Dosing regimens for cancer immunotherapy

Abstract

Some embodiments of the methods and compositions disclosed herein relate to immune cells engineered to express cytotoxic chimeric receptors, and various dosing regimens for administering such cells. In some embodiments, the immune cells express chimeric receptors that target ligands of NKG2D on tumor cells. In some embodiments, the cancer is a blood cancer, such as acute myeloid leukemia (e.g., relapsed / refractory acute myeloid leukemia) or myelodysplastic syndrome. In some embodiments, the tumor is a solid tumor, such as intrahepatic cholangiocarcinoma or other liver tumor, such as secondary metastasis from colon cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 201,792, filed May 13, 2021, and U.S. Provisional Patent Application No. 63 / 269,316, filed March 14, 2022, the entire contents of each of which are incorporated herein by reference.

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

[0003] As more is learned about different cancers and what properties cancerous cells have that can be used to specifically distinguish them from healthy cells, therapeutic agents are being developed that exploit the distinctive features of cancerous cells. Immunotherapy using engineered immune cells is one approach to treat cancer.

[0004] Incorporating information by reference in an ASCII text file This application incorporates by reference the sequence listing contained in the following ASCII text file submitted simultaneously: Filename: NKT.078WO_ST25.txt; created on May 7, 2022 and is 27,762 bytes in size. Summary of the Invention

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

[0006] In some embodiments, provided herein is a genetically engineered natural killer (NK) cell population for cancer immunotherapy, comprising a plurality of NK cells expanded in culture, wherein the plurality of NK cells are engineered to express a cytotoxic receptor complex comprising NKG2D, a ligand binding domain, a transmembrane domain, and a cytotoxic signaling complex.

[0007] In some embodiments, a dosing regimen for cancer immunotherapy is provided, comprising at least a first dosing cycle, the first dosing cycle comprising a first dose of engineered natural killer (NK) cells, a second dose of engineered NK cells, and a third dose of engineered NK cells, wherein the first dose is administered to a subject in need of cancer immunotherapy at a first time point, the second dose is administered to the subject 5-10 days after the first time point, and the third dose is administered to the subject 5-10 days after the second dose, each of the first, second, and third doses being at least 1.0×10 9 NK cells, at least a portion of the engineered NK cells are engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D). In some embodiments, additional doses can be added to a dosing cycle, e.g., the fourth, fifth or more doses.

[0008] In some embodiments, a method of treating cancer is provided that includes administering to a subject at least a first, second, and third dose of engineered NK cells, where the second dose is administered to the subject 6-8 days after the first dose and the third dose is administered to the subject 6-8 days after the second dose, and each of the first dose, second dose, and third dose is administered to the subject at a concentration of at least 1.0×10 9 The engineered NK cells comprise NK cells, the engineered NK cells being allogeneic to the subject and engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D).

[0009] In some embodiments, there is provided a use of an engineered NK cell population expressing a chimeric receptor targeting a ligand of the NKG2D receptor to treat cancer by administration of at least a first, second, and third dose of the engineered NK cells, the second dose being administered to the subject 6-8 days after the first dose, and the third dose being administered to the subject 6-8 days after the second dose, and the first, second, and third doses being at least 1.0×10 9 The engineered NK cells are included.

[0010] In some embodiments, the first, second and third doses are each about 1.5×10 9 In some embodiments, the first, second and third doses each comprise at least 1.5×10 NK cells. 9 In some embodiments, the first, second and third doses each contain a larger number of cells, e.g., 2×10 9 NK cells, 3 x 10 9 NK cells, 4 x 10 9 NK cells, 5 × 10 9 NK cells, or more, e.g., 1.5 x 10 9 Contains NK cells.

[0011] In some embodiments, the dosing cycle is from about 14 days to about 35 days, e.g., about 21 days or about 28 days. In some embodiments, the first, second and third doses of engineered NK cells are administered to the subject within about 21 days from the first time point. In some embodiments, the first, second and third doses of engineered NK cells are administered to the subject within about 14 days from the first time point.

[0012] According to some embodiments, the first dosing cycle is initiated after the subject undergoes lymphodepletion process to reduce the number of natural immune cells. Depending on the embodiment, the first dosing cycle may be followed by one or more additional dosing cycles, for example, 2, 3, 4 or more additional cycles. Additional cycles may be administered depending on the state of cancer in the subject, for example, in the case of progression or the occurrence of additional cancer. In some embodiments, if the subject shows a complete response (e.g., no cancer), additional cycles are not required.

[0013] In some embodiments, the dosing regimen and related methods and uses include administering to a subject having cancer a lymphodepletion regimen comprising at least two doses of fludarabine. In some embodiments, the lymphodepletion process comprises at least two doses of cyclophosphamide and at least two doses of fludarabine. In some embodiments, the lymphodepletion process comprises three doses of cyclophosphamide and three doses of fludarabine, where a first dose of cyclophosphamide and fludarabine is administered 5 days prior to the start of the dosing cycle, a second dose of cyclophosphamide and fludarabine is administered 4 days prior to the start of the dosing cycle, and a third dose of cyclophosphamide and fludarabine is administered 3 days prior to the start of the dosing cycle. In some embodiments, about two days can elapse between the third dose of cyclophosphamide and fludarabine and the start of the dosing cycle. In some embodiments, the cyclophosphamide is administered at a dose of about 50 to about 1000 mg / m 2 and fludarabine is administered in an amount of about 5 to about 100 mg / m 2In some embodiments, the cyclophosphamide is administered in an amount of about 100 to about 600 mg / m 2 and fludarabine is administered in an amount of about 10 to about 60 mg / m 2 In some embodiments, the cyclophosphamide is administered in an amount of about 200 to about 400 mg / m 2 and fludarabine is administered in an amount of about 20 to about 40 mg / m 2 In some embodiments, the cyclophosphamide is administered in an amount of about 300 mg / m 2 (For example, about 250 to 350 mg / m 2 ), and fludarabine is administered at a dose of about 30 mg / m 2 (For example, about 25 to 25 mg / m 2 ) in an amount of about 0.1 to about 20 g / m. In some embodiments, the lymphodepletion process includes at least two doses of cytosine arabinoside (Ara-C) and at least two doses of fludarabine. In some embodiments, the lymphodepletion process includes five daily doses of Ara-C and five daily doses of fludarabine, with the first dose of Ara-C and fludarabine being administered seven days prior to the start of the dosing cycle. In some embodiments, about two days can elapse between the final dose of Ara-C and fludarabine and the start of the dosing cycle. In some embodiments, Ara-C is administered at a dose of about 0.1 to about 20 g / m. 2 / day, and fludarabine is administered in an amount of about 5 to about 100 mg / m 2 In some embodiments, Ara-C is administered in an amount of about 0.5 to about 10 g / m 2 / day, and fludarabine is administered in an amount of about 10 to about 60 mg / m 2 In some embodiments, Ara-C is administered in an amount of about 1 to about 5 g / m 2 / day, and fludarabine is administered in an amount of about 20 to about 40 mg / m 2 In some embodiments, Ara-C is administered in an amount of about 2 g / m 2 / day (e.g., about 1.5 to 2.5 g / m 2 / day) and fludarabine is administered at approximately 30 mg / m 2 / day (e.g., about 25-35 mg / m2 / day) is administered.

[0014] In some embodiments, the first and second doses of engineered NK cells are administered to the subject before the subject's natural immune cell populations are restored from the lymphodepletion process. Depending on the embodiment, other lymphodepleting agents can be used in addition to or in place of cyclophosphamide, Ara-C and / or fludarabine, such as, for example, daunorubicin (daunomycin) or idarubicin, mycophenolate mofetil, and / or bendamustine.

[0015] In some embodiments, the dosing regimen and related methods and uses are configured to treat a subject whose cancer is a hematological cancer. In some embodiments, the cancer is relapsed / refractory (R / R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS). In some embodiments, the subject has R / R AML and has had at least one but no more than two lines of previous standard anti-leukemia therapy prior to the dosing regimen. In some embodiments, the subject has fms-like tyrosine kinase 3 (FLT3)-mutated and / or isocitrate dehydrogenase (IDH)1 / 2-mutated disease and has had at least one but no more than three lines of previous therapy prior to the dosing regimen. In some embodiments, the subject is classified as intermediate, high, or very high risk MDS and has relapsed and / or refractory MDS. In some embodiments, the subject has had at least one but no more than two lines of previous standard anti-MDS therapy prior to the dosing regimen. In some embodiments, the subject has less than about 5-10%, less than about 5-8%, or less than about 5% blasts in a blood sample prior to the dosing regimen. In some embodiments, the subject has a white blood cell count of 30×10 or less prior to the dosing regimen. 9 Less than or equal to 28 × 10 WBC / L 9 Less than or equal to 10 WBC / L, or approximately 25 × 10 9In some embodiments, the subject has no evidence of leukemic meningitis or known active central nervous system disease and / or no peripheral leukocytosis greater than or equal to 20,000 blasts / μL.

[0016] In some embodiments, the dosing regimen and related methods and uses are configured to treat a subject whose cancer is a solid tumor. In some embodiments, the cancer is a liver tumor, including intrahepatic cholangiocarcinoma and liver tumors that are secondary metastases from colorectal cancer.

[0017] In some embodiments, the engineered NK cells express a chimeric receptor encoded by a polynucleotide having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 33. In some embodiments, the engineered NK cells express a chimeric receptor having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 34. In some embodiments, the engineered NK cells are also engineered to express membrane-bound interleukin 15 (mbIL15). In some embodiments, mbIL15 has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 36 and / or 38. In some embodiments, the engineered NK cells are allogeneic to the subject. In some embodiments, the dosing regimen, related methods and / or uses result in one or more of a decrease in blast cell count, an increase in platelet count, and an increase in neutrophil count.

[0018] In some embodiments, there is provided a use of a population of engineered NK cells expressing a chimeric receptor that targets a ligand of the NKG2D receptor to treat cancer in a subject by intravenously administering at least three consecutive doses of the engineered NK cells, wherein a first dose of the engineered NK cells is administered to the subject at a first time point and comprises at least 1.0×10 9the second dose is administered to the subject 6 to 8 days after the first dose and comprises at least 1.0 x 10 genetically engineered NK cells. 9 the third dose is administered to the subject 6 to 8 days after the second dose and comprises at least 1.0 × 10 genetically engineered NK cells. 9 the first time point comprises (i) about 300 mg / m cyclophosphamide; 2 and fludarabine at approximately 30 mg / m 2 or (ii) Ara-C administered at a dose of about 2 g / m 2 / day, with fludarabine at approximately 30 mg / m 2 and about 2 days after completion of a lymphodepletion process comprising either a daily dose of Ara-C for 5 days and a daily dose of fludarabine for 5 days administered in an amount of 1.5×10 / day, and the engineered NK cells express a chimeric receptor having at least 95% sequence identity to SEQ ID NO: 34. In some embodiments, each of the first, second and third doses is at least 1.5×10 9 Contains NK cells.

[0019] In some embodiments, a dosing regimen for cancer immunotherapy is provided that includes at least a first dosing cycle, the first dosing cycle including a first dose of engineered natural killer (NK) cells, a second dose of engineered NK cells, and a third dose of engineered NK cells. In some embodiments, the first dose is administered to a subject in need of cancer immunotherapy at a first time point, the second dose is administered to the subject 5-10 days after the first time point, and the third dose is administered to the subject 5-10 days after the second dose. In some embodiments, each of the first, second, and third doses is administered to a subject in need of cancer immunotherapy at a dose of at least 1.5×10 9 NK cells (or at least 3 × 10 for subjects weighing less than 50 kg) 7cells / kg), where at least a portion of the engineered NK cells are engineered to express a chimeric receptor comprising a domain that binds a ligand of natural killer group 2D (NKG2D), a transmembrane domain, and a cytotoxic signaling complex. In some embodiments, the cytotoxic signaling complex comprises an OX40 subdomain and a CD3 zeta subdomain. In some embodiments, the engineered NK cells also express membrane-bound interleukin 15 (mbIL15). In some embodiments, the first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers, and may be followed by one or more additional dosing cycles.

[0020] Also provided herein is a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, the first dosing cycle comprising a first dose of engineered NK cells, a second dose of engineered NK cells, and a third dose of engineered NK cells, wherein the first dose is administered to the subject at a first time point, the second dose is administered to the subject 5-10 days after the first time point, and the third dose is administered to the subject 5-10 days after the second dose, each of the first, second, and third doses being administered at a dose of 1.5×10 9 NK cells, at least some of which express a chimeric receptor that includes a domain that binds to a ligand of natural killer group 2D (NKG2D). In some embodiments, a first dosing cycle is initiated after the subject has undergone a lymphodepletion process, and may be followed by one or more additional dosing cycles.

[0021] In some embodiments, the dosing cycle is about 14 days to about 35 days. In some embodiments, the dosing cycle is about 21 days or about 28 days, including 21, 22, 23, 24, 25, 26, 27, or 28 days. In some embodiments, the lymphodepletion process includes at least two doses of cytosine arabinoside (Ara-C) and at least two doses of fludarabine. In some embodiments, the lymphodepletion process includes 3, 4, or 5 days of daily doses of Ara-C and 3, 4, or 5 days of daily doses of fludarabine, with the first dose of Ara-C and fludarabine being administered 5 to 7 days prior to the start of the dosing cycle. In some embodiments, 1 day, 2 days, or 3 days can elapse between the final dose of Ara-C and fludarabine and the start of the dosing cycle. In some embodiments, the Ara-C is administered at a dose of about 0.2 to 20 g / m 2 / day (e.g., 0.5-10g / m 2 / day), and fludarabine is administered at a dose of approximately 5 to 75 mg / m 2 / day (e.g., 10-60 mg / m 2 / day). In some embodiments, Ara-C is administered in an amount of about 1-5 g / m 2 Fludarabine is administered at a dose of approximately 20-40 mg / m 2 In some embodiments, Ara-C is administered in an amount of about 2 g / m 2 / day, and fludarabine is administered at approximately 30 mg / m 2 It is administered in an amount of 100 mg / day.

[0022] In some embodiments, the lymphodepletion process includes at least two doses of cyclophosphamide and at least two doses of fludarabine. In some embodiments, the lymphodepletion process includes three doses of cyclophosphamide and three doses of fludarabine, where a first dose of cyclophosphamide and fludarabine is administered 5 days prior to the start of the dosing cycle, a second dose of cyclophosphamide and fludarabine is administered 4 days prior to the start of the dosing cycle, and a third dose of cyclophosphamide and fludarabine is administered 3 days prior to the start of the dosing cycle. In some embodiments, one, two, or three days can elapse between the third dose of cyclophosphamide and fludarabine and the start of the dosing cycle. In some embodiments, cyclophosphamide is administered at a dose of about 10 to 1000 mg / m 2 (For example, about 100 to 600 mg / m 2 ) and fludarabine is administered at a dose of about 5 to 100 mg / m 2 (about 10-60 mg / m3). In some embodiments, cyclophosphamide is administered in an amount of about 200-400 mg / m 2 Fludarabine is administered at a dose of approximately 20-40 mg / m 2 In some embodiments, the cyclophosphamide is administered in an amount of about 500 mg / m 2 and fludarabine is administered in an amount of about 30 mg / m 2 is administered in an amount of

[0023] In some embodiments, the first and second doses of engineered NK cells are administered to the subject before the subject's natural immune cell populations have recovered from the lymphodepletion process. In some embodiments, the first, second, and third doses of engineered NK cells are administered to the subject within about 21 days of the first time point. In some embodiments, the first, second, and third doses of engineered NK cells are administered to the subject within about 14 days of the first time point.

[0024] Further provided is a dosing regimen comprising at least a first dosing cycle, the first dosing cycle being comprised of first, second, and third doses of genetically engineered NK cells, at least a portion of the engineered NK cells being engineered to express a chimeric receptor comprising a ligand binding domain for natural killer group 2D (NKG2D), a transmembrane domain, and a cytotoxic signaling complex, the first dose cycle being administered to a subject in need of cancer immunotherapy at a first time point, the second dose being administered to the subject 5-10 days after the first time point, and the third dose being administered to the subject 11-16 days after the first time point, the first, second, and third doses combined resulting in the administration of about 4 billion engineered NK cells. In some embodiments, the first, second, and third doses are each about 1.5×10 9 Contains NK cells.

[0025] In some embodiments of the dosing regimens provided herein, the engineered NK cells express a chimeric receptor encoded by a polynucleotide having at least about 90%, 95%, or 98% sequence identity to SEQ ID NO: 33. In some embodiments, the engineered NK cells express a chimeric receptor having at least about 90%, 95%, or 98% sequence identity to SEQ ID NO: 34. In some embodiments, the mbIL15 expressed by the NK cells has at least about 90%, 95%, or 98% sequence identity to SEQ ID NO: 36. Another embodiment utilizes mbIL15 having at least 95% sequence identity to SEQ ID NO: 38.

[0026] In some embodiments, the first and second doses are administered 6-8 days apart, and the second and third doses are administered 6-8 days apart. In some embodiments, each dose is 1.5×10 9 the second dose is administered about 7 days after the first dose and the third dose is administered about 7 days after the second dose.

[0027] In some embodiments, the dosing regimens provided herein are for the treatment of cancer, such as blood cancer. In some embodiments, the cancer is relapsed / refractory (R / R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS). In some embodiments, the subject has R / R AML and has had at least one but no more than two lines of previous standard anti-leukemia therapy. In some embodiments, the subject has fms-like tyrosine kinase 3 (FLT3)-mutated and / or isocitrate dehydrogenase (IDH)1 / 2-mutated disease and has had at least one but no more than three lines of previous therapy. In some embodiments, the subject qualifies to be classified as intermediate, high, or very high risk MDS and has relapsed and / or refractory MDS. In some embodiments, the subject has had at least one but no more than two lines of previous standard anti-MDS therapy. In some embodiments, the subject has less than about 5% blasts in a blood sample. In some embodiments, the subject has at least 25×10 9 In some embodiments, the subject has a white blood cell count of less than or equal to 10,000 WBC / L. In some embodiments, the subject does not show evidence of leukemic meningitis or known active central nervous system disease and / or does not have a peripheral leukocytosis of more than or equal to 20,000 blasts / μL.

[0028] In some embodiments, the dosing regimen provided herein is for the treatment of cancer, such as solid tumors. In some embodiments, the cancer is a liver tumor, including intrahepatic cholangiocarcinoma and liver tumors that are secondary metastases from colorectal cancer.

[0029] In some embodiments of the dosing regimens provided herein, the engineered NK cells are allogeneic to the subject. In some embodiments, the dosing regimen results in one or more of a decrease in blast cell count, an increase in platelet count, and an increase in neutrophil count.

[0030] In some embodiments, a dosing regimen for treating relapsed / refractory (R / R) acute myelodysplastic syndrome (AML) or high-risk myelodysplastic syndrome (MDS) is provided, comprising, consisting of, or consisting essentially of at least a first dosing cycle, the first dosing cycle comprising a first dose of engineered natural killer (NK) cells, a second dose of engineered NK cells, and a third dose of engineered NK cells, wherein the first dose is administered to a subject in need of cancer immunotherapy at a first time point, the second dose is administered to the subject 5-10 days after the first time point, and the third dose is administered to the subject 5-10 days after the second dose, and each of the first, second, and third doses is administered to the subject at a dose of at least 1.5×10 9 the engineered NK cells are allogeneic to the subject and are engineered to express a chimeric receptor that binds a ligand for natural killer cluster 2D (NKG2D) and has at least about 80% sequence identity to SEQ ID NO: 34, the first dosing cycle is initiated after the subject has received at least three doses of cytosine arabinoside (Ara-C) and at least three doses of fludarabine, and the first dosing cycle may be followed by one or more additional dosing cycles.

[0031] In some embodiments, a method is provided for treating relapsed / refractory (R / R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS), comprising administering at least three doses of cytosine arabinoside (Ara-C) and at least three doses of fludarabine to a subject with R / R AML or MDS, wherein the amount of Ara-C administered is about 1-5 g / m 2 / day, and the amount of fludarabine administered is approximately 20-40 mg / m 2 / day, comprising administering to the subject at least a first, second, and third dose of the engineered NK cells, wherein the first dose is administered to the subject after the final dose of Ara-C and fludarabine, the second dose is administered to the subject 6-8 days after the first time point, and the third dose is administered to the subject 6-8 days after the second dose, each of the first, second, and third doses being at least 1.5 x 10 9NK cells, wherein the engineered NK cells are allogeneic to the subject and are engineered to express a chimeric receptor that binds a ligand for natural killer group 2D (NKG2D) and has at least about 80% sequence identity to SEQ ID NO:34, and the first dosing cycle may be followed by one or more additional dosing cycles.

[0032] In some embodiments, the method further comprises evaluating at least one metric associated with progression or regression of R / R AML or MDS status to determine whether to administer an additional dosing cycle. In some embodiments, the subject has relapsed and / or refractory acute myeloid leukemia and has received at least one but not more than two lines of previous standard anti-leukemia therapy. In some embodiments, the subject has fms-like tyrosine kinase 3 (FLT3)-mutated and / or isocitrate dehydrogenase (IDH)1 / 2-mutated disease and has received at least one but not more than three lines of previous therapy. In some embodiments, the subject is eligible to be classified as intermediate, high, or very high risk MDS and has relapsed and / or refractory MDS. In some embodiments, the subject has received at least one but not more than two lines of previous standard anti-MDS therapy. In some embodiments, the subject has less than about 5% blasts in a blood sample and has received at least 25×10 9 have a white blood cell count less than or equal to 10,000 WBC / L and / or have no evidence of leukemic meningitis or known active central nervous system disease and / or have a peripheral leukocytosis greater than or equal to 20,000 blasts / μL.

[0033] In some embodiments, provided herein is a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, the first dosing cycle consisting of a first dose of engineered natural killer (NK) cells provided herein and a second dose of engineered NK cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point and the second dose being administered to the subject 5-10 days after the first time point, each of the first and second doses being at least 1.5×10 8 NK cells (or at least 3 × 10 for subjects weighing less than 50 kg) 6 cells / kg), where a first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers, and the first dosing cycle may be followed by a second or more dosing cycles.

[0034] Further embodiments provide a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, the first dosing cycle consisting of a first dose of engineered natural killer (NK) cells and a second dose of engineered NK cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point and the second dose being administered to the subject 5-10 days after the first time point, the first dose and the second dose being administered to the subject at a dose of at least 1.5×10 8 NK cells, the first dosing cycle being initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers, and the first dosing cycle may be followed by a second or more dosing cycles.

[0035] According to some embodiments, the dosing cycle is about 14 days to about 35 days or longer, and the subject is evaluated for at least one metric of cancer at the end of the dosing cycle or at a more distant time point (e.g., continuously) as appropriate to determine whether to start an additional dosing cycle.In some embodiments, the dosing cycle is about 21 days.In some embodiments, the dosing cycle is about 28 days.

[0036] According to some embodiments, the first and second doses of engineered NK cells are administered to the subject before the subject's natural immune cell population has recovered from the lymphodepletion process. This advantageously allows for a larger effector:target cell ratio based on the subject's natural immune cell population that has not yet recovered and is not diluted with the engineered NK cell population. In some embodiments, the first and second doses of engineered NK cells are administered to the subject within about 14-21 days from the first time point. In some embodiments, the first and second doses of engineered NK cells are administered to the subject within about 14 days from the first time point.

[0037] In some embodiments, a dosing regimen for cancer immunotherapy is provided, comprising at least a first dosing cycle, the first dosing cycle consisting of a first dose of engineered natural killer (NK) cells and a second dose of engineered NK cells, the first dose cycle being administered to a subject in need of cancer immunotherapy at a first time point and the second dose being administered to the subject 5-10 days after the first time point, each of the first and second doses being at least 1.5×10 8 NK cells, or at least 3 × 10 for subjects weighing less than 50 kg 6 per kg, wherein a first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers, and a second dose of the first dosing cycle is administered before the subject's natural immune cell population has recovered from the lymphodepletion process.

[0038] Also provided herein is a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, the first dosing cycle consisting of a first dose of genetically engineered natural killer (NK) cells and a second dose of genetically engineered NK cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point and the second dose being administered to the subject 5-10 days after the first time point, in combination, the first and second doses result in the administration of about 300 million to about 3 billion engineered NK cells, the first dosing cycle being initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers, and the second dose of the first dosing cycle being administered within about 14-21 days of the first time point. In some embodiments, the first and second doses each comprise about 1.5×10 8 NK cells, and the first and second doses each comprise about 4.5×10 8 or the first and second doses each contain about 1.5×10 NK cells. 9 Contains NK cells.

[0039] In some embodiments, the lymphodepletion process is a standard chemotherapy lymphodepletion process. In some embodiments, the lymphodepletion process includes at least two doses of cyclophosphamide and at least two doses of fludarabine. In some embodiments, the lymphodepletion process includes three doses of cyclophosphamide and three doses of fludarabine, where a first dose of cyclophosphamide and fludarabine is administered 5 days prior to the start of a dosing cycle, a second dose of cyclophosphamide and fludarabine is administered 4 days prior to the start of a dosing cycle, and a third dose of cyclophosphamide and fludarabine is administered 3 days prior to the start of a dosing cycle. In some embodiments, about two days can elapse between the third dose of cyclophosphamide and fludarabine and the start of a dosing cycle. In some embodiments, cyclophosphamide is administered at a dose of about 100-600 mg / m 2 Fludarabine is administered at a dose of approximately 10-60 mg / m 2In some embodiments, the cyclophosphamide is administered in an amount of about 200-400 mg / m 2 Fludarabine is administered at a dose of approximately 20-40 mg / m 2 In some embodiments, the cyclophosphamide is administered in an amount of about 300 mg / m 2 and fludarabine is administered in an amount of about 30 mg / m 2 is administered in an amount of

[0040] According to embodiments provided herein, a dosing regimen is provided in which at least a portion of the engineered NK cells are engineered to express a chimeric receptor comprising a domain that binds to a ligand of natural killer group 2D (NKG2D), a transmembrane domain, and a cytotoxic signaling complex. In some embodiments, the cytotoxic signaling complex comprises an OX40 subdomain and a CD3 zeta subdomain. In some embodiments, the engineered NK cells also express membrane-bound interleukin 15 (mbIL15).

[0041] In some embodiments, the engineered NK cells express a chimeric receptor encoded by a polynucleotide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 33. In some embodiments, the engineered NK cells express a chimeric receptor having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 34. In some embodiments, mbIL15 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 36 and / or 38.

[0042] In some embodiments, the first and second doses are at least 1.5×10 8 NK cells, and the first and second doses are administered 6-8 days apart. In some embodiments, the first and second doses comprise 1.5×10 8NK cells, and the first and second doses are administered about 7 days apart. In some embodiments, the first and second doses comprise at least 4.5×10 NK cells. 8 NK cells, and the first and second doses are administered 6-8 days apart. In some embodiments, the first and second doses comprise 4.5 x 10 8 NK cells, and the first and second doses are administered about 7 days apart. In some embodiments, the first and second doses comprise at least 1.5×10 9 NK cells, and the first and second doses are administered 6-8 days apart. In some embodiments, the first and second doses comprise 1.5×10 9 The first and second doses comprise NK cells, and are administered about 7 days apart.

[0043] In some embodiments, the dosing regimen is configured to treat a hematological cancer, in some embodiments, the hematological cancer is relapsed / refractory (R / R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS).

[0044] In some embodiments, the dosing regimen is configured to treat solid tumors. In some embodiments, the cancer is a liver tumor. In some embodiments, the liver tumor is intrahepatic cholangiocarcinoma. In some embodiments, the liver tumor is one or more secondary metastases from colorectal cancer.

[0045] In some embodiments, the engineered NK cells are allogeneic to the subject. In some embodiments, the dosing regimen results in one or more of a decrease in blast cell count, an increase in platelet count, and an increase in neutrophil count.

[0046] In some embodiments, a dosing regimen for cancer immunotherapy is provided, comprising at least a first dosing cycle, the first dosing cycle consisting of at least one dose of engineered natural killer (NK) cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point, the first dose being at least 1×108 NK cells, or 2 × 10 for subjects weighing less than 50 kg 6 cells / kg, wherein at least a portion of the engineered NK cells are engineered to express a chimeric receptor comprising a domain that binds a ligand for natural killer group 2D (NKG2D), a transmembrane domain, and a cytotoxic signaling complex, wherein the cytotoxic signaling complex comprises an OX40 subdomain and a CD3 zeta subdomain, and wherein the engineered NK cells also express membrane-bound interleukin 15 (mbIL15), and the first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers, and the first dosing cycle may be followed by subsequent dosing cycles.

[0047] Also provided herein is a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, the first dosing cycle consisting of at least a first dose of engineered natural killer (NK) cells, a second dose of engineered NK cells, and a second dose of engineered NK cells, wherein the first dose is administered to a subject in need of cancer immunotherapy at a first time point, the second dose is administered to the subject 5-10 days after the first time point, and the third dose is administered to the subject 5-10 days after the second time point, each of the first, second, and third doses being administered at a dose of at least 1×10 8 NK cells, or at least 2 × 10 for subjects weighing less than 50 kg 6 cells / kg, wherein at least a portion of the engineered NK cells are engineered to express a chimeric receptor comprising a domain that binds a ligand for natural killer group 2D (NKG2D), a transmembrane domain, and a cytotoxic signaling complex, wherein the cytotoxic signaling complex comprises an OX40 subdomain and a CD3 zeta subdomain, and wherein the engineered NK cells also express membrane-bound interleukin 15 (mbIL15), and the first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers, and the first dosing cycle may be followed by a second or more dosing cycles.

[0048] In some embodiments, the lymphodepletion process is a standard chemotherapy lymphodepletion process. In some embodiments, the lymphodepletion process includes at least two doses of cyclophosphamide and at least two doses of fludarabine. In some embodiments, the lymphodepletion process includes three doses of cyclophosphamide and three doses of fludarabine, where a first dose of cyclophosphamide and fludarabine is administered 5 days prior to the start of a dosing cycle, a second dose of cyclophosphamide and fludarabine is administered 4 days prior to the start of a dosing cycle, and a third dose of cyclophosphamide and fludarabine is administered 3 days prior to the start of a dosing cycle. In some embodiments, about two days can elapse between the third dose of cyclophosphamide and fludarabine and the start of a dosing cycle. In some embodiments, cyclophosphamide is administered at a dose of about 100-600 mg / m 2 Fludarabine is administered at a dose of approximately 10-60 mg / m 2 In some embodiments, the cyclophosphamide is administered in an amount of about 200-400 mg / m 2 Fludarabine is administered at a dose of approximately 20-40 mg / m 2 In some embodiments, the cyclophosphamide is administered in an amount of about 300 mg / m 2 and fludarabine is administered in an amount of about 30 mg / m 2 is administered in an amount of

[0049] In some embodiments, the dosing cycle ranges from about 14 to about 28 days, and the subject is evaluated for at least one metric of cancer at or after the completion of the dosing cycle to determine whether an additional dosing cycle is warranted, as appropriate.

[0050] In some embodiments, the engineered NK cells express a chimeric receptor encoded by a polynucleotide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 33. In some embodiments, the engineered NK cells express a chimeric receptor having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 34. In some embodiments, mbIL15 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 36 and / or 38.

[0051] In some embodiments, the first, second, and third doses are 1.0×10 8 NK cells and are administered 6-8 days apart. In some embodiments, the first, second, and third doses are about 1.0 x 10 8 NK cells and are administered about 7 days apart. In some embodiments, the first, second, and third doses are about 3×10 8 NK cells, administered 6-8 days apart. In some embodiments, the first, second, and third doses are about 3×10 8 NK cells and are administered about 7 days apart. In some embodiments, the first, second, and third doses are about 1×10 9 NK cells, administered 6-8 days apart. In some embodiments, the first, second, and third doses are about 1 x 10 9 The NK cells are administered approximately seven days apart.

[0052] In some embodiments, the dosing regimen is for the treatment of a hematological cancer. In some embodiments, the hematological cancer is relapsed / refractory (R / R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS).

[0053] In some embodiments, the dosing regimen is for the treatment of solid tumors. In some embodiments, the cancer is a liver tumor. In some embodiments, the liver tumor is an intrahepatic cholangiocarcinoma. In some embodiments, the liver tumor is a secondary metastasis from colorectal cancer. [Brief description of the drawings]

[0054] [Figure 1] FIG. 1 shows a non-limiting schematic diagram of a polynucleotide encoding a cytotoxic receptor construct comprising a binding portion for a ligand of NKG2D and either encoding mbIL15 (receptor B) or not encoding mbIL15 (receptor A). [Diagram 2] FIG. 2 shows a non-limiting schematic diagram of a polynucleotide encoding a cytotoxic receptor construct comprising an NKG2D receptor domain (e.g., a fragment) and either encoding mbIL15 (receptor B) or not encoding mbIL15 (receptor A). [Diagram 3] 3A-3B show non-limiting schematic diagrams of dosing cycles according to embodiments disclosed herein. Fig. 3A shows a 28-day cycle including three dosing events. Fig. 3B shows a 28-day cycle including two dosing events. [Figure 4] Figures 4A-4B show early patient response data. Figure 4A shows the change in blast count in a subject before treatment and after a three-dose regimen. Figure 4B shows the trace of NK cell count in an exemplary subject during and after a three-dose regimen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Some embodiments of the methods and compositions provided herein relate to engineered immune cells and combinations thereof for use in immunotherapy. In some embodiments, the engineered cells are engineered in multiple ways, for example, to express cytotoxicity-inducing receptor complexes. As used herein, the term "cytotoxicity receptor complex" shall be given its ordinary meaning and (unless otherwise specified) shall refer to chimeric antigen receptors (CARs), chimeric receptors (in the case of NKG2D chimeric receptors, also referred to as activating chimeric receptors). In some embodiments, the cells are further engineered to achieve modification of the responsiveness of the cells to non-tumor tissues and / or other therapeutic cells.

[0056] The term "anti-cancer effect" refers to a biological effect that may be manifested by various means, including, but not limited to, reduction in tumor volume, reduction in cancer cell number, reduction in the number of metastases, increase in average life span, reduction in cancer cell proliferation, reduction in cancer cell survival, and / or amelioration of various physiological symptoms associated with a cancerous condition.

[0057] cell type Some embodiments of the methods and compositions provided herein refer to cells, such as immune cells, e.g., immune cells, such as NK cells or T cells, that can be engineered to contain a chimeric receptor, such as an NKG2D ligand-directed chimeric receptor, or can be engineered to contain a nucleic acid encoding said chimeric receptor, as described herein. Further embodiments relate to engineering a second set of cells to express another cytotoxic receptor complex, such as the NKG2D-chimeric receptor complex disclosed herein.

[0058] Traditional anti-cancer therapies have relied on surgical approaches, radiation therapy, chemotherapy, or a combination of these methods. As research has led to a better understanding of some of the mechanisms of certain cancers, this knowledge has been used to develop targeted cancer therapies. Targeted therapy is a cancer treatment that uses specific drugs to target specific genes or proteins found in cancer cells or cells that support cancer growth (such as blood vessel cells) to suppress or block the growth of cancer cells. More recently, genetic engineering has made it possible to develop approaches that harness specific aspects of the immune system to fight cancer. In some cases, a patient's own immune cells are modified to specifically eradicate that patient's type of cancer. As described in more detail below, various types of immune cells can be used, such as T cells, natural killer (NK cells), or a combination thereof.

[0059] Also provided herein are polynucleotides, polypeptides, and vectors encoding chimeric receptors comprising a target binding moiety (e.g., an extracellular binding portion of a ligand expressed by a cancer cell) and a cytotoxic signaling complex to facilitate cancer immunotherapy. For example, some embodiments include polynucleotides, polypeptides, or vectors encoding activating chimeric receptors comprising an NKG2D extracellular domain directed against, among others, tumor markers, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, to facilitate immune cell targeting to cancer and exert a cytotoxic effect on cancer cells. Also provided are engineered immune cells (e.g., NK cells and / or T cells) expressing such chimeric receptors. Also provided herein are polynucleotides, polypeptides, and vectors encoding constructs comprising two or more subdomains, e.g., an extracellular domain comprising a first and a second ligand-binding receptor and a cytotoxic signaling complex, in some embodiments. Also provided are engineered immune cells (e.g., NK cells and / or T cells) expressing such bispecific constructs (in some embodiments, the first and second ligand binding domains target the same ligand). Methods of treating cancer and other uses of such cells for cancer immunotherapy are also provided herein.

[0060] Engineered Cells for Immunotherapy In some embodiments, cells of the immune system are engineered to have an increased cytotoxic effect on target cells, such as tumor cells. For example, cells of the immune system can be engineered to include tumor-directed chimeric receptors and / or tumor-directed CARs, as described herein. In some embodiments, white blood cells or leukocytes are used, as their natural function is to defend the body against abnormal cell proliferation and infections. There are various types of white blood cells that play specific roles in the human immune system, and are therefore the preferred starting point for the engineering of the cells disclosed herein. White blood cells include granulocytes and agranulocytes (the 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 the following cells or other cells described herein can be engineered to include chimeric antigen receptors, such as NKG2D ligand-directed chimeric receptors, or nucleic acids encoding chimeric receptors. In some embodiments, the cells may be engineered to co-express a membrane-bound interleukin 15 (mbIL15) domain. As discussed in more detail below, in some embodiments, the therapeutic cells are further genetically modified to increase cytotoxicity and / or cellular persistence. In some embodiments, the genetic modification increases the ability of the cells to resist signals emanating from the tumor microenvironment that cause a decrease in efficacy or shortened lifespan of the therapeutic cells.

[0061] Monocytes for immunotherapy Monocytes are a subtype of white blood cells. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are associated with the adaptive immune system and are responsible for the major functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of ingesting cellular material, or whole cells, followed by digestion and destruction of the ingested cellular material. In some embodiments, monocytes are used in association with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally membrane-bound interleukin 15 (mbIL15) domains.

[0062] Lymphocytes for immunotherapy Lymphocytes are the other primary subtype of white blood cells and include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity). B cells are engineered according to some embodiments disclosed herein, although some embodiments also relate to engineered T cells or engineered NK cells (mixtures of T cells and NK cells are used in some embodiments, either from the same donor or from different donors). Some embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally membrane-bound interleukin 15 (mbIL15) domains.

[0063] T Cells for Immunotherapy T cells can be distinguished from other lymphocyte subtypes (e.g., B cells or NK cells) based on the presence of T cell receptors on the cell surface. T cells are divided into various subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. In some embodiments, T cells of a specific subtype are engineered. In some embodiments, a mixed pool of T cell subtypes is engineered. In some embodiments, there is no specific selection of T cell types that are engineered to express the cytotoxic receptor complexes disclosed herein. In some embodiments, specific techniques, such as the use of cytokine stimulation, are used to increase the expansion / collection of T cells with specific marker profiles. For example, in some embodiments, activation of specific human T cells, e.g., CD4+ T cells, CD8+ T cells, is achieved by the use of CD3 and / or CD28 as stimulatory molecules. In some embodiments, methods of treating or preventing cancer or infectious diseases are provided that include administering a therapeutically effective amount of T cells expressing a cytotoxic receptor complex and / or a homing moiety as described herein. In some embodiments, the engineered T cells are autologous cells, while in some embodiments, the T cells are allogeneic cells. Some embodiments of the methods and compositions disclosed herein relate to T cells engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0064] NK Cells for Immunotherapy In some embodiments, methods of treating or preventing cancer or infectious diseases are provided that include administering a therapeutically effective amount of natural killer (NK) cells expressing a cytotoxic receptor complex and / or a homing moiety as described herein. In some embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In some embodiments, NK cells are preferred because of the relatively high natural cytotoxic potential of NK cells. In some embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, resulting in more effective activity against target cells (e.g., tumor or other disease cells). Some embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, immortalized NK cells are used and subjected to engineering as disclosed herein. In some embodiments, the NK cells are derived from the cell line NK-92. NK-92 cells are derived from NK cells, but lack the major inhibitory receptors displayed by normal NK cells while retaining most of the activating receptors. Some embodiments of NK-92 cells described herein in connection with NK-92 cells engineered to silence certain additional inhibitory receptors, e.g., SMAD3, allowing for upregulation of interferon-gamma (IFNγ), granzyme B, and / or perforin production. Further information regarding NK-92 cell lines is disclosed in International Publication No. WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, which are incorporated herein by reference in their entireties. NK-92 cells are used in some 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 the NK cells disclosed herein.In an additional embodiment, NK-92 cells are used in combination with the T cells disclosed herein.

[0065] Hematopoietic stem cells for cancer immunotherapy In some embodiments, hematopoietic stem cells (HSCs) are used in the immunotherapy methods disclosed herein. In some embodiments, the cells are engineered to express homing moieties and / or cytotoxic receptor complexes. HSCs are used in some embodiments to take advantage of their ability to engraft for long-term blood cell production, which can provide a sustained source of targeted anti-cancer effector cells, for example, to combat cancer remission. In some embodiments, this continued production helps to counteract anergy or exhaustion of other cell types, for example, by the tumor microenvironment. In some embodiments, allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In some embodiments, HSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally include a membrane-bound interleukin 15 (mbIL15) domain.

[0066] induced pluripotent stem cells In some embodiments, induced pluripotent stem cells (iPSCs) are used in the immunotherapy methods disclosed herein. iPSCs are used in some embodiments to take advantage of their ability to differentiate and induce non-pluripotent cells, including, but not limited to, CD34 cells, hemogenic endothelial cells, HSCs (hematopoietic stem cells and hematopoietic progenitors), hematopoietic pluripotent progenitors, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells, that contain one or several genetic modifications at selected sites, through differentiation of iPSCs or less differentiated cells that contain the same genetic modifications at selected sites. In some embodiments, iPSCs are used to generate iPSC-derived NK or T cells. In some embodiments, the cells are engineered to express homing moieties and / or cytotoxic receptor complexes. In some embodiments, iPSCs are used in combination with one or more additional engineered cell types disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cells engineered to express activating chimeric receptors that target ligands on tumor cells, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), and optionally a membrane-bound interleukin 15 (mbIL15) costimulatory domain.

[0067] Extracellular domain (tumor binding factor) Some embodiments of the compositions and methods described herein relate to chimeric receptors that include an extracellular domain that includes a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain) as described herein. Some embodiments of the compositions and methods described herein relate to chimeric receptors that include an extracellular domain that includes a ligand-binding domain that binds to a ligand expressed by a tumor cell (also referred to as an activating chimeric receptor) as described herein. The ligand-binding domain targets, for example, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 (among others), depending on the embodiment.

[0068] In some embodiments, the antigen binding domain is derived from or comprises a wild type or non-wild type sequence of an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (SDAB), vH or vL domain, camelid VHH domain, or a non-immunoglobulin scaffold, such as a DARPIN, an affibody, an affilin, an adnectin, an affitin, a repebody, a finomer, an alphabody, an avimer, an atrimer, a sentinin, 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 comprises more than one antigen binding domain.

[0069] Antigen-binding proteins In some embodiments, an antigen-binding protein is provided. As used herein, the term "antigen-binding protein" shall be given its ordinary meaning and shall also refer to an antigen-binding fragment that binds to an antigen, and, where appropriate, a protein that includes a scaffold or framework portion that allows the antigen-binding fragment to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen or a fragment thereof. In some embodiments, the antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment comprises all three CDRs from the heavy chain of the antibody that binds to the antigen, or from the light chain of the antibody that binds to the antigen. Furthermore, in some embodiments, the antigen-binding fragment comprises all six CDRs from the antibody that binds to the antigen (three from the heavy chain and three from the light chain). In some embodiments, the antigen-binding fragment comprises one, two, three, four, five, or six CDRs from the antibody that binds to the antigen, and in some 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.

[0070] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, single chain variable fragments (scFv), nanobodies (e.g., the VH domain of a camelid heavy chain antibody; VHH fragments), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity determining region (CDR) fragments. These molecules can be derived from any mammalian source, such as human, mouse, rat, rabbit, or pig, dog, or camel. Antibody fragments can compete with intact (e.g., natural) antibodies for binding to a target antigen, and fragments can be synthesized de novo using modification of intact antibodies (e.g., enzymatic or chemical cleavage) or recombinant DNA technology or peptide synthesis. Antigen-binding proteins can include, for example, alternative protein frameworks or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds that contain, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds that contain, for example, biocompatible polymers. Additionally, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimetics that utilize fibronectin components as a scaffold.

[0071] In some embodiments, the antigen binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, antigen binding proteins can include, but are not limited to, diabodies; intracellular antibodies; domain antibodies (a single VL or VH domain, or two or more VH domains connected by a peptide linker); maxibodies (two scFvs fused to an Fc region); triabodies; tetrabodies; minibodies (scFvs fused to a CH3 domain); peptibodies (one or more peptides bound to an Fc region); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions with complementary light chain polypeptides); small modular immunopharmaceuticals; and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0072] 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 refer to a tetrameric molecule, each tetramer comprising two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 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.

[0073] Within light and heavy chains, the variable (V) and constant (C) regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 more amino acids. The variable regions of each light / heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

[0074] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0075] 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 (λ) light chains refer to the two major antibody light chain isotypes. A light chain can comprise a polypeptide that includes, from the amino terminus to the carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL).

[0076] Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. An antibody "heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation and typically determines the class to which the antibody belongs. A heavy chain can comprise, from amino terminus to carboxyl terminus, a polypeptide comprising 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).

[0077] The IgG class is further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4. The IgA class is further divided into subclasses, namely IgA1 and IgA2. IgM has subclasses including, but not limited to, IgM1 and IgM2. The heavy chains of IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (e.g., between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0078] In some embodiments, the antigen-binding protein is an antibody. The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. An antibody may be a monoclonal, or polyclonal, multiple or single chain, or intact immunoglobulin, and may be derived from a natural or recombinant source. An antibody may be a tetramer of an immunoglobulin molecule. An antibody may be "humanized," "chimeric," or non-human. An antibody may include intact immunoglobulins of any isotype, including, for example, chimeric, humanized, human, and bispecific antibodies. An intact antibody generally includes at least two full-length heavy chains and two full-length light chains. An antibody sequence may be derived only from a single species, or may be "chimeric," i.e., different portions of the antibody may be derived from two different species, as further described below. Unless otherwise indicated, the term "antibody" also includes antibodies comprising two substantially full-length heavy chains and two substantially full-length light chains, provided that the antibody retains the same or similar binding and / or function as an antibody composed of two full-length light and heavy chains. For example, antibodies with substitutions, insertions or deletions of 1, 2, 3, 4 or 5 amino acid residues at the N-terminus and / or C-terminus of the heavy and / or light chains are included in the definition, provided that the antibody retains the same or similar binding and / or function as an antibody comprising two full-length heavy chains and two full-length light chains. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies. In some embodiments, monoclonal and polyclonal antibodies are provided. As used herein, the term "polyclonal antibody" shall be given its ordinary meaning and shall refer to an antibody population that typically varies widely in composition and binding specificity. As used herein, the term "monoclonal antibody" ("mAb") shall be given its ordinary meaning and shall refer to one or more of a population of antibodies having identical sequence. A monoclonal antibody binds to an antigen at a specific epitope on the antigen.

[0079] In some embodiments, the antigen-binding protein is an antibody fragment or antigen-binding fragment. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either vL or vH), camelid vHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see, e.g., U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). Antibody fragments can include Fab, Fab', F(ab')2, and / or Fv fragments that contain at least one CDR of an immunoglobulin sufficient to confer specific antigen binding to a cancer antigen (e.g., CD19). Antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.

[0080] In some embodiments, Fab fragments are provided. Fab fragments are monovalent fragments with VL, VH, CL and CH1 domains; F(ab')2 fragments are bivalent fragments with two Fab fragments linked by disulfide bridges at the hinge region; Fd fragments have VH and CH1 domains; Fv fragments have VL and VH domains of a single arm of an antibody; and dAb fragments have VH domain, VL domain, or antigen-binding fragments of VH or VL domain. In some embodiments, these antibody fragments can be incorporated into single domain antibodies, single chain antibodies, maxibodies, minibodies, intrabodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs. In some embodiments, the antibody comprises at least one CDR as described herein.

[0081] Also provided herein, in some embodiments, are single-chain variable fragments. As used herein, the term "single-chain variable fragment" ("scFv") shall be given its ordinary meaning and shall refer to a fusion protein in which the VL and VH regions are connected to form a continuous protein chain via a linker (e.g., a synthetic sequence of amino acid residues), which is long enough for the protein chain to fold back on itself and form a monovalent antigen-binding site. For the sake of clarity, unless otherwise indicated, a "single-chain variable fragment" is not an antibody or antibody fragment as defined herein. A diabody is a bivalent antibody comprising two polypeptide chains, each polypeptide chain comprising a VH and a VL domain connected by a linker configured to reduce or not allow pairing between the two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain. According to some embodiments, when the two polypeptide chains of a diabody are identical, the diabody resulting from their pairing has two identical antigen-binding sites. Polypeptide chains with different sequences can be used to generate diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, respectively, which can be the same or different.

[0082] In some embodiments, an 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 regions (also called "minimal recognition units" or "hypervariable regions") within an antibody variable sequence. CDRs allow an antigen binding protein to specifically bind to a particular antigen of interest. There are three heavy chain variable region CDRs (CDR-H1, CDR-H2 and CDR-H3) and three light chain variable region CDRs (CDR-L1, CDR-L2 and CDR-L3). The CDRs in each of the two chains are typically aligned by framework regions to form a structure that specifically binds to a particular epitope or domain on the target protein. From N-terminus to C-terminus, both naturally occurring light and heavy chain variable regions typically conform to the following order of these elements: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. For the heavy chain variable region, the order is typically from N-terminus to C-terminus: FW-H1, CDR-H1, FW-H2, CDR-H2, FW-H3, CDR-H3, and FW-H4. For the light chain variable region, the order is typically from N-terminus to C-terminus: FW-L1, CDR-L1, FW-L2, CDR-L2, FW-L3, CDR-L3, FW-L4. A numbering system has been devised that numbers the 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. The complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system.Other numbering systems for amino acids 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 can utilize CDRs defined according to any of these systems. For any given embodiment containing more than one CDR, the CDRs can be defined according to any of the Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or conformational definitions, or any combination of the above. Any CDR can be interpreted under any of these numbering systems, separately or within the context of a variable domain, as appropriate by one of skill in the art. One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an antigen binding protein.

[0083] 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 one or more CDR(s) to another polypeptide chain. In some embodiments, the antigen binding proteins incorporate one or more CDRs non-covalently. 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 sufficient to form a conformationally stable structural support, or framework, or scaffold, capable of presenting one or more sequences of amino acids that bind to antigens (e.g., CDRs, variable regions, etc.) at a localized surface region. Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs) or may have one or more modifications, such as amino acid additions, deletions, and / or substitutions, relative to a naturally occurring polypeptide or fold. Depending on the embodiment, the scaffold can be derived from polypeptides of a variety of different species (or more than one species), such as humans, non-human primates or other mammals, other vertebrates, invertebrates, plants, bacteria or viruses.

[0084] Depending on the embodiment, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. In some such embodiments, these framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain and / or tendamistat domain.

[0085] In some embodiments, antigen binding proteins with more than one binding site are also provided. In some embodiments, the binding sites are identical to each other, while in some embodiments, the binding sites are different from each other. 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 bind to two different epitopes that may be present on the same or different protein targets. In some embodiments, this is particularly advantageous because the bispecific chimeric antigen receptor can confer the engineered cells the ability to target multiple tumor markers. For example, bispecific antibodies can bind to MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, among others, and additional tumor markers, such as CD70, CD123, CD19, Her2, mesothelin, claudin 6, BCMA, EGFR, or any other marker disclosed herein or recognized in the art as a tumor-specific or tumor-associated antigen.

[0086] Natural killer group domains that bind to tumor ligands In some embodiments, engineered immune cells such as NK cells are utilized for their ability to recognize and destroy tumor cells. NK cells express both inhibitory and activating receptors on the cell surface. Inhibitory receptors bind to self-molecules expressed on the surface of healthy cells (thus preventing an immune response against "self" cells), while activating receptors bind to ligands expressed on abnormal cells such as tumor cells. When the balance between inhibitory and activating receptor activation is in favor of activating receptors, NK cell activation occurs and the target (e.g., tumor) cells are lysed.

[0087] Natural killer group 2 member D (NKG2D) is an NK cell activating receptor that recognizes various ligands expressed on cells. Surface expression of various NKG2D ligands is generally low on healthy cells, but is upregulated, for example, by malignant transformation. Non-limiting examples of ligands recognized by NKG2D include, but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, as well as other molecules expressed on target cells that control the cytolytic or cytotoxic function of NK cells. In some embodiments, T cells are engineered to express an extracellular domain to bind one or more tumor ligands and activate the T cells. For example, in some embodiments, T cells are engineered to express an NKG2D receptor as a binding agent / activating moiety. In some embodiments, the engineered cells disclosed herein are engineered to express another member of the NKG2 family, for example, NKG2A, NKG2C, and / or NKG2E. In some embodiments, a combination of such receptors is engineered. Additionally, in some embodiments, other receptors are expressed, such as killer cell immunoglobulin-like receptors (KIRs).

[0088] In some embodiments, the cells are engineered to express a cytotoxicity receptor complex that includes full-length NKG2D as an extracellular component for recognizing a ligand on the surface of tumor cells (e.g., hepatocytes). In one embodiment, the full-length NKG2D has the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the full-length NKG2D, or a functional fragment thereof, is human NKG2D. Further information regarding the chimeric receptors for use in the methods and compositions of the present disclosure is described in PCT Patent Publication No. 2018 / 183385, which is incorporated herein by reference in its entirety.

[0089] In some embodiments, cells are engineered to express a cytotoxicity receptor complex that includes a functional fragment of NKG2D as an extracellular component for recognizing a ligand on the surface of tumor cells or other diseased cells. In one embodiment, the functional fragment of NKG2D has the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, the fragment of NKG2D has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with full-length wild-type NKG2D. In some embodiments, the fragment can have one or more additional mutations from SEQ ID NO: 25, but retains ligand binding function, or in some embodiments, has enhanced ligand binding function. In some embodiments, the functional fragment of NKG2D comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the NKG2D fragment is provided as a dimer, trimer, or other concatemeric format, such embodiments providing enhanced ligand binding activity. In some embodiments, the sequence encoding the NKG2D fragment may be fully or partially codon optimized. In one embodiment, the sequence encoding the codon-optimized NKG2D fragment comprises the sequence of SEQ ID NO: 28. According to some embodiments, advantageously, the functional fragment lacks its native transmembrane or intracellular domain, but retains its ability to bind to NKG2D's ligand, as well as its ability to transmit an activation signal upon ligand binding. An additional advantage of such a fragment is that it is not necessary to express DAP10 to localize NKG2D to the cell membrane. Thus, in some embodiments, the cytotoxic receptor complex encoded by the polypeptide disclosed herein does not include DAP10.In some embodiments, immune cells such as NK or T cells (e.g., non-alloreactive T cells engineered according to embodiments disclosed herein) are engineered to express one or more chimeric receptors targeting, for example, CD70, CD19, CD123, Her2, mesothelin, claudin 6, BCMA, EGFR, and NKG2D ligands, e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. Such cells, in some embodiments, also co-express mbIL15.

[0090] In some embodiments, the cytotoxic receptor complex is configured to dimerize. Dimerization may include homodimerization or heterodimerization depending on the embodiment. In some embodiments, dimerization results in improved ligand recognition by the cytotoxic receptor complex (and thus the NK cell expressing the receptor), resulting in a reduction (or absence) of harmful toxic effects. In some embodiments, the cytotoxic receptor complex employs an internal dimer, or a repeat of one or more component subunits. For example, in some embodiments, the cytotoxic receptor complex may include a first NKG2D extracellular domain bound to a second NKG2D extracellular domain, and a transmembrane / signaling region (or a separate transmembrane region with a separate signaling region).

[0091] In some embodiments, the various domains / subdomains are separated by a linker, for example a GS3 linker (SEQ ID NOs: 15 and 16, nucleotide and protein, respectively) (or a GSn linker) is used. 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 some embodiments, other linkers comprise a peptide sequence of one of SEQ ID NOs: 18, 20, 22, 24. This provides the possibility to separate the various component parts of the receptor complex along with a polynucleotide that can increase the expression, stability, and / or functionality of the receptor complex.

[0092] Cytotoxicity Signaling Complex Some embodiments of the compositions and methods described herein relate to chimeric receptors, e.g., chimeric receptors directed against NKG2D ligands, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6, which comprise a cytotoxic signaling complex. As disclosed herein, according to some embodiments, the cytotoxic receptor complexes provided include one or more transmembrane and / or intracellular domains that initiate a cytotoxic signaling cascade upon the extracellular domain(s) that bind to a ligand on the surface of a target cell.

[0093] In some embodiments, the cytotoxic signaling complex comprises at least one transmembrane domain, at least one costimulatory domain, and / or at least one signaling domain. In some embodiments, more than one component moiety comprises a given domain - for example, a costimulatory domain can comprise two subdomains. Furthermore, in some embodiments, a domain can serve multiple functions, for example, a transmembrane domain can serve to provide a signaling function.

[0094] Transmembrane domain Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or ligand-directed chimeric receptors) that include a transmembrane domain. Some embodiments include a transmembrane domain from NKG2D or another transmembrane protein. In some embodiments where a transmembrane domain is employed, the portion of the transmembrane protein employed retains at least a portion of its normal transmembrane domain.

[0095] However, in some embodiments, the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells. In some embodiments, the transmembrane domain comprises CD8α. In some embodiments, the transmembrane domain is referred to as a "hinge." In some embodiments, the "hinge" of CD8α has the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the CD8α hinge is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a CD8α having the sequence of SEQ ID NO:1. In some embodiments, the "hinge" of CD8α comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the CD8α can be truncated or modified to have 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:2.

[0096] In some embodiments, the transmembrane domain comprises a CD8α transmembrane region. In some embodiments, the CD8α transmembrane domain has the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the CD8α hinge is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a CD8α having the sequence of SEQ ID NO:3. In some embodiments, the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the CD8α hinge is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a CD8α having the sequence of SEQ ID NO:4.

[0097] In some embodiments, together, the CD8 hinge / transmembrane complex is encoded by the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the CD8 hinge / transmembrane complex is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a CD8 hinge / transmembrane complex having the sequence of SEQ ID NO: 13. In some embodiments, the CD8 hinge / transmembrane complex comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD8 hinge / transmembrane complex hinge is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a CD8 hinge / transmembrane complex having the sequence of SEQ ID NO: 14.

[0098] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CD28 transmembrane domain complex hinge is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a CD28 transmembrane domain having the sequence of SEQ ID NO: 30.

[0099] Costimulatory domain Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors) that include costimulatory domains. In addition, in some embodiments, various transmembrane and signaling domains (and transmembrane / signaling domain combinations), additional coactivator molecules can be provided. These can be, for example, specific molecules that further increase the activity of immune cells. Cytokines can be used in some embodiments. For example, as a non-limiting example, specific interleukins such as IL-2 and / or IL-15 are used. In some embodiments, immune cells for treatment are engineered to express such molecules in a secreted form. In further embodiments, such costimulatory domains are engineered to be membrane-bound, acting as autocrine stimulatory molecules (or even as paracrine stimulators to neighboring cells).

[0100] In some embodiments, the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15). In some embodiments, IL15 is expressed from a separate cassette on a construct comprising any one of the CARs disclosed herein. In some embodiments, IL15 is expressed on the same cassette as any one of the CARs disclosed herein, optionally separated by a cleavage site, e.g., a proteolytic cleavage site or a T2A, P2A, E2A, or F2A autocleaving peptide cleavage site. In some embodiments, IL15 is membrane-bound IL15 (mbIL15). In some embodiments, mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain. In some embodiments, the native IL15 sequence is encoded by a sequence having at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO:11. In some embodiments, the native IL15 sequence comprises a peptide sequence having at least 85%, at least 90%, at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the at least one transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, mbIL15 may comprise additional components such as a leader sequence and / or a hinge sequence. In some embodiments, the leader sequence is a CD8 leader sequence. In some embodiments, the hinge sequence is a CD8 hinge sequence.

[0101] In some embodiments, the tumor antigen-directed CAR and / or the tumor ligand-directed chimeric receptor are encoded by a polynucleotide that encodes one or more cytoplasmic protease cleavage sites. Such sites can be recognized and cleaved by cytoplasmic proteases, resulting in the separation (and separate expression) of the various component parts of the receptor encoded by the polynucleotide. In some embodiments, the tumor antigen-directed CAR and / or the tumor ligand-directed chimeric receptor are encoded by a polynucleotide that encodes one or more self-cleaving peptides, such as a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and / or an F2A cleavage site. As a result, depending on the embodiment, various components of the engineered cytotoxic receptor complex can be delivered to NK cells or T cells by a single vector or multiple vectors. Thus, as shown diagrammatically in the figure, the construct can be encoded by a single polynucleotide, but also includes a cleavage site, so that downstream elements of the construct are expressed by the cell as separate proteins (as is the case in some embodiments with IL-15). In some embodiments, a T2A cleavage site is used. In some embodiments, the T2A cleavage site has the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the T2A cleavage site may be truncated or modified to have 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: 9. In some embodiments, the T2A cleavage site comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the T2A cleavage site is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the T2A cleavage site having the sequence of SEQ ID NO: 10.

[0102] In some embodiments, NK cells are engineered to express membrane-bound interleukin 15 (mbIL15). In such embodiments, mbIL15 expression on NK increases the cytotoxic effect of engineered NK cells by increasing the proliferation and / or life span of NK cells. In some embodiments, mbIL15 is encoded by the same polynucleotide as the CAR. In some embodiments, mbIL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 11 and a sequence encoding a transmembrane domain. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 12 operably linked to the amino acid sequence of the transmembrane domain. In some embodiments, mbIL15 has the nucleic acid sequence of SEQ ID NO: 1188. In some embodiments, mbIL15 can be truncated or modified to have 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: 1188. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 1189. In some embodiments, mbIL15 is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to mbIL15 having the sequence of SEQ ID NO: 1189. Membrane-bound IL15 sequences are found in PCT Publication Nos. 2018 / 183385 and 2020 / 056045, each of which is expressly incorporated herein by reference in its entirety, and relate to membrane-bound IL15 sequences.

[0103] Signaling domains Some embodiments of the compositions and methods described herein relate to chimeric receptors (e.g., tumor antigen-directed CARs and / or tumor ligand-directed chimeric receptors) that include a signaling domain. For example, an immune cell engineered according to some embodiments disclosed herein may include at least one subunit of the CD3 T cell receptor complex (or a fragment thereof). In some embodiments, the signaling domain includes a CD3 zeta subunit. In some embodiments, the CD3 zeta is encoded by the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the CD3 zeta may be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with a CD3 zeta having the sequence of SEQ ID NO: 7. In some embodiments, the CD3 zeta domain includes the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CD3 zeta 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 a CD3 zeta domain having the sequence of SEQ ID NO:8.

[0104] In some embodiments, the unexpected increased signaling is achieved by the use of multiple signaling domains whose activities act synergistically. For example, in some embodiments, the signaling domain further comprises an OX40 domain. In some embodiments, the OX40 domain is an intracellular signaling domain. In some embodiments, the OX40 intracellular signaling domain has a nucleic acid sequence of SEQ ID NO:5. In some embodiments, the OX40 intracellular signaling domain can be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with OX40 having a sequence of SEQ ID NO:5. In some embodiments, the OX40 intracellular signaling domain comprises an amino acid sequence of SEQ ID NO:6. In some embodiments, the OX40 intracellular signaling domain is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with OX40 intracellular signaling domain having a sequence of SEQ ID NO:6. In some embodiments, OX40 is used as the only transmembrane / signaling domain in the construct, but in some embodiments, OX40 can be used with one or more other domains. For example, in some embodiments, a combination of OX40 and CD3 zeta is used. As a further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD zeta is used.

[0105] In some embodiments, the signaling domain comprises a 4-1BB domain. In some embodiments, the 4-1BB domain is an intracellular signaling domain. In some embodiments, the 4-1BB domain is encoded by the nucleic acid sequence of SEQ ID NO:29. In some embodiments, the 4-1BB domain can be truncated or modified to have 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 domain having the sequence of SEQ ID NO:29. In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the 4-1BB intracellular signaling domain can be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the 4-1BB intracellular signaling domain having the sequence of SEQ ID NO:30. In some embodiments, 4-1BB is used as the only transmembrane / signaling domain in the construct, although in some embodiments, 4-1BB can be used with one or more other domains. For example, in some embodiments, a combination of 4-1BB and CD3 zeta is used. By way of further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0106] In some embodiments, the signaling domain comprises a CD28 domain. In some embodiments, the CD28 domain is an intracellular signaling domain. In some embodiments, the CD28 intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the CD28 intracellular signaling domain can be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD28 intracellular signaling domain having the sequence of SEQ ID NO: 32. In some embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CD28 intracellular signaling domain is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the CD28 intracellular signaling domain having the sequence of SEQ ID NO: 32. In some embodiments, CD28 is used as the only transmembrane / signaling domain in the construct, although in some embodiments, CD28 may be used in conjunction with one or more other domains. For example, in some embodiments, a combination of CD28 and CD3 zeta is used. By way of further example, in some embodiments, a combination of CD28, OX40, 4-1BB, and / or CD3 zeta is used.

[0107] Cytotoxicity Receptor Complex Construct Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as the activating chimeric receptors (ACRs) that target the ligands of NKG2D.The expression of these cytotoxic receptor complexes in immune cells, such as genetically modified non-allo-reactive T cells and / or NK cells, allows the targeting and destruction of specific target cells, such as cancerous cells.Non-limiting examples of such cytotoxic receptor complexes are discussed in more detail below.

[0108] In some embodiments, a polynucleotide is provided that encodes a tumor-binding agent / CD8 hinge-CD8TM / OX40 / CD3 zeta chimeric receptor complex (see FIG. 1, Chimeric Receptor A). The polynucleotide comprises or consists of an NKG2D ligand-binding portion, a CD8a hinge, a CD8a transmembrane domain, an OX40 domain, and a CD3 zeta domain. In some embodiments, the polynucleotide further encodes a 2A cleavage site, and an mbIL-15 domain, as described herein (see FIG. 1, Chimeric Receptor B, where a single polynucleotide represents a polynucleotide structure encoding both the receptor and mbIL15). In some embodiments, the receptor complex is encoded by a nucleic acid molecule that comprises a sequence obtained from a combination of sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence or amino acid sequence comprises a sequence according to one or more SEQ ID NOs described herein, such as those included herein as examples of constituent parts. In some embodiments, the encoding nucleic acid or amino acid sequence comprises a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with a sequence resulting from a combination of one or more SEQ ID NOs described herein. It is recognized that certain sequence changes, extensions, and / or truncations of the disclosed sequences may occur when combining sequences, for example, as a result of ease or efficiency of cloning (e.g., for creation of restriction sites). In some embodiments, the chimeric receptor comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to one or more of the SEQ ID NOs provided herein, or a portion thereof (e.g., a portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence), or a range defined by any two of the foregoing percentages.

[0109] In some embodiments, a polynucleotide is provided that encodes an NKG2D / CD8a hinge / CD8a transmembrane domain / OX40 / CD3 zeta activating chimeric receptor complex (see FIG. 2, NKG2D ACR A). The polynucleotide comprises or consists of a fragment of the NKG2D receptor that can bind to the ligand of the NKG2D receptor, the CD8α hinge, the CD8a transmembrane domain, the OX40 domain, and the CD3 zeta domain, as described herein. In some embodiments, the receptor complex is encoded by a nucleic acid molecule that comprises the nucleic acid sequence of SEQ ID NO: 33. In yet another embodiment, the chimeric receptor is encoded by the amino acid sequence of SEQ ID NO: 34. In some embodiments, the sequence of the chimeric receptor can differ from SEQ ID NO: 32 or 33, but remains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 33 or 34, depending on the embodiment. In some embodiments, the chimeric receptor can differ from SEQ ID NO: 33 or 34, but the chimeric receptor retains, or in some embodiments has enhanced, NK cell activation and / or cytotoxicity functions. Additionally, in some embodiments, the construct may be co-expressed with mbIL15, such as mbIL15 encoded by SEQ ID NO: 35 or 37 (FIG. 2, NKG2D ACR B, where a single polynucleotide represents a polynucleotide structure encoding both the receptor and mbIL15). In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 36 or 38. In some embodiments, the sequence of mbIL15 can differ from SEQ ID NO: 36 or 38, but remains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 36 or 38, depending on the embodiment.

[0110] Further information regarding chimeric receptors for use in the methods and compositions of the present disclosure can be found in PCT Patent Publication No. 2018 / 183385, filed March 27, 2018, which is incorporated by reference in its entirety.

[0111] Treatment Some embodiments relate to methods of treating, ameliorating, inhibiting, or preventing cancer using cells or immune cells comprising chimeric antigen receptors and / or activated chimeric receptors as disclosed herein. In some embodiments, the methods include treating or preventing cancer. In some embodiments, the methods include administering a therapeutically effective amount of immune cells expressing tumor-targeting chimeric antigen receptors and / or tumor-targeting chimeric receptors as described herein. Examples of types of cancer that can be treated in this way are described herein.

[0112] Disclosed herein is a method for treating cancer in a subject.In some embodiments, the method comprises administering to 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.

[0113] In certain embodiments, treatment of a subject with the genetically engineered cell(s) described herein achieves one, two, three, four, or more of the following effects, including, for example, (i) reduction or amelioration of the severity of a disease or a symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against progression of a disease or a symptom associated therewith; (iv) regression of a disease or a symptom associated therewith; (v) protection against the onset or onset of a symptom associated with a disease; (vi) protection against recurrence of a symptom associated with a disease; (vii) reduction in hospitalization of a subject; (viii) reduction in length of hospitalization; (ix) increase in survival of a subject with a disease; (x) reduction in the number of symptoms associated with a disease; (xi) enhancement, improvement, complement, supplement, or augmentation of the prophylactic or therapeutic effect of another therapy. Advantageously, the non-allo-reactive engineered T cells disclosed herein further increase one or more of the above. Administration can be by various routes, including, but not limited to, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal, and / or localized delivery to the affected tissue.

[0114] Also disclosed herein is the use of any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, any one of the cells disclosed herein, or any combination thereof for the treatment of cancer.

[0115] Also disclosed herein is the use of any one of the NKG2D ligand binding domains disclosed herein, any one of the chimeric receptors disclosed herein, any one of the cells disclosed herein, or any combination thereof in the manufacture of a medicament for the treatment of cancer.

[0116] Administration and Dosage Further provided herein is a method for treating a subject with cancer, comprising administering to the subject a composition comprising immune cells (e.g., NK cells and / or T cells) engineered to express the cytotoxic receptor complex disclosed herein.For example, some embodiments of the compositions and methods described herein relate to the use of tumor-targeting chimeric antigen receptors and / or tumor-targeting chimeric receptors, or the use of cells expressing tumor-targeting chimeric antigen receptors and / or tumor-targeting chimeric receptors, to treat cancer patients.Also provided is the use of such engineered immune cells to treat cancer.

[0117] In certain embodiments, treatment of a subject with the genetically engineered cell(s) described herein achieves one, two, three, four, or more of the following effects, including, for example, (i) reduction or amelioration of the severity of a disease or a symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against progression of a disease or a symptom associated therewith; (iv) regression of a disease or a symptom associated therewith; (v) protection against the onset or onset of a symptom associated with a disease; (vi) protection against recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the length of hospitalization; (ix) increase in survival of a subject with a disease; (x) reduction in the number of symptoms associated with a disease; (xi) enhancement, improvement, complement, supplement, or augmentation of the prophylactic or therapeutic effect of another therapy. Each of these comparisons is against a different therapy for a disease, including, for example, a cell-based immunotherapy for a disease using cells that do not express a construct disclosed herein. Advantageously, the non-allo-reactive engineered T cells disclosed herein further increase one or more of the above.

[0118] Dosing can be by a variety of routes, including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal, and / or localized delivery to the affected tissue. Cells (particularly NK cells and / or T cells) engineered to express the chimeric receptor complexes described herein can be formulated for parenteral dosing by injection, e.g., bolus injection or infusion.

[0119] The dose of immune cells, such as NK and / or T cells, can be readily determined for a given subject based on their weight, disease type and condition, and the desired aggressiveness of the treatment, but may range from about 10 to about 20% depending on the embodiment. 5 cells / kg~about 10 12 cells / kg (e.g., 10 5 ~10 7 , 10 7 ~10 10 , 10 10 ~10 12 and overlapping ranges therein). In one embodiment, a dose escalation regimen is used. In some embodiments, various immune cells, such as NK and / or T cells, are administered at a dose of, for example, about 1×10 6 cells / kg ~ approx. 1×10 8 cells / kg.

[0120] In some embodiments, 1×10 8 NK cells are administered three times over a 28-day cycle (2 × 10 for subjects weighing less than 50 kg). 6 In some embodiments, 3×10 8 NK cells are administered three times over a 28 day cycle. In some embodiments, 1 x 10 9 The NK cells are administered three times over a 28-day cycle.

[0121] In some embodiments, 1.5×10 8 NK cells are administered twice over a 28-day cycle (3 × 10 for subjects weighing less than 50 kg). 6 In some embodiments, 4.5×10 8NK cells are administered twice over a 28 day cycle. In some embodiments, 1.5×10 9 NK cells will be administered twice over a 28-day cycle.

[0122] In some embodiments, 1.5×10 9 NK cells are administered three times over a 28-day cycle (3 × 10 for subjects weighing less than 50 kg). 7 In some embodiments, 3×10 9 NK cells are administered three times over a 28 day cycle. In some embodiments, 1.5×10 10 NK cells are dosed three times over a 28 day cycle. In some embodiments, at least 4.5×10 9 NK cells are administered throughout the cycle.

[0123] In some embodiments, administration of the engineered NK cells is preceded by one or more preparatory treatments. In some embodiments, administration of the engineered NK cells is preceded by lymphodepletion. In some embodiments, a combination of chemotherapeutic agents is used for lymphodepletion. In some embodiments, a single chemotherapeutic agent is used for lymphodepletion. In some embodiments, when a combination of chemotherapeutic agents is used, agents with different mechanisms may be used. In some embodiments, agents from different classes may be used. In some embodiments, an antimetabolite is used. In some embodiments, an antimetabolite inhibits and / or prevents cell replication. In some embodiments, an antimetabolite is a modified nucleotide that disrupts DNA replication and is effective in targeting rapidly dividing tumor cells (such as those in AML or myelodysplastic syndromes (MDS)). In some embodiments, cytosine arabinoside (Ara-C) is used. In some embodiments, about 0.2 to about 10 g / m 2 A dose of Ara-C of approximately 0.2 g / m 2 , about 0.5g / m 2 , about 1.0g / m 2 , about 1.5g / m2 , about 2.0g / m 2 , about 2.5g / m 2 , about 3.0g / m 2 , about 3.5g / m 2 , about 4.0g / m 2 , about 5.0g / m 2 , about 6.0g / m 2 , about 7.0g / m 2 , about 8.0g / m 2 , about 9.0g / m 2 , about 10.0g / m 2 , about 10.5g / m 2 , or any dose therebetween. In some 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 some embodiments, the dose can be divided, for example, given twice daily, if necessary. In some embodiments, an additional agent is used in combination with Ara-C. In some embodiments, the additional agent is also an antimetabolite. In some embodiments, the additional agent inhibits one or more of DNA polymerase alpha, ribonucleotide reductase, and / or DNA primase, thus inhibiting DNA synthesis. In some embodiments, the additional agent is fludarabine. In some embodiments, the additional agent is about 5.0 mg / m 2 ~about 200mg / m 2 A dose of fludarabine of approximately 5.0 mg / m 2 , about 10.0mg / m 2 , about 15.0mg / m 2 , about 20.0mg / m 2 , about 25.0mg / m 2 , about 30.0mg / m 2 , about 35.0mg / m 2 , about 40.0mg / m 2 , about 45.0mg / m 2 , about 50.0mg / m 2 , about 60.0mg / m 2 , about 70.0mg / m 2 , about 80.0mg / m 2 , about 90.0mg / m 2 , about 100.0mg / m 2, about 125.0mg / m 2 , about 150.0mg / m 2 , about 175.0mg / m 2 , about 200.0mg / m 2 or any dose therebetween. In some 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 some embodiments, the dose can be divided, for example, given twice daily, if necessary. In some embodiments, the combination of fludarabine and Ara-C is about 20 mg / m 2 ~40mg / m 2 daily dose of fludarabine, and approximately 1.5 g / m 2 ~2.5g / m 2 In some embodiments, the combination of fludarabine and Ara-C (or any other agent or agents disclosed herein) is administered for at least about 5 days, with administration beginning about 7 days (e.g., days -7 to -3) prior to the first dose of engineered NK cells. In some embodiments, lymphodepletion begins on day -5 prior to the dose of engineered NK cells. In some embodiments, this combination advantageously functions not only as a lymphodepleting regimen, but also as an anti-cancer agent (in addition to the engineered NK cells). In some embodiments, the lymphodepleting regimen acts synergistically with the engineered NK cells to provide effective reduction and / or elimination of cancerous cells.

[0124] In certain embodiments, a dose of the genetically engineered cells or compositions described herein is administered to a subject every day, every other day, every few days, every third day, once a week, twice a week, three times a week, or once every two weeks. In other embodiments, 2, 3, or 4 doses of the genetically engineered cells or compositions described herein are administered to a subject every day, every second day, every third day, once a week, or once every two weeks. In some embodiments, a dose of the genetically engineered cells or compositions described herein is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, a dose of the genetically engineered cells or compositions described herein is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or more.

[0125] In some embodiments, the subject is subjected to lymphodepletion at least once prior to administration of the genetically engineered cells as disclosed herein. In some embodiments, lymphodepletion is performed prior to administration of one or more additional doses of engineered cells. In some embodiments, following lymphodepletion, a dosing cycle is used that includes at least two doses of engineered cells as disclosed herein, with the two doses separated by a fixed time interval. In some embodiments, the time interval is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or more (including the time interval that marks the exact time interval from the last administration, e.g., 84 hours or 3.5 days). In some embodiments, the dosing cycle itself is about 14, 21, 28, 35, 42 days or more. In some embodiments, the three doses are administered at about one week intervals from each other. In some embodiments, the two doses are administered at about one week intervals from each other. In some embodiments, the subject receives a first dose on day 0 of the cycle, a second dose on day 7 of the cycle, and a third dose on day 14 of the cycle. In some such embodiments, a 28-day cycle is used with the primary outcome measure evaluated on day 28 (see, e.g., FIG. 3A). In some embodiments, the subject receives a first dose on day 0 of the cycle, and a second dose on day 7 of the cycle. In some such embodiments, a 28-day cycle is used with the primary outcome measure evaluated on day 28 (see, e.g., FIG. 3B). In some embodiments, lymphodepletion is performed before the start of each dosing cycle if a subsequent dosing cycle is required (e.g., if the subject requires further treatment). For example, in some embodiments, the subject undergoes lymphodepletion, receives multiple doses of engineered cells according to the cycle, is evaluated at the end of the cycle time, and if deemed necessary, undergoes a second lymphodepletion, followed by a second dosing cycle. In some embodiments, fludarabine / cyclophosphamide is used to achieve lymphodepletion. 2) and fludarabine (30 mg / m 2 ) is administered daily for three days. Depending on the embodiment, different concentrations can be used. For example, in some embodiments, 500 mg / m 2 A dose of cyclophosphamide is used with fludarabine. In such an embodiment where multiple dosing cycles are used, the first and second dosing cycles do not have to be the same (e.g., the first cycle may have 2 doses, and the second cycle uses 3 doses). Depending on the subject, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dosing cycles are performed.

[0126] Depending on the embodiment, various types of cancers can be treated. In some embodiments, the cancer treated is acute myeloid leukemia (AML). In some embodiments, the cancer treated is myelodysplastic syndrome. In some embodiments, hepatocellular carcinoma is treated. In some embodiments, intrahepatic cholangiocarcinoma or secondary metastasis from other liver tumors, such as colorectal cancer, is treated. Further embodiments provided herein include the treatment or prevention of the following non-limiting examples of cancers, including but not limited to acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (e.g., but not limited to, astrocytoma, spinal cord tumor, brain stem glioma, glioblastoma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloblastoma), breast cancer, bronchial tumor, Burkitt's lymphoma, cervical cancer, and pulmonary tumors. These include intestinal cancer, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cavity cancer, nasopharyngeal cancer, liver cancer, lung cancer (e.g., but not limited to, non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer.

[0127] In some embodiments, the present disclosure provides methods for identifying nucleic acid or amino acid sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges therebetween) sequence identity and / or homology to each of the nucleic acid or amino acid sequences of SEQ ID NOs: 1-38 (or a combination of two or more of SEQ ID NOs: 1-38), and that exhibit, but are not limited to, (i) increased proliferation, (ii) increased activation, (iii) increased activity, (iv) increased activity, (v) increased activity, (vi) increased activity, (vii) increased activity, (v ... Also provided are nucleic acid and amino acid sequences that also exhibit one or more of the following functions, including: (i) increased cytotoxic activity against cells presenting a ligand bound by NK cells; (ii) increased homing to tumors or sites of infection; (iii) decreased off-target cytotoxic effects; (iv) increased secretion of immunostimulatory cytokines and chemokines (including, but not limited to, IFNg, TNFa, IL-22, CCL3, CCL4, and CCL5); (v) increased ability to stimulate further innate and adaptive immune responses; and (vi) combinations thereof.

[0128] Additionally, in some embodiments, amino acid sequences corresponding to any of the nucleic acids disclosed herein are provided, taking into account the degeneracy of the nucleic acid code. Additionally, sequences (either nucleic acid or amino acid) that differ from those explicitly disclosed herein but have functional similarity or equivalence are also contemplated within the scope of the present disclosure. The above includes mutations, truncations, substitutions, or other types of modifications.

[0129] In some embodiments, the polynucleotide encoding the disclosed cytotoxic receptor complex is mRNA.In some embodiments, the polynucleotide is DNA.In some embodiments, the polynucleotide is operably linked to at least one regulatory element for the expression of the cytotoxic receptor complex.

[0130] Furthermore, according to some embodiments, a vector is provided that comprises a polynucleotide encoding any of the polynucleotides provided herein, and the polynucleotide may be operably linked to at least one regulatory element for expression of a cytotoxic receptor complex. In some embodiments, the vector is a retrovirus.

[0131] Further provided herein are engineered immune cells (e.g., NK and / or T cells) that comprise a polynucleotide, vector, or cytotoxic receptor complex disclosed herein. Further provided herein are compositions that include a mixture of engineered immune cells (e.g., NK cells and / or engineered T cells), each population comprising a polynucleotide, vector, or cytotoxic receptor complex disclosed herein.

[0132] Type of Cancer Some embodiments of the compositions and methods described herein relate to administering immune cells comprising a tumor-tropic chimeric antigen receptor and / or a tumor-tropic chimeric receptor to a patient with cancer. Various embodiments provided herein include the treatment or prevention of the following non-limiting examples of cancers. Examples of cancers include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (e.g., but not limited to, astrocytoma, spinal cord tumor, brain stem glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloblastoma), breast cancer, bronchial tumor, Burkitt's lymphoma, cervical cancer, colon cancer, chronic lymphocytic leukemia (CLL), and other non-limiting examples of cancers. LL), chronic myeloid leukemia (CML), chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (e.g., but not limited to, non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer.

[0133] Cancer Targeting Some embodiments of the compositions and methods described herein relate to immune cells that contain chimeric receptors that target cancer antigens, such as MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. Further non-limiting examples of target antigens include CD70, CD5, CD19; CD123; CD22; CD30; CD171; CS1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); TNF receptor family member B cell maturation (BCMA); CD38; DLL3; G protein-coupled receptor class C group 5, member D (GPRC5D); epidermal growth factor receptor (EGFR) CD138; prostate-specific membrane antigen (PSMA); tyrosine kinase. Fms-like tyrosine kinase 3 (FLT3); KREMEN2 (kringle-containing transmembrane protein 2), ALPPL2, claudin 4, claudin 6, C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer); Tn antigen ((TnAg) or (Gal NAca-Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not hematopoietic progenitor cells, glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD27 6);KIT (CD117);Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2);Mesothelin;Interleukin-11 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 fetal 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)bDClalp(l-4)bDGlcp(ll)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMA A);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);G protein-coupled receptor class C group 5, member D (GPRC5D);chromosome X open reading frame 61 (CXORF61);CD97;CD179a;anaplastic lymphoma kinase (ALK);polysialic acid;placenta-specific 1 (PLAC1);hexasaccharide moiety of globoH glycoceramide (GloboH);mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ES0-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1);ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCT Al or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); rat sarcoma (Ras) mutant; human telomerase; reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myeloid cell tumor viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 IB1 (CYPIB1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Proteins) 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); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPVE6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1);Fc fragment of 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); Immunoglobulin lambda-like polypeptide 1 (IGLLl), MPL, biotin, c-MYC epitope Tag, CD34, LAMP1, TROP2, GFR alpha 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; sialyl Lewis antigen);Fucosyl-GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, ILl lRa, IL13Ra2, CD179b-IGLll, TCR gamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Tim1- / HVCR1, CSF2RA (GM-CSFR-alpha), TGF beta R2, Lewis Ag, TCR-beta chain, TCR-beta 2 chain, TCR-gamma chain, TCR-delta chain, FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV These include antigens recognized by K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, guanylate cyclase C (GCC), autoantibodies to desmoglein 3 (Dsg3), autoantibodies 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, claudin 8.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Liv1, nectin-4, Cripto, gpA33, BST1 / CD157, small conductance chloride channel, and TNT antibodies. ; EXAMPLES

[0134] The following is a non-limiting description of the experimental methods and materials used in the examples disclosed below. [Example 1]

[0135] Initial dosing regimens for NK cell immunotherapy As discussed in more detail herein, certain cancer types express selected markers in an elevated manner. In some embodiments, cytotoxic receptor constructs are generated according to sequences disclosed herein to specifically target a given cancer. For example, many cancers express elevated levels of ligands for the NKG2D receptor. Thus, as discussed in detail above, in some embodiments, NKG2D-ligand-directed cytotoxic receptor constructs are provided. In some embodiments, the polynucleotides encoding these constructs are engineered to bicistronic express mbIL15. In some embodiments, dosing regimens are tested to evaluate the efficacy of cells expressing such constructs. In some embodiments, the cells engineered to express the constructs are NK cells. In some embodiments, the NK cells are pre-made allogeneic engineered NK cells (derived from unrelated donors) and compared to a matched dose of engineered NK cells derived from haplomatched related donors. In some embodiments, the engineered NK cells express a cytotoxic receptor encoded by SEQ ID NO: 33 (including degenerate or codon optimized versions of SEQ ID NO: 33). In some embodiments, the engineered NK cells express a cytotoxic receptor comprising the amino acid sequence of SEQ ID NO: 34, and mbIL15 comprising the amino acid sequence of SEQ ID NO: 36 or 38.

[0136] The dosing regimen is designed to evaluate three doses of engineered NK cells administered three times in a 28-day dosing cycle. Prior to the dosing cycle, subjects will undergo lymphodepletion (cyclophosphamide (300 mg / m 2 ) and fludarabine (30 mg / m 2 On day 0, subjects receive the first of three different doses: 1×10 8 NK cells (2 × 10 for subjects weighing less than 50 kg) 6 pieces / kg), 3×10 8 NK cells, or 1 x 10 9Patients will receive one of 10 doses of NK cells. Dose 2 will be administered on day 7 and Dose 3 will be administered on day 14. On day 28, outcome measures will be assessed.

[0137] Primary endpoints include: (1) the incidence, nature, and severity of treatment-related adverse events will be evaluated. An adverse event is any untoward and unintended manifestation, including clinically significant laboratory abnormalities, symptoms, or illness. This should be measured 30 days after the last dose of NK cells, and (2) the proportion of subjects who experience NK cell dose-limiting toxicity (DLT). A DLT is defined as a treatment-emergent adverse event that occurs during cycle 1 and meets protocol-specified criteria. This should be measured 28 days after the first dose of NK cells.

[0138] Secondary outcome measures include: (1) assessment of NK cell half-life measured as the time required for a 50% decline from the maximum amount of circulating engineered NK cells. This should be measured 28 days after the first dose of NK cells; (2) NK cell persistence by measuring the amount of engineered NK cells in peripheral blood every 3 months after dosing to determine persistence. This is measured for up to 2 years after the final dose of NK cells; (3) assessment of the host immune response to the engineered NK cells via serum samples measured for antibodies to the engineered NK cells. This is measured for up to 2 years after the final dose of NK cells; (4) objective response rate to the engineered NK cells by measuring the percentage of subjects who achieved complete and partial responses. AML subjects will be evaluated for the antitumor activity of the engineered NK cells based on the latest ELN criteria (Doehner 2017). MDS patients will be evaluated for the antitumor activity of the engineered NK cells based on the IWG criteria with MDS (Cheson 2006). This will be measured 28 days after the first dose of engineered NK cells and followed up to two years after the final dose.

[0139] Studies will begin with the lowest dose, and if a dose clears the regimen with less than 33% toxicity, the next higher dose will be studied.

[0140] Administration of three doses of engineered NK cells expressing chimeric receptor constructs targeting NKG2D ligands and expressing mbIL15 is expected to be well tolerated and show limited adverse events. Administration of three doses of engineered NK cells expressing chimeric receptor constructs targeting NKG2D ligands and expressing mbIL15 is also expected to result in limited DLT. NK cells are expected to show extended half-life and prolonged duration. NK cells are expected to induce limited host immune responses and clinically meaningful objective response rates (e.g., reduced tumor burden). [Example 2]

[0141] Secondary dosing regimens for NK cell immunotherapy As discussed in more detail herein, certain cancer types express selected markers in an elevated manner. In some embodiments, cytotoxic receptor constructs are generated according to sequences disclosed herein to specifically target a given cancer. For example, many cancers express elevated levels of ligands for the NKG2D receptor. Thus, as discussed in detail above, in some embodiments, NKG2D-ligand-directed cytotoxic receptor constructs are provided. In some embodiments, the polynucleotides encoding these constructs are engineered to bicistronic express mbIL15. In some embodiments, dosing regimens are tested to evaluate the efficacy of cells expressing such constructs. In some embodiments, the cells engineered to express the constructs are NK cells. In some embodiments, the NK cells are pre-made allogeneic engineered NK cells (derived from unrelated donors) and compared to a matched dose of engineered NK cells derived from haplomatched related donors. In some embodiments, the engineered NK cells express a cytotoxic receptor encoded by SEQ ID NO: 33 (including degenerate or codon optimized versions of SEQ ID NO: 33). In some embodiments, the engineered NK cells express a cytotoxic receptor comprising the amino acid sequence of SEQ ID NO: 34, and mbIL15 comprising the amino acid sequence of SEQ ID NO: 36 or 38.

[0142] The dosing regimen is designed to evaluate three different doses of engineered NK cells, administered twice in a 28-day dosing cycle. Prior to the dosing cycle, subjects will undergo lymphodepletion (cyclophosphamide (300 mg / m 2 ) and fludarabine (30 mg / m 2 The conditioning phase will involve 1.5×10 mAb (using 1.5×10 mAb) on days −5, −4, and −3. On day 0, subjects will receive the first of three different doses: 1.5×10 mAb (using 1.5×10 mAb) on days −5, −4, and −3. 8 NK cells (3 × 10 in subjects weighing less than 50 kg) 6 pieces / kg), 4.5×10 8 NK cells, or 1.5 x 10 9Patients will receive one of 10 doses of NK cells. Dose 2 will be administered on day 7. Outcome measures will be assessed on day 28.

[0143] Primary endpoints include: (1) the incidence, nature, and severity of treatment-related adverse events will be evaluated. An adverse event is any untoward and unintended manifestation, including clinically significant laboratory abnormalities, symptoms, or illness. This should be measured 30 days after the last dose of NK cells, and (2) the proportion of subjects who experience NK cell dose-limiting toxicity (DLT). A DLT is defined as a treatment-emergent adverse event that occurs during cycle 1 and meets protocol-specified criteria. This should be measured 28 days after the first dose of NK cells.

[0144] Secondary outcome measures include: (1) assessment of NK cell half-life measured as the time required for a 50% decline from the maximum amount of circulating engineered NK cells. This should be measured 28 days after the first dose of NK cells; (2) NK cell persistence by measuring the amount of engineered NK cells in peripheral blood every 3 months after dosing to determine persistence. This is measured for up to 2 years after the final dose of NK cells; (3) assessment of the host immune response to the engineered NK cells via serum samples measured for antibodies to the engineered NK cells. This is measured for up to 2 years after the final dose of NK cells; (4) objective response rate to the engineered NK cells by measuring the percentage of subjects who achieved complete and partial responses. AML subjects will be evaluated for the antitumor activity of the engineered NK cells based on the latest ELN criteria (Doehner 2017). MDS patients will be evaluated for the antitumor activity of the engineered NK cells based on the IWG criteria with MDS (Cheson 2006). This will be measured 28 days after the first dose of engineered NK cells and followed up to two years after the final dose.

[0145] Studies will begin with the lowest dose, and if a dose clears the regimen with less than 33% toxicity, the next higher dose will be studied.

[0146] Three doses of engineered NK cells expressing chimeric receptor constructs targeting NKG2D ligands and expressing mbIL15 are expected to be well tolerated and show limited adverse events. Three doses of engineered NK cells expressing chimeric receptor constructs targeting NKG2D ligands and expressing mbIL15 are also expected to result in limited DLT. NK cells are expected to show extended half-life and prolonged duration. NK cells are expected to induce limited host immune responses and clinically meaningful objective response rates (e.g., reduced tumor burden).

[0147] It is also believed that a two-dose cycle improves outcomes compared to a three-dose cycle. Without being bound by theory, the greater initial loading of engineered NK cells in a two-dose cycle allows for a greater effector:target cell ratio. Studies have demonstrated that the host NK cell population begins to recover approximately 14-21 days after Flu / Cy lymphodepletion, which may result in the simultaneous clearing of the infused allogeneic cells. Thus, a two-dose cycle allows for the delivery of the greatest number of engineered cells administered in the post-lymphodepletion window, resulting in enhanced therapeutic outcomes. [Example 3]

[0148] A third dosing regimen for NK cell immunotherapy As discussed in more detail herein, certain cancer types express selected markers in an elevated manner, such as ligands for the NKG2D receptor. Thus, as discussed in detail above, in some embodiments, NKG2D-ligand-directed cytotoxic receptor constructs are provided. In some embodiments, dosing regimens are tested to evaluate the efficacy of cells expressing such constructs. In some embodiments, the cells engineered to express the constructs are NK cells. In some embodiments, the NK cells are pre-made allogeneic engineered NK cells (derived from an unrelated donor) and may be compared to a matched dose of engineered NK cells from a haplomatched related donor. In some embodiments, the engineered NK cells express a cytotoxic receptor encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO:33 (including degenerate or codon-optimized versions of SEQ ID NO:33). In some embodiments, the engineered NK cells express a cytotoxic receptor comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:34. In some embodiments, immune cells (eg, NK cells) are also engineered to express mbIL15 comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:36 or 38.

[0149] In some embodiments, patients receiving the NK cell immunotherapy regimen have relapsed and / or refractory acute myeloid leukemia (according to standard European LeukemiaNet (ELN) criteria). In some embodiments, patients have received at least one but no more than three, preferably no more than two lines of conventional standard antileukemic therapy. In some embodiments, subjects in complete remission with minimal residual disease (e.g., less than about 5% blasts) can receive the NK cell immunotherapy regimen. In some embodiments, patients can have fms-like tyrosine kinase 3 (FLT3)-mutated and / or isocitrate dehydrogenase (IDH)1 / 2-mutated disease and have received at least one prior respective targeted therapy, but no more than four, preferably no more than three lines of prior therapy. In some embodiments, patients can have a white blood cell count of 25×10 9 In some embodiments, patients will not receive the NK cell immunotherapy regimen if they show evidence of leukemic meningitis or known active central nervous system disease and / or have a peripheral leukocytosis greater than or equal to 20,000 blasts / μL (or other evidence of rapidly progressive disease that prevents the patient from completing at least one cycle of treatment).

[0150] In some embodiments, patients receiving the NK cell immunotherapy regimen have intermediate, high, or very high risk MDS classified according to the World Health Organization Classification and the Revised International Prognostic Scoring System, and have relapsed and / or refractory MDS. In some embodiments, the patient has received at least one, but not more than three, preferably not more than two, prior standard anti-MDS therapies.

[0151] The dosing regimen is designed to include three doses of engineered NK cells administered within a 28-day dosing cycle. Prior to the dosing cycle, subjects undergo lymphodepletion (cytosine arabinoside (Ara-C) (2.0 g / m 2 / day) and fludarabine (30 mg / m 2A conditioning phase is performed with 1.5×10 / day (used on days −7, −6, −5, −4, and −3). On day 0, subjects are dosed with the first 3 doses of NK cells engineered to express a chimeric receptor-targeted ligand for NKG2D as well as mbIL15. Each dose contains 1.5×10 9 Dose 2 is administered on day 7 and dose 3 on day 14. Outcome measures are assessed on day 28.

[0152] Primary endpoints include: (1) the incidence, nature, and severity of treatment-related adverse events will be evaluated. An adverse event is any untoward and unintended manifestation, including clinically significant laboratory abnormalities, symptoms, or illness. This should be measured 30 days after the last dose of NK cells, and (2) the proportion of subjects who experience NK cell dose-limiting toxicity (DLT). A DLT is defined as a treatment-emergent adverse event that occurs during cycle 1 and meets protocol-specified criteria. This should be measured 28 days after the first dose of NK cells.

[0153] Secondary outcome measures include: (1) assessment of NK cell half-life measured as the time required for a 50% decline from the maximum amount of circulating engineered NK cells. This should be measured 28 days after the first dose of NK cells; (2) NK cell persistence by measuring the amount of engineered NK cells in peripheral blood every 3 months after dosing to determine persistence. This is measured for up to 2 years after the final dose of NK cells; (3) assessment of the host immune response to the engineered NK cells via serum samples measured for antibodies to the engineered NK cells. This is measured for up to 2 years after the final dose of NK cells; (4) objective response rate to the engineered NK cells by measuring the percentage of subjects who achieved complete and partial responses. AML subjects will be evaluated for the antitumor activity of the engineered NK cells based on the latest ELN criteria (Doehner 2017). MDS patients will be evaluated for the antitumor activity of the engineered NK cells based on the IWG criteria with MDS (Cheson 2006). This will be measured 28 days after the first dose of engineered NK cells and followed up to two years after the final dose.

[0154] Administration of three doses of engineered NK cells expressing a chimeric receptor construct targeting an NKG2D ligand and expressing mbIL15 is believed to be well tolerated and exhibit limited adverse events. Additionally, administration of three doses of engineered NK cells expressing a chimeric receptor construct targeting an NKG2D ligand and expressing mbIL15 is believed to result in limited DLTs. The NK cells are believed to exhibit extended half-life and prolonged duration. The NK cells are believed to induce limited host immune responses and clinically meaningful objective response rates (e.g., reduced tumor burden). Combination of lymphodepletion with Ara-C and fludarabine is believed to further enhance the anti-cancer effect of engineered NK cells due to the combined effect of lymphodepletion and anti-tumor effect. [Example 4]

[0155] Preliminary outcomes from a three-dose regimen Subjects were treated according to either Example 1 or Example 3 as discussed above. Additional subjects received 3 doses of either 100 million or 300 million engineered NK cells on each of days 0, 7, and 14 (this dosing cycle is disclosed in Examples 1 or 3). Alternative dosing / timing can be used depending on the embodiment. The median age of subjects was 60 years, with 17 subjects diagnosed with AML and 4 subjects diagnosed with MDS. The median time since diagnosis was 13 months. The median baseline blast percentage was 27%, and 15 subjects had a neutrophil count (ANC) of 1×10 9 The median number of prior therapies was 3. Among the treated subjects, the median number of prior therapies was 3. Among the AML patients, each patient had been previously treated with a BCL-2 inhibitor compound. Four patients (whether AML or MDS) had previously undergone allogeneic cell transplantation.

[0156] As discussed, subjects underwent lymphodepletion (cyclophosphamide (300 mg / m 2 ) and fludarabine (30 mg / m 2) on days -5, -4, and -3). Subjects received three doses of engineered NK cells expressing a chimeric receptor construct targeting NKG2D ligands and expressing mbIL15. Outcomes were measured at day 28. The three doses of engineered NK cells expressing a chimeric receptor construct targeting NKG2D ligands and expressing mbIL15 were well tolerated at each dose level tested across subjects dosed. No dose-limiting toxicities were observed. Lymphodepletion and myelosuppression and infections consistent with underlying disease were detected, with high-grade responses being the most common. Advantageously, no CAR T-like toxicity was observed at any dose. No subjects experienced cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) or other neurotoxicity, or graft-versus-host disease. The overall response rate for subjects receiving any of the four doses of NK cells was greater than 60%. Patients who received 100 or 300 million cells in each of the three doses demonstrated an overall response rate (complete response; complete response with poor hematologic recovery; morphological leukemia-free status; or partial response) of approximately 70%. 9 pcs or 1.5×10 9 In patients who received three doses of either of these cells, 60% had a complete response, with two of the three complete responders being minimally residually negative (i.e., no disease detected after treatment). Figure 4A shows the change in blast count from baseline (most recent blast count from subject prior to first dose). As can be seen, there is a significant trend toward decreased blast counts regardless of dose. As noted above, a 1.0 / 1.5 x 10 9 Three of the subjects who received cells had a complete response, one of which was observed to have positive minimal residual disease and two were observed to have negative minimal residual disease. One subject (male, age 68) diagnosed with AML (IDH1 mutated) was refractory to four lines of prior therapy with 8% blasts (by morphology) and 25% blasts (by FISH) at baseline. After dosing cycles (non-limiting examples include 1.0 × 10 cells on days 0, 7, and 14), 9(Figure 4B shows a graph of the detection of engineered NK cells (by measuring engineered NK cell DNA) over time for this subject. After each of the three doses (dotted line), engineered NK cells were detected in the blood. The administered cells showed the expected NK cell-like pharmacokinetic profile and clearance by day 20. After the first cycle, the subject was observed to have a complete response and to be negative for minimal residual tumor. Observations of the subject's bone marrow showed a normal cell phenotype. The engineered NK cells were well tolerated, with minor hematological effects of anemia, neutropenia, and decreased platelet count. At 6 months of follow-up, the subject underwent a consolidative hematopoietic cell transplant and maintained a complete response. These initial data demonstrate that engineered NK cells expressing NKG2D-targeted chimeric receptors and expressing mbIL15 are safe and effective cancer immunotherapeutic agents, especially when administered to patients in a three-dose regimen as provided in the embodiments herein.

[0157] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above can be made and still fall within the scope of one or more inventions. Moreover, any particular feature, aspect, method, property, characteristic, quality, attribute, element, etc. disclosed herein in relation to an embodiment can be used in all other embodiments described herein. Thus, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another to form various modes of the disclosed invention. Thus, it is intended that the scope of the invention disclosed herein should not be limited by the specific disclosed embodiments described above. Moreover, the invention is susceptible to various modifications and alternative forms, examples of which are shown in the drawings and described in detail herein. However, the invention is not limited to the particular forms or methods disclosed, but on the contrary, it should be understood that the invention covers all modifications, equivalents and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any method disclosed herein need not be performed in the order shown. The methods disclosed herein include specific actions taken by a practitioner; however, they may also include, explicitly or implicitly, third-party direction of those actions. Moreover, when features or aspects of the disclosure are described in terms of a Markush group, one of skill in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0158] Ranges disclosed herein also encompass any and all overlaps, subranges, and combinations thereof. Terms such as "up to," "at least," "greater than," "less than," "between" and the like include the indicated number. Numbers preceding terms such as "about" or "approximately" include the indicated number. 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 a reference sequence. Furthermore, when a sequence is disclosed as "comprising" a nucleotide or amino acid sequence, such reference is also intended to include that the sequence "comprises," "consists of," or "consists essentially of" the indicated sequence, unless otherwise indicated. Any titles or subheadings used herein are for organizational purposes and should not be used to limit the scope of the embodiments disclosed herein.

[0159] array In some embodiments, amino acid sequences corresponding to any of the nucleic acids disclosed herein (and / or contained in the attached sequence listing) are provided, taking into account the degeneracy of the nucleic acid code. Additionally, sequences (either nucleic acid or amino acid) that differ from the sequences explicitly disclosed herein (and / or contained in the attached sequence listing), but have functional similarity or equivalence, are also contemplated within the scope of the present disclosure. This includes mutations, truncations, substitutions, codon optimization, or other types of modifications.

[0160] In accordance with some embodiments described herein, any of the sequences may be used, or truncated or mutated forms of any of the sequences disclosed herein (and / or contained in the attached sequence listing) may be used, in any combination.

[0161] A sequence listing in electronic format may be submitted herewith. Some of the sequences provided in the sequence listing may be designated as artificial sequences by being non-naturally occurring fragments or parts of other sequences, including naturally occurring sequences. Some of the sequences provided in the sequence listing may be designated as artificial sequences by being combinations of sequences from different sources, such as humanized antibody sequences.

Claims

1. A pharmaceutical composition comprising genetically engineered natural killer (NK) cells for use in treating cancer in a subject, comprising: The genetically engineered NK cells are administered using a dosing regimen comprising at least a first dosing cycle; the first dosing cycle comprises a first dose of engineered natural killer (NK) cells, a second dose of engineered NK cells, and a third dose of engineered NK cells; a first dose is administered to the subject at a first time point; a second dose is administered to the subject 5 to 10 days after the first time point; a third dose is administered to the subject 5 to 10 days after the second dose; Each of the first, second and third doses is at least 1.0 x 10 ９ comprising genetically engineered NK cells, At least a portion of the genetically engineered NK cells are engineered to express a chimeric receptor that binds to a ligand for natural killer group 2D (NKG2D); The first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce natural immune cell numbers; The first dosing cycle may be followed by one or more additional dosing cycles. Pharmaceutical compositions.

2. The first, second, and third doses are each about 1.5×10 ９ 2. The pharmaceutical composition of claim 1, comprising the genetically engineered NK cells.

3. The pharmaceutical composition of claim 1, wherein the first dosing cycle is from about 14 days to about 35 days.

4. 2. The pharmaceutical composition of claim 1, wherein the lymphodepletion process comprises the administration of at least two doses of cyclophosphamide and at least two doses of fludarabine.

5. 5. The pharmaceutical composition of claim 4, wherein the lymphodepletion process comprises administration of three doses of cyclophosphamide and three doses of fludarabine, wherein a first dose of cyclophosphamide and fludarabine is administered 5 days prior to the start of a dosing cycle, a second dose of cyclophosphamide and fludarabine is administered 4 days prior to the start of a dosing cycle, and a third dose of cyclophosphamide and fludarabine is administered 3 days prior to the start of a dosing cycle.

6. 2. The pharmaceutical composition of claim 1, wherein the lymphodepletion process comprises the administration of at least two doses of cytosine arabinoside (Ara-C) and at least two doses of fludarabine.

7. 7. The pharmaceutical composition of claim 6, wherein the lymphodepletion process comprises administration of a daily dose of Ara-C for 5 consecutive days and a daily dose of Fludarabine for 5 consecutive days, the first dose of Ara-C and Fludarabine being administered 7 days prior to the start of a dosing cycle.

8. 8. The pharmaceutical composition of claim 7, wherein about 2 days are allowed to lapse between the final dose of Ara-C and fludarabine and the start of the dosing cycle.

9. Ara-C is about 0.5 to 10 g / m ２ / day, and fludarabine is administered at about 10-60 mg / m ２ The pharmaceutical composition of claim 6, wherein the composition is administered in an amount of about 100 mg / day.

10. Ara-C is about 1 to 5 g / m ２ / day, and fludarabine is administered at about 20-40 mg / m ２ The pharmaceutical composition of claim 6, wherein the composition is administered in an amount of about 100 mg / day.

11. Ara-C is about 2 g / m ２ / day, and fludarabine is administered at about 30 mg / m ２ The pharmaceutical composition of claim 6, wherein the composition is administered in an amount of about 100 mg / day.

12. The pharmaceutical composition of claim 1 , wherein the cancer is a blood cancer.

13. The pharmaceutical composition of claim 12, wherein the cancer is relapsed / refractory (R / R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS).

14. The pharmaceutical composition of claim 1 , wherein the cancer is a solid tumor.

15. 15. The pharmaceutical composition of claim 14, wherein the cancer comprises liver tumors, including intrahepatic cholangiocarcinoma and liver tumors that are secondary metastases from colorectal cancer.

16. The pharmaceutical composition of claim 1, wherein the second and third doses of the genetically engineered NK cells are administered to the subject within about 21 days or within about 14 days of the first time point.

17. The pharmaceutical composition of claim 1, wherein the chimeric receptor comprises an intracellular signaling domain comprising an OX40 subdomain and a CD3 zeta subdomain.

18. A pharmaceutical composition comprising a population of engineered natural killer (NK) cells expressing a chimeric receptor that targets a ligand of the NKG2D receptor for treating cancer in a subject, the pharmaceutical composition being administered by administration of at least first, second, and third doses of the engineered NK cells; The first dose of engineered NK cells is administered to the subject after the final dose of a lymphodepletion process comprising at least two doses of fludarabine; the second dose is administered to the subject 6 to 8 days after the first dose; a third dose is administered to the subject 6 to 8 days after the second dose; Each of the first, second and third doses is at least 1.0 x 10 ９ A pharmaceutical composition comprising the genetically engineered NK cells.

19. Each of the first, second and third doses is at least 1.5×10 ９ 20. The pharmaceutical composition of claim 18, comprising the genetically engineered NK cells.

20. 20. The pharmaceutical composition of claim 18, wherein the lymphodepleting regimen further comprises administration of at least two doses of cyclophosphamide.

21. 21. The pharmaceutical composition of claim 20, wherein the lymphodepleting regimen comprises administration of three doses of cyclophosphamide and three doses of fludarabine, wherein a first dose of cyclophosphamide and fludarabine is administered 5 days prior to the start of a dosing cycle, a second dose of cyclophosphamide and fludarabine is administered 4 days prior to the start of a dosing cycle, and a third dose of cyclophosphamide and fludarabine is administered 3 days prior to the start of a dosing cycle.

22. 19. The pharmaceutical composition of claim 18, wherein the lymphodepletion regimen further comprises administration of at least two doses of cytosine arabinoside (Ara-C).

23. 23. The pharmaceutical composition of claim 22, wherein the lymphodepletion regimen comprises administration of a daily dose of Ara-C for 5 consecutive days and a daily dose of Fludarabine for 5 consecutive days, with a first dose of Ara-C and Fludarabine being administered 7 days prior to the start of a dosing cycle.

24. 24. The pharmaceutical composition of claim 23, wherein about 2 days are allowed to lapse between the final dose of Ara-C and fludarabine and the start of the dosing cycle.

25. Ara-C is about 0.5 to 10 g / m ２ / day, and fludarabine is administered at about 10-60 mg / m ２ The pharmaceutical composition of claim 22, wherein the composition is administered in an amount of about 100 mg / day.

26. Ara-C is about 1 to 5 g / m ２ / day, and fludarabine is administered at about 20-40 mg / m ２ The pharmaceutical composition of claim 22, wherein the composition is administered in an amount of about 100 mg / day.

27. Ara-C is about 2 g / m ２ / day, and fludarabine is administered at about 30 mg / m ２ The pharmaceutical composition of claim 22, wherein the composition is administered in an amount of about 100 mg / day.

28. 19. The pharmaceutical composition of claim 18, wherein the cancer comprises relapsed / refractory (R / R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS).

29. 29. The pharmaceutical composition of claim 28, wherein the subject has relapsed and / or refractory acute myeloid leukemia and has received at least one but not more than two lines of prior standard anti-leukemia treatment.

30. The pharmaceutical composition of claim 18, wherein the chimeric receptor comprises an intracellular signaling domain comprising an OX40 subdomain and a CD3 zeta subdomain.

31. 1. A pharmaceutical composition comprising a population of engineered natural killer (NK) cells expressing a chimeric receptor that targets a ligand of the NKG2D receptor for treating cancer in a subject by intravenously administering at least three consecutive doses of the engineered NK cells, A first dose of the genetically engineered NK cells is administered to the subject at a first time point and comprises at least 1.0×10 ９ comprising genetically engineered NK cells, The second dose is administered to the subject 6-8 days after the first dose and contains at least 1.0 x 10 ９ comprising genetically engineered NK cells, The third dose is administered to the subject 6 to 8 days after the second dose and contains at least 1.0 x 10 ９ comprising genetically engineered NK cells, The engineered NK cells express a chimeric receptor having at least 95% sequence identity to SEQ ID NO:

34. Pharmaceutical compositions.

32. Each of the first, second and third doses is at least 1.5×10 ９ 32. The pharmaceutical composition of claim 31 , comprising the genetically engineered NK cells.

Images