental methods and materials that will be used in examples disclosed below.

Example 1 - Treatment of Acute Myeloid Leukemia with Alternative Dosing of NKG2D Chimeric Receptor- Expressing NK Cells

[00276] Subjects having relapsed/refractory (r/r) acute myeloid leukemia (AML) and treated with one or more prior lines of therapy are treated with allogeneic natural killer (NK) cells expressing a NKG2D chimeric receptor (e.g., SEQ ID NO:39, encoded by SEQ ID NO:33).

[00277] Primary NK cells are isolated by immunoaffinity-based selection from leukapheresis samples from healthy donors. Isolated NK cells are transduced with a viral vector (e.g., retroviral vector) encoding a non-limiting example of a NKG2D chimeric receptor, expanded in culture, and cryopreserved. The NKG2D chimeric receptor contains a NKG2D-based extracellular domain (e.g., SEQ ID NO:26), a CD8alpha hinge (e.g., SEQ ID NO:2) and transmembrane domain (e.g., SEQ ID NO:4), and an intracellular signaling region having an 0X40 co-stimulatory domain (e.g., SEQ ID NO:6) and a CD3zeta domain (e.g., SEQ ID NO:8). Polynucleotides encoding the NKG2D chimeric receptor construct are engineered to bicistronically express membrane-bound interleukin 15 (mblL15; e.g., SEQ ID NO: 40, encoded by SEQ ID NO: 35); the sequences encoding the chimeric receptor and mblL15 are separated by a sequence encoding a T2A ribosomal skip sequence (e.g., SEQ ID NO:10).

[00278] The genetically engineered NK cells are administered in a dosing regimen in which the NK cells are administered three times in a 28-day dosing cycle. The dosing cycle is preceded by a lymphodepleting therapy of fludarabine (Flu: 30 mg/m²) and cytosine arabinoside (Ara-C; 2 g/m²) on each of Days -7, -6, -5, -4, and -3. Alternatively, the dosing cycle is preceded by a lymphodepleting therapy of fludarabine (Flu; 30 mg/m²) and cyclophosphamide (Cy; 500 mg/m²) on each of Days -5, -4, and -3. Subjects are optionally administered decitabine at a dose of 20 mg/m² on each of Days -5, -4, -3, -2, and - 1 .

[00279] On each of days 0, 3, and 7, subjects are administered 1 .5 x 10⁹, 2 x 10⁹, or 2.5 x 10⁹ genetically engineered NK cells. Alternatively, on each of days 0, 3, and 7, subjects are administered 1 x 10⁹ genetically engineered NK cells. Efficacy is assessed at the end of the 28-day dosing cycle. Subjects who exhibit a clinical response are allowed treatment with an additional dosing cycle as consolidation treatment. Subjects who initially exhibit a clinical response and subsequently progress are allowed retreatment with an additional dosing cycle. Each cycle is optionally preceded by the lymphodepleting therapy.

[00280] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present inventions herein disclosed should not be limited by the particular disclosed embodiments described above. Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00281] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between," and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “about 90%” includes “90%. ” In some embodiments, at least 95% sequence identity or homology includes 96%, 97%, 98%, 99%, and 100% sequence identity or homology to the reference sequence. In addition, when a sequence is disclosed as “comprising” a nucleotide or amino acid sequence, such a reference shall also include, unless otherwise indicated, that the sequence “comprises”, “consists of” or “consists essentially of” the recited sequence. Any titles or subheadings used herein are for organization purposes and should not be used to limit the scope of embodiments disclosed herein. Sequences

[00282] In several embodiments, there are provided amino acid sequences that correspond to any of the nucleic acids disclosed herein (and/or included in the accompanying sequence listing), while accounting for degeneracy of the nucleic acid code. Furthermore, those sequences (whether nucleic acid or amino acid) that vary from those expressly disclosed herein (and/or included in the accompanying sequence listing), but have functional similarity or equivalency are also contemplated within the scope of the present disclosure. The foregoing includes mutants, truncations, substitutions, codon optimization, or other types of modifications.

[00283] In accordance with some embodiments described herein, any of the sequences may be used, or a truncated or mutated form of any of the sequences disclosed herein (and/or included in the accompanying sequence listing) may be used and in any combination.

Claims

WHAT IS CLAIMED IS:

(i) a first dose of the genetically engineered NK cells;

(iii) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

(i) a first dose of the genetically engineered NK cells;

(iii) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

3. The method of claim 1 , wherein: all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

6. The method of claim 4, wherein the second dose of the genetically engineered NK cells is administered to the subject 2-4 days after administration of the first dose of the genetically engineered cells; and the third dose of the genetically engineered NK cells is administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells.

7. The method of claim 1 , wherein the second dose of the genetically engineered NK cells is administered to the subject about 3 days after administration of the first dose of the genetically engineered cells.

8. The method of claim 1 , wherein the third dose of genetically engineered NK cells is administered to the subject about 4 days after administration of the second dose of the genetically engineered NK cells.

9. The method of claim 1 , wherein each dose of the genetically engineered NK cells comprises between about 1 x 10⁹ and about 5x 10⁹ genetically engineered NK cells.

10. The method of claim 1 , wherein each dose of the genetically engineered NK cells comprises about 1 .5 x 10⁹ genetically engineered NK cells.

1 1 . The method of claim 1 , wherein each dose of the genetically engineered NK cells comprises about 2 x 10⁹ genetically engineered NK cells.

12. The method of claim 1 , wherein each dose of the genetically engineered NK cells comprises about 2.5 x 10⁹ genetically engineered NK cells.

13. The method of claim 1 , wherein all three doses of the genetically engineered NK cells are administered to the subject within about 10 days of conclusion of administration of the lymphodepleting therapy.

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

15. The method of claim 14, wherein each dose of Flu comprises between about 10 mg/m² and about 40 mg/m².

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

17. The method of claim 1 , wherein the lymphodepleting therapy comprises administration cytosine arabinoside (Ara-C).

18. The method of claim 17, wherein each dose of Ara-C comprises between about 1 g/m² and about 4 g/m².

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

20. The method of claim 19, wherein the first doses of Flu and Ara-C are each given 7 days prior to initiation of the dosing cycle; the second doses of Flu and Ara-C are each given 6 days prior to initiation of the dosing cycle; the third doses of Flu and Ara-C are each given 5 days prior to initiation of the dosing cycle; the fourth doses of Flu and Ara-C are each given 4 days prior to initiation of the dosing cycle; and the fifth doses of Flu and Ara-C are each given 3 days prior to initiation of the dosing cycle.

21 . The method of claim 17, wherein each dose of Flu comprises about 30 mg/m² and each dose of Ara-C comprises about 2 g/m².

22. The method of claim 1 , wherein the cancer is a relapsed/refractory acute myeloid leukemia (r/r AML).

23. A method of treating a relapsed/refractory acute myeloid leukemia (r/r AML) in a subject comprising administering to a subject having a cancer a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) a first dose of the genetically engineered NK cells;

(Hi) a third dose of genetically engineered NK cells administered to the subject 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m²fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

(i) a first dose of the genetically engineered NK cells;

25. The method of any one of claims 1 -24, wherein the dosing cycle is between about 21 days and about 35 days.

26. The method of any one of claims 1 -25, wherein the dosing cycle is about 28 days.

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

28. The method of any one of claims 1 -27, wherein, if the subject exhibits a clinical response following the dosing cycle and subsequently exhibits disease progression, the method comprises administering an additional dosing cycle as retreatment.

29. The method of any one of claims 1 -28 wherein the method comprises administration of between one dosing cycle and five dosing cycles.

30. The method of any one of claims 27-29, wherein the subject is administered the lymphodepleting therapy prior to each dosing cycle.

31 . The method of any one of claims 1 -30, wherein the subject has less than or equal to 5% peripheral blasts, optionally wherein the subject does not have evidence of extramedullary disease.

32. The method of any one of claims 1 -31 , further comprising:

(a) assessing the level or amount of a NKG2D ligand in a biological sample from the subject, wherein the level or amount of the NKG2D ligand is the level or amount of a protein or a polynucleotide encoded by the NKG2D ligand gene; and (b) selecting the subject for treatment with the genetically engineered NK cells if the level or amount of the NKG2D ligand is above a reference value, wherein the biological sample is obtained from the subject prior to the administration of the genetically engineered NK cells.

33. The method of claim 32, wherein the reference value is within 25%, within 20%, within 15%, within 10%, or within 5% of the average level or amount of the NKG2D ligand in a population of subjects having the cancer and administered the genetically engineered NK cells who did not exhibit a clinical response following administration of the genetically engineered NK cell.

34. The method of claim 32 or claim 33, wherein the NKG2D ligand comprises MICA, MICB, ULBP1 , ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or any combination thereof.

35. The method of any one of claims 1 -34, wherein the method further comprises administering to the subject a therapeutic agent selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, a NK cell engager, a therapeutic agent that increases expression of a NKG2D ligand in the subject, and any combination thereof, optionally wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a monoclonal antibody, and a NK cell engager.

36. The method of claim 35, wherein administration of the therapeutic agent is prior to administration of the first dose of the genetically engineered NK cells.

37. The method of claim any one of claims 1 -21 and 25-36, wherein the cancer is acute myeloid leukemia (AML).

38. The method of any one of claims 1 -37, wherein the cancer is relapsed/refractory (R/R).

39. The method of any one of claims 1 -38, wherein the subject has been treated with at least one prior line of therapy.

40. The method of any one of claims 1 -39, wherein the subject has been treated with one, two, three, or four prior lines of therapy.

41 . The method of any one of claims 1 -40, wherein the subject has an ECOG of 0-2, optionally 0 or 1 .

42. The method of any one of claims 1 -41 , wherein the subject is 18 years of age or older.

43. The method of any one of claims 1 -42, wherein the chimeric receptor comprises an extracellular binding domain, a transmembrane domain, and an intracellular signaling region.

44. The method of claim 43, wherein the extracellular binding domain has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42

45. The method of claim 43 or claim 44, wherein the transmembrane domain comprises a CD8 transmembrane domain.

46. The method of any one of claims 43-45, wherein the intracellular signaling region comprises a co-stimulatory domain and a CD3zeta.

47. The method of claim 46, wherein the co-stimulatory domain comprises an 0X40 domain.

48. The method of any one of claims 1 -47, wherein the chimeric receptor has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39.

49. The method of any one of claims 1 -48, wherein the genetically engineered NK cells express a membrane-bound interleukin 15 (mblL15).

50. The method of claim 49, wherein the mblL15 has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40.

51 . The method of any one of claims 1 -50, wherein the population of engineered NK cells are allogeneic to the subject, optionally wherein the population of engineered NK cells are derived from a donor not having a cancer.

52. The method of any one of claims 1 -51 , wherein a dose of the genetically engineered NK cells is administered to the subject on an outpatient basis, optionally wherein each dose of the engineered NK cells is administered to the subject on an outpatient basis.

53. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) administration of a first dose of the genetically engineered NK cells;

(Hi) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

54. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) administration of a first dose of the genetically engineered NK cells;

(Hi) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy.

55. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

56. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having a cancer, wherein: the genetically engineered NK cells are administered in a dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells and a third dose of genetically engineered NK cells; each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells; prior to administration of the first dose of the genetically engineered NK cells to the subject, the subject is administered a lymphodepleting therapy; and all three doses of the genetically engineered NK cells are administered to the subject within about 14 days of conclusion of administration of the lymphodepleting therapy.

57. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed/refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(I) administration of a first dose of the genetically engineered NK cells;

(iii) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 1 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m²fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

58. Use of a population of Natural Killer (NK) cells genetically engineered to express a chimeric receptor that binds a ligand of natural killer cell group 2D (NKG2D) for treating a subject having relapsed/refractory acute myeloid leukemia (r/r AML), wherein the genetically engineered NK cells are administered in a dosing cycle comprising:

(i) administration of a first dose of the genetically engineered NK cells;

(Hi) administration of a third dose of genetically engineered NK cells 2-4 days after administration of the second dose of the genetically engineered NK cells, wherein each dose of the genetically engineered NK cells comprises at least about 2 x 10⁹ genetically engineered NK cells, and wherein the subject is administered a lymphodepleting therapy comprising daily administration of about 30 mg/m²fludarabine (Flu) and about 2 g/m² cytosine arabinoside (Ara-C) on each of 7, 6, 5, 4, and 3 days prior to administration of the first dose of the genetically engineered NK cells.

59. The use of any one of claims 53-58, wherein all three doses of the genetically engineered NK cells are administered within about 10 days of conclusion of administration of the lymphodepleting therapy