Expansion of memory natural killer cells

Abstract

The present disclosure relates generally to natural killer (NK) cells, including, inter alia, memory-like and cytokine-induced memory-like (CIML) NK cells, methods for making and using same, for example, to treat cancer, and methods for increasing the anti-tumor properties of NK cells.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 222,306, filed July 15, 2021, the disclosure of which is incorporated by reference as if written in its entirety herein.

[0002] The present disclosure relates generally to natural killer (NK) cells, including, inter alia, memory / memory-like and cytokine-induced memory-like (CIML) NK cells, methods of making and using same, for example, to treat cancer, and methods of increasing the anti-tumor properties of NK cells. [Background technology]

[0003] Natural killer (NK) cells constitute a group of innate immune cells that are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cellular cytotoxicity by target-directed release of granzymes and perforin. Most NK cells possess a specific cell surface marker profile (e.g., CD3, CD56+, CD16+, CD57+, CD8+) as well as a diverse collection of activating and inhibitory receptors. Recently, NK cells have become an important component of certain cancer treatments, but the generation of large numbers of NK cells remains a major obstacle, as the proportion of NK cells in whole blood is relatively low. Summary of the Invention [Problem to be solved by the invention]

[0004] Various methods for generating memory NK cells are known in the art, all or nearly all of which suffer from various drawbacks such as low yields, the use of feeder cells, expensive reagents, etc. Consequently, there is a need to provide improved systems and methods for generating memory NK cells in large quantities. [Means for solving the problem]

[0005] Disclosed herein are compositions and methods that allow for the generation and expansion of memory / memory-like NK cells in a conceptually simple and efficient manner. Memory NK cells can be generated in a process that simultaneously primes NK cells to form memory NK cells and expands them to a desired volume. Alternatively, memory NK cells are formed by expanding NK cells to a desired volume and then priming.

[0006] A brief description of the sequence SEQ ID NOs: 1 to 48 in Tables 4 and 5 are the sequences of various components of the chimeric antigen receptor.

[0007] SEQ ID NOs:49-50 are sequences of exemplary extended fusion protein (EFP) chains that make up EFP 7t15-21s.

[0008] SEQ ID NOs:51-70 are sequences of exemplary cross-linker anti-tissue factor antibody ATF1.

[0009] SEQ ID NOs:71-72 are sequences of exemplary priming fusion protein (PFP) chains that make up PFP 18t15-12s. [Brief description of the drawings]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows the in vitro cancer cell (K562) killing rate by memory NK cells at days 6 / 7 at a given effector to target cell ratio for memory NK cells generated by a given combination of expansion and priming agents (7t15-21s+ATF1, 18t15-12s). [Diagram 2] Shown is the in vitro cancer cell (K562) killing rate by memory NK cells at day 13 at a given effector to target cell ratio for memory NK cells generated by a given combination of expansion and priming agents (7t15-21s+ATF1, 18t15-12s). [Diagram 3]FIG. 1 shows the in vitro cancer cell (K562) killing rate by memory NK cells at day 17 at a given effector to target cell ratio for memory NK cells generated by a given combination of expansion and priming agents (7t15-21s+ATF1, 18t15-12s). [Figure 4] The EC50 of the cell ratio for cancer cell (K562) killing by memory NK cells generated by a given combination of expansion and priming agents (7t15-21s+ATF1, 18t15-12s) is shown. [Diagram 5] The cumulative fold change in the number of stimulated NK cells over a given number of days in culture at a given cell density is shown. [Figure 6] Shown is the in vitro cancer cell (K562) killing rate at a given effector:target ratio for expanded cells primed with 250 nM priming agent for a given length of time. [Figure 7] Shown are in vitro cancer cell (K562) killing rates at an effector:target ratio of 20:1 on days 2, 4, 6, 8, 10, 12, 14 and 16 for expanded cells primed with 250 nM priming agent 18t15-12s for the given lengths of time. [Figure 8] Shown are in vitro cancer cell (K562) killing rates at an effector:target ratio of 4:1 on days 2, 4, 6, 8, 10, 12, 14 and 16 for expanded cells primed with 250 nM priming agent 18t15-12s for the given lengths of time. [Figure 9] Shown are in vitro cancer cell (K562) killing rates at an effector:target ratio of 0.8:1 on days 2, 4, 6, 8, 10, 12, 14 and 16 for expanded cells primed with 250 nM priming agent 18t15-12s for the given lengths of time. [Figure 10] Shown are in vitro cancer cell (K562) killing rates at an effector:target ratio of 0.16:1 on days 2, 4, 6, 8, 10, 12, 14 and 16 for expanded cells primed with 250 nM priming agent 18t15-12s for the given durations. [Figure 11] IFNg production in cell cultures (K562 only, expanded NK cells, and NK cells expanded and primed with 250 nM priming agent 18t15-12s for the given durations) after 24 hours of culture either alone or in the presence of K562 target cells is shown. [Figure 12] Fold expansion of NK cells generated by expansion alone, priming followed by expansion, and expansion followed by priming is shown. [Figure 13] Shown is the in vitro cancer cell (K562) killing rate at a given effector:target ratio for NK cells that were isolated, primed for 3 hours, primed overnight, primed overnight and expanded, expanded and primed for 3 hours, expanded and primed overnight, or expanded only. [Figure 14] The K562 killing EC50 of NK cells isolated and primed for 3 hours, primed overnight, primed overnight and expanded, expanded and primed for 3 hours, expanded and primed overnight, or expanded only is shown. [Figure 15] IFNg production in cell cultures (NK cells alone or with K562 cells) from NK cells that were isolated and primed for 3 hours, primed overnight, primed overnight and expanded, expanded and primed for 3 hours, expanded and primed overnight, or expanded only. [Figure 16] Fold change in number of NK cells generated by 3 hour priming, overnight priming, overnight priming followed by expansion, expansion only, or expansion followed by 3 hour priming in the blood of immunodeficient NSG mice is shown compared to background at 7 days post injection at t=0. [Figure 17] K562-Luc killing by expanded and post-expansion primed NK cells on day 14 is shown. [Figure 18] The EC50 for K562-Luc killing by expanded and primed NK cells after expansion is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description Provided herein are compositions and methods that allow for the generation and expansion of memory / memory-like NK cells in a conceptually simple and efficient manner. Memory NK cells can be generated in a process that simultaneously primes NK cells to form memory NK cells and expands them to a desired volume. Alternatively, memory NK cells are formed by expanding NK cells to a desired volume and then priming.

[0012] Thus, the present specification includes, in sequence, a) expanding a population of purified NK cells; and b) Priming NK cells The present invention provides memory natural killer (NK) cells produced by

[0013] Also provided herein are purified memory natural killer (NK) cells produced by simultaneously priming and expanding a population of purified NK cells.

[0014] In addition, the present specification sequentially includes the following: a) purifying a population of NK cells; b) expanding NK cells; and c) Priming NK cells Also provided are memory natural killer (NK) cells produced by

[0015] In addition, the present specification states: a) purifying a population of NK cells; and b) Simultaneous priming and expansion of NK cells Also provided are memory natural killer (NK) cells produced by

[0016] Further, the present specification states: a) expanding the purified NK cell population; and then b) Priming NK cells Disclosed is a method of generating memory NK cells comprising:

[0017] Further disclosed herein is a method for generating memory NK cells that involves simultaneously priming and expanding a purified NK cell population.

[0018] Further, the present specification states: a) purifying a population of NK cells; b) expanding NK cells; and then c) Priming NK cells Disclosed is a method of generating memory NK cells comprising:

[0019] Further, the present specification states: a) purifying a population of NK cells; and b) Simultaneous priming and expansion of NK cells Disclosed is a method of generating memory NK cells comprising:

[0020] The following embodiments are also provided:

[0021] In some embodiments, NK cell populations are purified starting from a blood donation or a fresh or previously cryopreserved leukapheresis product. In some embodiments, purification is performed via positive selection (e.g., with Miltenyi CliniMACS Prodigy). In some embodiments, purification is performed via negative selection (e.g., with StemCell EasySep NK Cell Enrichment Kit). In some embodiments, purification is performed using a combination of positive and negative selection. In some embodiments, NK cells are differentiated from lymphoid progenitor cells.

[0022] In some embodiments, the NK cells are expanded by exposure to an expansion agent comprising a combination of cytokines, or functional fragments thereof, and / or a fusion protein comprising a functional fragment thereof, or a combination of any of the foregoing, and optionally a cross-linking agent.

[0023] In some embodiments, the NK cells are one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or functional fragments thereof; or fusion proteins comprising functional fragments of one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and optionally a cross-linking agent; or · Microspheres functionalized with NK cell cross-linking antibodies and expanding cytokines; or any combination of the foregoing They are enlarged by exposure to enlarging agents, including

[0024] In some embodiments, the NK cells are expanded by exposure to an expansion agent comprising a combination of IL-7, IL-21, and IL-15, or functional fragments thereof, and / or fusion proteins comprising functional fragments thereof, or a combination of any of the foregoing.

[0025] In some embodiments, the NK cells are expanded by exposure to an expansion agent comprising a fusion protein comprising functional fragments of IL-7, IL-21, and IL-15.

[0026] In some embodiments, the NK cells are expanded by exposure to an expansion agent that comprises 7t15-21s.

[0027] In some embodiments, the spreading agent comprises a cross-linking agent. In some embodiments, the cross-linking agent is a cross-linking antibody. In some embodiments, the cross-linking antibody is ATF1.

[0028] In some embodiments, the NK cells are expanded by exposure to an expansion agent comprising 7t15-21s and ATF1.

[0029] In some embodiments, the NK cells are expanded by exposure to an expansion agent comprising an NK cell crosslinking antibody and microspheres functionalized with an expanding cytokine.

[0030] In some embodiments, the NK cells are expanded by exposure to an expansion agent for between 1 and 40 days. In some embodiments, the NK cells are expanded by exposure to an expansion agent for between 7 and 21 days. In some embodiments, the NK cells are expanded by exposure to an expansion agent for about 14 days.

[0031] In some embodiments, the broadening agent comprises 7t15-21s and ATF1. In some embodiments, the broadening agent comprises 7t15-21s at a concentration of 0.1-300 nm and ATF1 at a concentration of 0.01-200 nm. In some embodiments, the broadening agent comprises 7t15-21s at a concentration of 0.2-200 nm and ATF1 at a concentration of 0.01-100 nm. In some embodiments, the broadening agent comprises 7t15-21s at a concentration of about 50 nm and ATF1 at a concentration of about 25 nm.

[0032] In some embodiments, NK cells are expanded by exposure to 7t15-21s and ATF1 for about 14 days. In some embodiments, NK cells are expanded by exposure to 7t15-21s at a concentration of about 50 nM and ATF1 at a concentration of about 25 nM for about 14 days.

[0033] In some embodiments, the NK cells are primed by exposure to a priming agent selected from, for example, a combination of cytokines, or a functional fragment thereof, and / or a fusion protein comprising a functional fragment thereof, or a combination of any of the foregoing.

[0034] In some embodiments, the NK cells are one or more of IL-12, IL-23, IL-27, and IL-35; one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and one or more of IL-18, IL-1a, IL-1b, IL-36a, IL-36b, and IL-36g; or a functional fragment thereof, and / or a fusion protein comprising a functional fragment thereof, or any combination of the foregoing. The antibody is primed by exposure to a priming agent comprising:

[0035] In some embodiments, the NK cells are primed by exposure to a priming agent comprising a combination of IL-12, IL-15, and IL-18.

[0036] In some embodiments, the NK cells are primed by exposure to a priming agent comprising a fusion protein comprising functional fragments of IL-12, IL-15, and IL-18. In some embodiments, the NK cells are primed by exposure to a priming agent comprising the fusion protein 18t15-12s.

[0037] In some embodiments, NK cells are primed with 18t15-12s at a concentration of 200-300 nM. In some embodiments, NK cells are primed with 18t15-12s at a concentration of 250 nM.

[0038] In some embodiments, the NK cells are primed for 1 minute to 24 hours. In some embodiments, the NK cells are primed for 0.5 to 16 hours. In some embodiments, the NK cells are primed for 1 to 3 hours.

[0039] In some embodiments, the NK cells are cryopreserved.

[0040] In some embodiments, the NK cells are first expanded and then primed.

[0041] In some embodiments, the NK cells are expanded to more than 10-fold the starting number. In some embodiments, the NK cells are expanded to more than 100-fold the starting number. In some embodiments, the NK cells are expanded to more than 1000-fold the starting number.

[0042] In some embodiments, the NK cells are expanded and primed simultaneously.

[0043] In some embodiments, the cells have a memory-like (ML) NK phenotype.

[0044] In some embodiments, the memory-like phenotype is indicated by expression levels of cell surface CD69, CD25, CD16, and / or NKG2A.

[0045] In some embodiments, memory NK cells have a higher NK cell population than unprimed NK cells: a) Improved cytotoxicity against cancer cells; b) improved sustainability; c) improved antitumor activity; and / or d) increased cytokine production; One or more of the following are seen:

[0046] In some embodiments, the cancer cells are K562 cells.

[0047] In some embodiments, the cytokines produced are selected from IFNg, TNFa, GM-CSF, and combinations thereof.

[0048] In some embodiments, persistence is as measured over a period of 1-14 days in immunodeficient mice.

[0049] In some embodiments, the mouse is an NSG mouse.

[0050] In some embodiments, anti-tumor activity is measured as a reduction in K562 cell tumor growth in immunodeficient mice.

[0051] In some embodiments, the NK cells are cytokine-induced memory-like (CIML) NK cells.

[0052] In some embodiments, the memory NK cells additionally comprise: a) at least one extracellular ligand-binding domain that targets an antigen on a target cell; b) hinge domain; c) transmembrane domain; d) optionally, one or more costimulatory domains; and e) Cytoplasmic signaling domain The antibody comprises at least one chimeric antigen receptor (CAR),

[0053] Also provided herein is a method of treating a proliferative malignancy comprising administration of memory NK cells according to the above embodiments, or cells as produced by the method of the above embodiments, to a patient in need thereof.

[0054] In some embodiments, the cells are administered fresh to the patient.

[0055] In some embodiments, the proliferative malignancy is cancer.

[0056] In some embodiments, the cancer is a hematological cancer.

[0057] In some embodiments, the hematological cancer is selected from leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome.

[0058] In some embodiments, the hematological cancer is B-cell lymphoma.

[0059] In some embodiments, the B cell lymphoma is selected from diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL).

[0060] In some embodiments, the hematological cancer is T-cell lymphoma.

[0061] In some embodiments, the T cell lymphoma is selected from T cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T cell lymphoma (PTCL), T cell chronic lymphocytic leukemia (T-CLL), and Sézary syndrome.

[0062] In some embodiments, the hematological cancer is leukemia.

[0063] In some embodiments, the leukemia is selected from acute myeloid (or myeloid) leukemia (AML), chronic myeloid (or myeloid) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), and hairy cell leukemia.

[0064] In some embodiments, the hematological cancer is a plasma cell malignancy.

[0065] In some embodiments, the plasma cell malignancy is selected from lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0066] In some embodiments, the cancer is a solid tumor.

[0067] In some embodiments, the solid tumor is selected from a melanoma, neuroblastoma, glioma, sarcoma, or carcinoma.

[0068] In some embodiments, the solid tumor is a tumor of the brain, head, neck, breast, lung (e.g., non-small cell lung cancer, NSCLC), reproductive system (e.g., ovary), upper gastrointestinal tract, pancreas, liver, renal system (e.g., kidney), bladder, prostate, or colorectum.

[0069] Enumerated Embodiments The following embodiments are also provided herein:

[0070] Embodiment 1. Sequentially, a) expanding purified NK cells; and b) Priming NK cells A population of purified memory natural killer (NK) cells produced by

[0071] Embodiment 2. A population of purified memory natural killer (NK) cells generated by simultaneously priming and expanding purified NK cells.

[0072] Embodiment 3. The memory NK cells of embodiment 1 or 2, wherein the NK cells are enriched from fresh or frozen leukapheresis products or blood donations.

[0073] Embodiment 4. The memory NK cell of embodiment 1 or 2, wherein the NK cell is differentiated from a lymphoid progenitor cell.

[0074] Embodiment 5. The memory NK cells of embodiment 1 or 2, wherein the NK cells are purified by negative or positive selection, or a combination thereof.

[0075] Embodiment 6. The NK cells are one or more of IL-12, IL-23, IL-27, and IL-35; one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and one or more of IL-18, IL-1a, IL-1b, IL-36a, IL-36b, and IL-36g; or a functional fragment thereof, and / or a fusion protein comprising a functional fragment thereof, or any combination of the foregoing. The memory NK cell of embodiment 1 or 2, which is primed by exposure to

[0076] Embodiment 7. The memory NK cell of embodiment 6, wherein the NK cell is primed by exposure to 18t15-12s.

[0077] Embodiment 8. The memory NK cells of any one of embodiments 1 to 7, wherein the NK cells are primed for 1 minute to 24 hours.

[0078] Embodiment 9. The memory NK cell of embodiment 6, wherein the NK cell is primed by exposure to IL-12, IL-15, and IL-18.

[0079] Embodiment 10. The memory NK cells of embodiment 9, wherein the NK cells are primed for 1 minute to 24 hours.

[0080] Embodiment 11. The memory NK cells of any one of embodiments 1-10, wherein the NK cells are expanded by exposure to 7t15-21s and ATF1.

[0081] Embodiment 12. The memory NK cells of embodiment 11, wherein the NK cells are expanded for 1 to 40 days.

[0082] Embodiment 13. A memory NK cell according to any one of embodiments 1 to 12, wherein the memory NK phenotype is indicated by increased expression of CD69, CD25, and NKG2A, and sustained expression of CD16, as compared to untreated NK cells.

[0083] Embodiment 14. Memory NK cells, compared to untreated NK cells, Improved cytotoxicity against cancer cells; ·Improved sustainability; Improved antitumor activity; and / or Increased cytokine production The memory NK cells according to any one of embodiments 1 to 13, wherein one or more of the following are observed:

[0084] Embodiment 15. The memory NK cell of embodiment 14, wherein the cancer cell is a K562 cell.

[0085] Embodiment 16. The memory NK cell of embodiment 14, wherein the cytokines produced are selected from IFNg, TNFa, GM-CSF, and combinations thereof.

[0086] Embodiment 17. The memory NK cells of embodiment 14, wherein the persistence is as measured over a period of 1 to 14 days in immunodeficient mice.

[0087] Embodiment 18 The memory NK cell of embodiment 17, wherein the mouse is an NSG mouse.

[0088] Embodiment 19. The memory NK cells of embodiment 14, wherein the anti-tumor activity is measured as a reduction in tumor growth of cancer cells in immunodeficient mice.

[0089] Embodiment 20. The memory NK cell of any one of embodiments 1 to 19, wherein the NK cell is a cytokine-induced memory-like (CIML) NK cell.

[0090] Embodiment 21. In addition, a. at least one extracellular ligand binding domain that targets an antigen on a target cell; b. hinge domain; C. transmembrane domain; d. optionally, one or more costimulatory domains; and e. cytoplasmic signaling domain 21. The memory NK cell of any one of embodiments 1 to 20, comprising at least one chimeric antigen receptor (CAR).

[0091] Embodiment 22. A method for generating memory NK cells, comprising: a) purifying an enriched NK cell population; b) expanding NK cells; and c) Priming NK cells The method includes:

[0092] Embodiment 23. A method for generating memory NK cells, comprising: a) purifying an enriched NK cell population; and b) Simultaneous priming and expansion of NK cells The method includes:

[0093] Embodiment 24. The method of embodiment 22 or 23, wherein the NK cells are enriched from a fresh or frozen leukapheresis product or blood donation.

[0094] Embodiment 25 The method of embodiment 22 or 23, wherein the NK cells are differentiated from lymphoid progenitor cells.

[0095] Embodiment 26 The method of embodiment 22 or 23, wherein the NK cells are purified by negative or positive selection, or a combination thereof.

[0096] Embodiment 27. The NK cells are one or more of IL-12, IL-23, IL-27, and IL-35; one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and one or more of IL-18, IL-1a, IL-1b, IL-36a, IL-36b, and IL-36g; or a functional fragment thereof, and / or a fusion protein comprising a functional fragment thereof, or any combination of the foregoing. 24. The method of embodiment 22 or 23, wherein the antibody is primed by exposure to

[0097] Embodiment 28 The method of embodiment 27, wherein the NK cells are primed by exposure to 18t15-12s.

[0098] Embodiment 29. The method of embodiment 28, wherein the NK cells are primed for 1 minute to 24 hours.

[0099] Embodiment 30 The method of embodiment 27, wherein the NK cells are primed by exposure to IL-12, IL-15, and IL-18.

[0100] Embodiment 31 The method of embodiment 28, wherein the NK cells are primed for 2 to 40 days.

[0101] Embodiment 32 The method of embodiment 22 or 23, wherein the NK cells are expanded by exposure to 7t15-21s and ATF1.

[0102] Embodiment 33 The method of embodiment 22 or 23, wherein the NK cells are expanded for 1 to 40 days.

[0103] Embodiment 34. The method of any one of embodiments 1 to 33, wherein the memory NK phenotype is indicated by increased expression of CD69, CD25, and NKG2A, and sustained expression of CD16, compared to naive NK cells.

[0104] Embodiment 35. Memory NK cells, compared to untreated NK cells, Improved cytotoxicity against cancer cells; ·Improved sustainability; Improved antitumor activity; and / or Increased cytokine production The method according to any one of embodiments 22 to 34, wherein one or more of the following are observed:

[0105] Embodiment 36 The method of embodiment 35, wherein the cancer cells are K562 cells.

[0106] Embodiment 37. The method of embodiment 35, wherein the cytokines produced are selected from IFNg, TNFa, GM-CSF, and combinations thereof.

[0107] Embodiment 38. The method of embodiment 35, wherein the persistence is as measured over a period of 1 to 14 days in immunodeficient mice.

[0108] Embodiment 39 The method of embodiment 38, wherein the mouse is an NSG mouse.

[0109] Embodiment 40 The method of embodiment 35, wherein the improved anti-tumor activity is a reduction in tumor growth of cancer cells in immunocompromised mice.

[0110] Embodiment 41. The method of any one of embodiments 1 to 40, wherein the cells are cytokine-induced ML (CIML) NK cells.

[0111] Embodiment 42. A method of treating a proliferative malignancy comprising administering to a patient in need thereof memory NK cells as described in any one of embodiments 1-21, or memory NK cells as produced by the method of any one of embodiments 22-41.

[0112] Embodiment 43 The method of embodiment 42, wherein the cells are administered fresh to the patient.

[0113] Embodiment 44 The method of embodiment 42, wherein the proliferative malignancy is cancer.

[0114] Embodiment 45 The method of embodiment 44, wherein the cancer is a blood cancer.

[0115] Embodiment 46 The method of embodiment 44, wherein the hematological cancer is selected from leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome.

[0116] Embodiment 47. The method of embodiment 46, wherein the hematological cancer is B-cell lymphoma.

[0117] Embodiment 48. The method of embodiment 47, wherein the B-cell lymphoma is selected from diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL).

[0118] Embodiment 49. The method of embodiment 46, wherein the hematological cancer is T-cell lymphoma.

[0119] Embodiment 50. The method of embodiment 49, wherein the T-cell lymphoma is selected from T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), and Sezary syndrome.

[0120] Embodiment 51 The method of embodiment 46, wherein the blood cancer is leukemia.

[0121] Embodiment 52. The method of embodiment 51, wherein the leukemia is selected from acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL) and hairy cell leukemia.

[0122] Embodiment 53 The method of embodiment 46, wherein the hematological cancer is a plasma cell malignancy.

[0123] Embodiment 54. The method of embodiment 53, wherein the plasma cell malignancy is selected from lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0124] Embodiment 55. The method of embodiment 44, wherein the cancer is a solid tumor.

[0125] Embodiment 56 The method of embodiment 55, wherein the solid tumor is selected from melanoma, neuroblastoma, glioma, sarcoma, or carcinoma.

[0126] Embodiment 57. The method of embodiment 55, wherein the solid tumor is a tumor of the brain, head, neck, breast, lung (e.g., non-small cell lung cancer, NSCLC), reproductive system (e.g., ovary), upper gastrointestinal tract, pancreas, liver, renal system (e.g., kidney), bladder, prostate, or colorectum.

[0127] Methods for expanding and priming immune effector cells In vitro expansion of NK cells may be performed with an enrichment process using an expansion agent comprising a cytokine, or preferably an expansion fusion protein comprising a functional fragment of a cytokine, and a multi-chain complex thereof. For example, the expansion agent may comprise one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, or a combination thereof, e.g., a cocktail of IL-7, IL-21, and IL-15, in an amount sufficient to generate a desired quantity or expansion fold of NK cells. Such cytokines may be commercially available or may be made by methods known in the art. Or, for example, the expansion agent may comprise one or more expansion fusion proteins, e.g., 7t15-21s, which may be selected from among the multi-chain fusion protein complexes disclosed in WO2020047299, WO202047473, or WO2020257639, in an amount sufficient to expand NK cells. The sequence of 7t15-21s is disclosed in Table 1.

[0128] [Table 1]

[0129] The expansion is additionally facilitated by the use of a crosslinker, such as an antibody that targets the linking domain of the fusion protein disclosed above, such as an anti-tissue factor antibody. Examples of anti-tissue factor antibodies are known in the art. WO202047473 and WO2020257639 disclose the a-TF Ab used. See also U.S. Pat. No. 8,007,795 and WO2003037911, specifically the IgG1 humanized antibody incorporating the CDRs and humanized framework regions LC-08 (Figure 12) and HC-09 (Figure 13) of the H36 hybridoma. Table X below discloses the sequence of the a-TF Ab disclosed in US'795 and WO'911, obtained from HCW Biologics, as believed to be used in WO'473 and WO'639, and used in the following experiments unless otherwise specified, referred to herein as ATF1. ATF1 HCDR2 is one of the following two sequences: Thus, an expansion agent as disclosed herein may include a combination of one or more cytokines or EFPs as disclosed above, together with a crosslinker such as ATF1, one or more sequences of which are disclosed in Table 2.

[0130] [Table 2]

[0131] [Table 3]

[0132] Alternative cross-linking methods are known in the art and include functionalized microparticles (beads), feeder cells and cell membrane particles. In many cases, feeder-free systems are preferred. For example, R&D Systems' Cloudz Human NK Cell Expansion Kit, which utilizes dissolvable sodium alginate microspheres functionalized with anti-CD2 and anti-NKp46 antibodies, may be used with expansion cytokines (or fragments thereof, or fusion proteins containing same) and combinations thereof as disclosed herein, in addition to a release buffer to quickly dissolve the microparticles after expansion and facilitate cell recovery.

[0133] Priming to acquire memory-like properties is performed with a priming agent comprising a combination of stimulatory cytokines, such as one or more of IL-12, IL-23, IL-27, and IL-35; one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and one or more of IL-18, IL-1a, IL-1b, IL-36a, IL-36b, and IL-36g. Alternatively, the priming agent may comprise a priming fusion protein comprising a functional fragment of a cytokine, and a multi-chain complex thereof. For example, the fusion protein may be selected from among the multi-chain fusion protein complexes disclosed in WO2020047299, WO202047473, or WO2020257639, such as 18t15-12s (HCW-9201), the sequence of which is disclosed in Table 3.

[0134] [Table 4]

[0135] Chimeric antigen receptors (CARs) and CAR-bearing immune effector cells Also provided herein are chimeric antigen receptors (CARs) comprising the polypeptides as disclosed herein, and immune effector cells expressing same. CARs are recombinant fusion proteins that typically include 1) an extracellular ligand-binding domain, i.e., an antigen recognition domain, 2) a hinge domain, 3) a transmembrane domain, and 4) a cytoplasmic signaling domain, 5) and optionally a co-stimulatory domain.

[0136] Methods for the design, delivery and expression of CARs, as well as methods for producing clinical-grade cell populations expressing CARs, are known in the art. CAR design is generally tailored for each cell type.

[0137] The extracellular ligand-binding domain of the chimeric antigen receptor recognizes and specifically binds to an antigen, typically a surface-expressed antigen of a malignant cell. The extracellular ligand-binding domain may, for example, be a ligand that has an affinity constant or affinity of interaction (K) of about 0.1 pM to about 10 μM, or about 0.1 pM to about 1 μM, or about 0.1 pM to about 100 nM. D ) specifically binds to an antigen. Methods for determining the affinity of an interaction are known in the art. An extracellular ligand-binding domain can also be said to specifically bind when it selectively binds to a first polymorphic variant of an antigen relative to a second polymorphic variant of the same antigen.

[0138] The extracellular ligand-binding domain suitable for use in a CAR can be any antigen-binding polypeptide, a wide variety of which are known in the art. In some examples, the extracellular ligand-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelized antibody variable domain) and humanized versions thereof, lgNAR VH (shark antibody variable domain) and humanized versions thereof, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domains are suitable for use. In some instances, T cell receptor (TCR)-based recognition domains such as single chain TCR (scTv, single chain two domain TCR with VαVβ) are also suitable for use. In some embodiments, the extracellular ligand binding domain is constructed from a natural binding partner for the target antigen, or a functional fragment thereof. For example, a CAR can generally be constructed with a portion of the APRIL protein that targets the ligands of B cell maturation antigen (BCMA) and transmembrane activator-CAML interactor (TACI) to effectively co-target both BCMA and TACI for the treatment of multiple myeloma.

[0139] The targeted antigen that the CAR binds to through its extracellular ligand-binding domain can be an antigen expressed on malignant myeloid (AML) cells, T cells, or other cells. Antigens expressed on malignant myeloid (AML) cells include CD33, FLT3, CD123, and CLL-1. Antigens expressed on T cells include CD2, CD3, CD4, CD5, CD7, TCRα (TRAC), and TCRβ. Antigens expressed on malignant plasma cells include BCMA, CS1, CD38, CD79A, CD79B, CD138, and CD19. Antigens expressed on malignant B cells include CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, and CD45.

[0140] Typically, the extracellular ligand-binding domain is linked to the intracellular domain of the chimeric antigen receptor by a transmembrane (TM) domain. A peptide hinge connects the extracellular ligand-binding domain to the transmembrane domain. The transmembrane domain crosses the cell membrane, anchoring the CAR to the T cell surface and connecting the extracellular ligand-binding domain to the cytoplasmic signaling domain, thus influencing the expression of the CAR on the T cell surface.

[0141] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), IC OS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD1 9, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d , ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp. Alternatively, the transmembrane domain may be synthetic and comprise primarily hydrophobic amino acid residues (e.g., leucine and valine). In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the transmembrane domain is derived from the T cell surface glycoprotein CD8 α chain isoform 1 precursor (NP_001139345.1) or CD28. A small oligopeptide or polypeptide linker, such as 2-10 amino acids in length, may form the link between the transmembrane and endoplasmic domains of the CAR.In some embodiments, a CAR has two or more transmembrane domains, which may be repeats of the same transmembrane domain or may be different transmembrane domains.

[0142] NK cells express several transmembrane (TM) adaptors that send activation signals upon engagement with activating receptors, which results in the enhancement of NK cell-specific signals through the manipulation of TM domains from activating receptors, which add endogenous adaptors. The TM adaptor may be any endogenous TM adaptor capable of sending activation signals. In some embodiments, the TM adaptor may be selected from FceR1γ (ITAMx1), CD3ζ (ITAMx3), DAP12 (ITAMx1), or DAP10 (YxxM / YINM), NKG2D, FcγRIIIa, NKp44, NKp30, NKp46, actKIR, NKG2C, CD8α, and IL15Rb.

[0143] The CAR may further comprise a hinge region between the extracellular ligand-binding domain and the transmembrane domain. The term "hinge region" (equivalently, "hinge" or "spacer") generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the hinge region is used to provide a more flexible and accessible extracellular ligand-binding domain, and can confer stability for efficient CAR expression and activity. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The hinge region may be derived from all or a portion of a naturally occurring molecule, such as CD28, 4-1BB (CD137), OX-40 (CD134), CD3zeta, T cell receptor alpha or beta chain, CD45, CD4, CD5, CD8, CD8alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, ICOS, CD154, or all or a portion of an antibody constant region. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or the hinge region may be a completely synthetic hinge sequence. In one embodiment, the hinge domain comprises a portion of human CD8alpha (SEQ ID NO:2), FcγRIIIalpha receptor, or IgG1, with at least 80%, 90%, 95%, 97%, or 99% sequence identity thereto.

[0144] After antigen recognition, the cytoplasmic signaling domain transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell. The effector function of the NK cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Usually, the entire cytoplasmic signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function.

[0145] Cytoplasmic signaling sequences that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAMs with cytoplasmic signaling sequences include those derived from CD8, CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD32 (FcgammaRIIa), DAP10, DAP12, CD79a, CD79b, FcgammaRIgamma, FcgammaRIIIgamma, FcεRIβ (FCERIB), and FcεRIγ (FCERIG).

[0146] First generation CARs typically have a cytoplasmic signaling domain from the CD3 chain, which is primarily responsible for transmitting signals from the endogenous TCR. Second generation CARs provide additional signals to the cell by adding cytoplasmic signaling domains from various co-stimulatory protein receptors (e.g., CD28, 4-1BB, ICOS) to the cytoplasmic signaling domain of the CAR.

[0147] The "costimulatory domain" is derived from the intracellular signaling domain of a costimulatory protein that enhances cytokine production, proliferation, cytotoxicity, and / or persistence in vivo. Preclinical studies indicate that second generation CARs are designed to improve antitumor activity. Recently, third and subsequent generation CARs have further enhanced efficacy by combining multiple costimulatory domains. These CAR-grafted cells have demonstrated improved expansion, activation, persistence, and tumor eradication efficiency independent of costimulatory receptor / ligand interactions.

[0148] For example, the cytoplasmic signaling domain of a CAR can be designed to include a signaling domain (e.g., CD3ζ) itself, or can be combined with any other desired cytoplasmic domain or domains useful in the context of a CAR. For example, the cytoplasmic domain of a CAR includes a signaling domain (e.g., CD3ζ) chain portion and a costimulatory signaling region. A costimulatory signaling region refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.

[0149] In some embodiments, the cytoplasmic signaling domain is a CD3 zeta (CD3ζ) signaling domain. In some embodiments, the costimulatory domain comprises the cytoplasmic domain of CD28, 4-1BB, or a combination thereof. In some cases, the costimulatory signaling region has 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules.

[0150] The one or more costimulatory signaling domains may have one or more mutations in the cytoplasmic domain of CD28 and / or 4-1BB that enhance signaling. In some embodiments, the disclosed CARs comprise a costimulatory signaling region that includes a cytoplasmic domain of a mutant form of CD28 with altered phosphorylation at Y206 and / or Y218. In some embodiments, the disclosed CARs comprise an attenuating mutation at Y206, which reduces the activity of the CAR. In some embodiments, the disclosed CARs comprise an attenuating mutation at Y218, which reduces the expression of the CAR. To achieve attenuation, any amino acid residue can be substituted for tyrosine, such as alanine or phenylalanine. In some embodiments, the tyrosine at Y206 and / or Y218 is substituted with a phosphomimetic residue. In some embodiments, the substitution of Y206 with a phosphomimetic residue in the disclosed CARs increases the activity of the CAR. In some embodiments, the disclosed CARs comprise a substitution of Y218 with a phosphomimetic residue, which increases the expression of the CAR. For example, the phosphomimetic residue may be phosphotyrosine. In some embodiments, a CAR may contain a combination of a phosphomimetic amino acid and one or more substitutions with non-phosphorylatable amino acids at different residues of the same CAR. For example, a CAR may contain an alanine or phenylalanine substitution at Y209 and / or Y191, as well as a phosphomimetic substitution at Y206 and / or Y218.

[0151] In some embodiments, the disclosed CARs comprise one or more 4-1BB domains with mutations that enhance binding to a specific TRAF protein, such as TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or any combination thereof. In some cases, the 41BB mutation enhances TRAF1-dependent and / or TRAF2-dependent proliferation and survival of T cells, e.g., through NF-kB. In some cases, the 4-1BB mutation enhances TRAF3-dependent anti-tumor efficacy, e.g., through IRF7 / INFβ. Thus, the disclosed CARs can comprise one or more cytoplasmic domains of 4-1BB with at least one mutation in these highlighted sequences that enhances TRAF binding and / or enhances NFκB signaling.

[0152] Also as disclosed herein, TRAF proteins can optionally enhance CAR T cell function independent of NFκB and 4-1BB. For example, TRAF proteins can optionally enhance CD28 costimulation in T cells. Thus, also disclosed herein are immune effector cells co-expressing a CAR with one or more TRAF proteins, such as TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or any combination thereof. In some cases, the CAR is any CAR that targets a tumor antigen. For example, first generation CARs typically had an intracellular domain from the CD3 chain, while second generation CARs provided additional signals to T cells by adding intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 4-1BB, ICOS) to the cytoplasmic signaling domain of the CAR. In some cases, the CAR is a disclosed CAR with enhanced 4-1BB activation.

[0153] Depending on the cell type expressing the CAR, mutations in the CAR components may be advantageous.

[0154] For example, in NK cells, in some embodiments, the transmembrane domain can be a sequence associated with NKG2D, FcγRIIIa, NKp44, NKp30, NKp46, actKIR, NKG2C, or CD8α. In certain embodiments, the NK cells are ML-NK or CIML-NK cells and the TM domain is CD8α. Certain TM domains that do not function well in NK cells generally may function in a subset; CD8α, for example, functions in ML-NK but does not function generally in NK cells.

[0155] Similarly, in NK cells, in some embodiments, the one or more intracellular signaling domains can be any one or more co-activating receptors capable of functioning in NK cells, such as, for example, CD28, CD137 / 41BB (TRAF, NFkB), CD134 / OX40, CD278 / ICOS, DNAM-1 (Y-motif), NKp80 (Y-motif), 2B4 (SLAMF)::ITSM, CRACC (CS1 / SLAMF7)::ITSM, CD2 (Y-motif, MAPK / Erk), CD27 (TRAF, NFkB), or an integrin (e.g., multiple integrins).

[0156] Similarly, in NK cells, in some embodiments, the intracellular signaling domain can be a cytokine receptor capable of functioning in NK cells. For example, the cytokine receptor can be a cytokine receptor associated with persistence, survival, or metabolism, such as IL-2 / 15Rbyc::Jak1 / 3, STAT3 / 5, PI3K / mTOR, MAPK / ERK. As another example, the cytokine receptor can be a cytokine receptor associated with activation, such as IL-18R::NFkB. As another example, the cytokine receptor can be a cytokine receptor associated with IFN-γ production, such as IL-12R::STAT4. As another example, the cytokine receptor can be a cytokine receptor associated with cytotoxicity or persistence, such as IL-21R::Jak3 / Tyk2, or STAT3. As another example, the intracellular signaling domain can be a TM adaptor, such as FceR1γ (ITAMx1), CD3ζ (ITAMx3), DAP12 (ITAMx1), or DAP10 (YxxM / YINM). As another example, the CAR intracellular signaling domain (also known as the endodomain) can be derived from costimulatory molecules from the CD28 family (such as CD28 and ICOS) or the tumor necrosis factor receptor (TNFR) gene family (such as 4-1BB, OX40, or CD27). TNFR family members signal through the recruitment of TRAF proteins and are associated with cell activation, differentiation, and survival. Certain signaling domains may not function well in all NK cells generally, but may function in subsets; CD28 or 4-1BB, for example, function in ML-NK.

[0157] Any domain of the CAR may also contain a heterodimerization domain in order to separate the key signaling and antigen recognition modules of the CAR.

[0158] The CAR may be designed to contain any portion or parts of the above-mentioned domains as described herein in any combination that results in a functional CAR.

[0159] Methods for producing CARs and CAR-bearing cells Chimeric antigen receptor (CAR) constructs encoding chimeric receptors can be prepared by conventional methods. Natural sequences are generally utilized, and natural genes are isolated and manipulated as necessary to allow the various components to be appropriately joined together (e.g., when using type II receptors, immune signaling receptor components may have to be inverted). Thus, by utilizing polymerase chain reaction (PCR), nucleic acid sequences encoding N-terminal and C-terminal proteins of chimeric receptors can be isolated using appropriate primers that cause deletion of undesired parts of the gene. Alternatively, restriction digestion of cloned genes can be used to generate chimeric constructs. In either case, sequences can be selected to provide blunt-ended or complementary overlapping restriction sites.

[0160] Various manipulations for preparing chimeric constructs can be performed in vitro, and in a specific embodiment, the chimeric constructs are introduced into vectors for cloning and expression in a suitable host using standard transformation or transfection methods. Thus, after each manipulation, the construct obtained by joining DNA sequences is cloned, the vector is isolated, and the sequence is screened to ensure that the sequence encodes the desired chimeric receptor. The sequence can be screened by restriction analysis, sequencing, etc.

[0161] The chimeric construct can be introduced into immune effector cells as naked DNA or in a suitable vector.Methods for stably transfecting immune effector cells by electroporation using naked DNA are known in the art.Naked DNA generally refers to the DNA encoding the chimeric receptor contained in a plasmid expression vector in the proper orientation for expression.

[0162] Alternatively, the chimeric construct can be introduced into immune cells, e.g., T cells, using a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector). A suitable vector is non-replicative in the immune effector cells of the subject. A number of viral-based vectors are known in which the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell. Exemplary vectors include the pFB-neo vector (STRATAGENE™) as well as vectors based on HIV, SV40, EBV, HSV, or BPV. Once it has been established that the chimeric receptor is capable of being expressed as a surface membrane protein with the desired regulation and at the desired level by the transfected or transduced immune effector cells, it can be determined whether the chimeric receptor is functional in the host cell and provides the desired signal induction (e.g., production of RANTES, 1-alpha, GM-CSF upon stimulation with the appropriate ligand).

[0163] The engineered CAR may be introduced into the CAR-bearing immune effector cells using a retrovirus that efficiently and stably integrates a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. Other methods known in the art include, but are not limited to, lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR / Cas systems (e.g., Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas1Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (or CasA), Csel (or CasB), Csel (or CasC), Csel (or CasD ... 2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders DN, "Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives," Genes Cells 2015 January; 20(1):1-10. Base-editing CRISPR systems including Cas-CRISPR proteins fused to base-editing proteins such as deaminases (e.g., from Beam Therapeutics) can also be used.

[0164] The amino acid sequences of selected components that can be used to construct a CAR are disclosed in Tables 4 and 5 below.

[0165] [Table 5]

[0166] [Table 6]

[0167] Table 5 below lists V antigens targeting the antigens listed. H and V L The sequences of the domains are disclosed. These sequences may be incorporated into a CAR with elements from Table 4 or as disclosed herein.

[0168] [Table 7]

[0169] [Table 8]

[0170] [Table 9]

[0171] Cell-specific mutations The CAR components and construction methods disclosed above are generally suitable for use in T cells and other immune effector cells, but are not limited thereto. In cell subsets, certain mutations may be useful and are known in the art.

[0172] For example, in NK cells, the TM domain may be selected or adapted from NKG2D, FcγRIIIa, NKp44, NKp30, NKp46, actKIR, NKG2C, or CD8α. NK cells also express a number of transmembrane adaptors that are triggered by engagement with activating receptors, thereby providing enhanced NK cell-specific signals. For example, the TM adaptor may be selected or adapted from FceR1γ (ITAMx1), CD3ζ (ITAMx3), DAP12 (ITAMx1), or DAP10 (YxxM / YINM). In certain embodiments, TM domains and adaptors may be paired, for example: NKG2D and DAP10, FcγRIIIa and CD3ζ or FceR1γ, NKp44 and DAP12, NKp30 and CD3ζ or FceR1γ, NKp46 and CD3ζ or FceR1γ, actKIR and DAP12, and NKG2C and DAP12.

[0173] In certain embodiments, in NK cells, the hinge domain may be selected or adapted from, for example, NKG2, TMα, or CD8.

[0174] In certain embodiments, in NK cells, the intracellular signaling and / or costimulatory domain is selected from the group consisting of CD137 / 41BB (TRAF, NFkB), DNAM-1 (Y-motif), NKp80 (Y-motif), 2B4 (SLAMF)::ITSM, CRACC (CS1 / SLAMF7)::ITSM, CD2 (Y-motif, MAPK / Erk), CD27 (TRAF, NFkB); one or more integrins (e.g., multiple integrins); IL-2 / 15Rbyc::Jak1 / 3 , cytokine receptors associated with persistence, survival, or metabolism, such as STAT3 / 5, PI3K / mTOR, and MAPK / ERK; cytokine receptors associated with activation, such as IL-18R::NFkB, cytokine receptors associated with IFN-γ production, such as IL-12R::STAT4; cytokine receptors associated with cytotoxicity or persistence, such as IL-21R::Jak3 / Tyk2, or STAT3; and one or more of the TM adaptors as disclosed above. In some embodiments, the NK cell CAR comprises three signaling domains, a TM domain, and optionally a TM adaptor.

[0175] The choice of costimulatory domain may also depend on the phenotype or subtype of the NK cell; for example, in some experiments, 4-1BB may be effective as a costimulatory domain in memory-like (ML) NK cells (including CIML), but less effective in NK cells. In addition, signaling domains that are more selectively expressed in ML NK cells may be utilized, including DNAM-1, CD137, and CD2.

[0176] Immune effector cells Immune effector cells as disclosed herein include NK cells and subtypes thereof, such as memory NK cells, memory-like (ML) NK cells, and cytokine-induced memory-like (CIML) NK cells, and variants thereof, any of which may be derived from a variety of sources, including peripheral blood or umbilical cord blood cells, stem cells, induced pluripotent stem cells (iPSCs), and immortalized NK cells, such as NK-92 cells.

[0177] NK cells Natural killer (NK) cells are traditionally considered innate immune effector lymphocytes that mediate host defense against pathogens and antitumor immune responses by targeting and eliminating abnormal or stressed cells through the integration of signals from activating and inhibitory receptors, rather than through antigen recognition or presensitization. Natural killer (NK) cells are an alternative to T cells for allogeneic cellular immunotherapy because they are safely administered without significant toxicity, do not cause graft-versus-host disease (GvHD), naturally recognize and eliminate malignant cells, and are amenable to cell engineering.

[0178] Memory, memory-like, and CIML NK cells In addition to their natural cytotoxic and immunostimulatory activities, NK cells constitute a heterogeneous and versatile cell subset that generates robust recall responses, including a long-lasting memory NK population, sometimes referred to as memory-like or cytokine-induced memory-like (CIML) NK cells. Memory NK cells can be generated by stimulation with proinflammatory cytokines or activating receptor pathways, either natural or artificially stimulated ("primed"). Memory NK cells generated by cytokine activation have been used clinically in the setting of leukemia immunotherapy.

[0179] Increases in CD56, Ki-67, NKG2A, and activating receptors NKG2D, NKp30, and NKp44 have been observed in in vivo differentiated memory NK cells. In addition, in vivo differentiation showed a slight decrease in median expression of CD16 and CD11b. Increases in the frequency of TRAIL-, CD69-, CD62L-, NKG2A-, and NKp30-positive NK cells were observed in ML NK cells compared to both ACT and BL NK cells, whereas the frequency of CD27+ and CD127+ NK cells was decreased. Finally, unlike in vitro differentiated ML NK cells, in vivo differentiated ML NK cells did not express CD25.

[0180] Cytokine-induced memory-like natural killer cells (CIML-NK) For example, NK cells can be induced to acquire a memory-like phenotype by priming (preactivation) with a combination of cytokines, such as interleukin-12 (IL-12), IL-15, and IL-18. These cytokine-induced memory-like (CIML) NK cells (CIML-NK or CIML) exhibit enhanced responses upon restimulation with cytokines or triggering by activating receptors. CIML NK cells can be generated by activation with cytokines such as IL-12, IL-15, and IL-18 and / or related family members thereof, or functional fragments thereof, or fusion proteins comprising functional fragments thereof.

[0181] Memory NK cells typically exhibit differences in cell surface protein expression patterns when compared to conventional NK cells. Such expression patterns are known in the art and can include, for example, increased expression of CD56, CD56 subset CD56dim, CD56 subset CD56bright, CD16, CD94, NKG2A, NKG2D, CD62L, CD25, NKp30, NKp44, and NKp46 (compared to control NK cells) in CIML NK cells (see, e.g., Romee et al. Sci Transl Med. 2016 Sep 21;8(357):357). Memory NK cells may also be identified by observed in vitro and in vivo properties, such as enhanced effector functions, such as cytotoxicity, improved persistence, and increased IFN-γ production, when compared to heterogeneous NK cell populations.

[0182] Pharmaceutical Compositions Also disclosed are pharmaceutical compositions comprising the disclosed molecules in a pharma- ceutically acceptable carrier. Pharmaceutical carriers are known to those of skill in the art. They will most typically be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffer solutions at physiological pH. Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharma- ceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, and more preferably about 7 to about 7.5. The solution should be RNase-free. Additional carriers include sustained release formulations, such as semipermeable matrices of solid hydrophobic polymers containing antibodies, in the form of tangible articles, e.g., films, liposomes, or microparticles. It will be apparent to those of skill in the art that certain carriers may be more preferable, for example, depending on the route of administration and concentration of the composition being administered.

[0183] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surface active agents, etc. Pharmaceutical compositions may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

[0184] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral solvents include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous solvents include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases.

[0185] therapeutic application The NK cells disclosed herein can be used to treat or prevent the progression of proliferative diseases such as cancer and myelodysplastic syndromes. Cancer can be a hematological malignancy or a solid tumor. Hematological malignancies include leukemia, lymphoma, multiple myeloma, and subtypes thereof. Lymphomas can be classified in various ways, often based on the type of underlying malignant cell, and include Hodgkin's lymphoma (often a cancer of Reed-Sternberg cells, but sometimes also occurs in B cells; all other lymphomas are non-Hodgkin's lymphomas), non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, and others as defined herein and known in the art. Myelodysplastic syndromes include a group of diseases in which immature white blood cells and / or hematopoietic stem cells (HSCs) are affected; MDS can progress to AML.

[0186] B-cell lymphomas include, but are not limited to, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and others as defined herein and known in the art.

[0187] T-cell lymphomas include T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), Sezary syndrome, and others as defined herein and known in the art.

[0188] Leukemias include acute myeloid (or myeloid) leukemia (AML), chronic myeloid (or myeloid) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (sometimes classified as a lymphoma), and others as defined herein and known in the art.

[0189] Plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0190] Solid tumors include melanoma, neuroblastoma, glioma, or carcinoma, such as tumors of the brain, head and neck, breast, lung (e.g., non-small cell lung cancer, NSCLC), reproductive system (e.g., ovary), upper gastrointestinal tract, pancreas, liver, renal system (e.g., kidney), bladder, prostate, and colorectum.

[0191] The methods described herein are generally performed on a subject in need thereof. A subject in need of the treatment methods described herein may be a subject who has, has been diagnosed with, is suspected of having, or is at risk of developing or progressing to a later stage of cancer. The determination of the need for treatment will typically be determined by a medical history, physical examination, or diagnostic tests consistent with the disease or condition in question. Diagnosis of various conditions treatable by the methods described herein is within the skill of the art. The subject may be an animal subject, including mammals such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans, or other animals such as chickens. For example, the subject may be a human subject.

[0192] Generally, a safe and effective amount of a therapy, e.g., an antibody or functional antigen-binding fragment thereof, a CAR-bearing immune effector cell, or an antibody-drug conjugate, is an amount that will produce a desired therapeutic effect in a subject, while minimizing undesirable side effects, for example.

[0193] According to the methods described herein, administration may be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, intraocular, buccal, or rectal. If the formulation is, for example, a biologic or cell therapy, the mode of administration will likely be by injection or infusion.

[0194] Conditioning regimens for standard of care and immunotherapy Standard care treatment for cancers such as AML can involve anti-cancer pharmaceutical therapy, including chemotherapy and targeted therapies.

[0195] For example, the combination of cytarabine (cytosine arabinoside or ara-C) with an anthracycline such as daunorubicin (daunomycin) or idarubicin is the chemotherapy of first choice for AML. Other chemotherapy drugs that may be used to treat AML include cladribine (Leustatin, 2-CdA), fludarabine (Fludara), mitoxantrone, etoposide (VP-16), 6-thioguanine (6-TG), hydroxyurea, corticosteroids such as prednisone or dexamethasone, methotrexate (MTX), 6-mercaptopurine (6-MP), azacitidine (Vidaza), and decitabine (Dacogen). In addition, targeted therapies may be used in appropriate patients, such as midostaurin (Rydapt) or gilteritinib (Xospata) in patients with FLT-3 mutations; gemtuzumab ozogamicin (Mylotarg) in CD33-positive AML; BCL-2 inhibitors such as venetoclax (Venclexta); IDH inhibitors such as ivosidenib (Tibsovo) or enasidenib (Idhifa); and hedgehog pathway inhibitors such as glasdegib (Daurismo). Although complete remission rates can be as high as 80% after initial induction chemotherapy, the majority of AML patients will eventually progress to relapsed or refractory (RR) disease, with 5-year survival rates of approximately 35% in those under 60 years of age and 10% in those over 60 years of age. See Walter RB et al., “Resistance prediction in AML: analysis of 4601 patients from MRC / NCRI, HOVON / SAKK, SWOG and MD Anderson Cancer Center,” Leukemia 29(2):312-20 (2015) and Doehner, Het al., “Acute Myeloid Leukemia,” NEJM 373 (12): 1136-52 (2015).

[0196] In the treatment of cancer, adoptive cell transfer (ACT) therapy, which may or may not be accompanied by a conditioning regimen, is possible. Typically, when ACT, such as HSCT, is performed in patients with malignant disorders, a pretreatment or conditioning regimen is administered as part of the procedure to induce immune ablation to prevent graft rejection and to reduce tumor burden. Traditionally, these goals were achieved by using essentially supra-lethal total body irradiation (TBI) and chemotherapy agents with no overlapping toxicity, so-called "high intensity" ACT pre-conditioning. However, since it was recognized that the efficacy of ACT is substantially contributed by the immunological response of donor cells against malignant host cells (i.e., graft-versus-tumor effect), low intensity non-myeloablative conditioning regimens have been developed, allowing the application of ACT to a wider range of patients, including elderly and medically frail patients.

[0197] Conditioning regimens are known in the art. See, for example, Gyurkocza and Sandmaier BM, "Conditioning regimens for hematopoietic cell transplantation: one size does not fit all," Blood 124(3): 344-353 (2014). Conditioning regimens can be classified as high-dose (myeloablative), low-intensity, and non-myeloablative according to the Reduced-Intensity Conditioning Regimen Workshop held by the Center for International Blood and Marrow Transplant Research (CIBMTR) during the Bone Marrow Transplantation Tandem Meeting in 2006.

[0198] definition Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art.

[0199] As used herein, the term "antibody" refers to a polypeptide that contains sufficient standard immunoglobulin sequence elements to confer specific binding or, for example, is immunoreactive and / or directed to a particular target antigen. As is known in the art, intact antibodies as produced in nature are tetrameric agents of about 150 kD composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) associated with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain contains at least four domains (each about 110 amino acids long) - the amino-terminal variable (V H ) domain followed by three constant domains: C H 1. C H 2, and the carboxy-terminal C H 3. A short region known as the "switch" connects the heavy chain variable region to the constant region. The "hinge" is C H 2 and C H In an intact antibody, two disulfide bonds in this hinge region link the two heavy chain polypeptides together. Each light chain contains two domains separated from each other by another "switch" - the amino-terminal variable (V L ) domain followed by a carboxy-terminal constant (C L) domains. An intact antibody tetramer is composed of two heavy-light chain dimers, in which the heavy and light chains are linked together by a single disulfide bond; two other disulfide bonds link the heavy chain hinge regions together, causing these dimers to be linked together to form a tetramer. Naturally produced antibodies also typically consist of C H The variable domains are glycosylated on two domains. Each domain of a natural antibody has a structure characterized by an "immunoglobulin fold" formed by two β-sheets (e.g., three-, four-, or five-stranded sheets) packed together into a flattened antiparallel β-barrel. Each variable domain has three hypervariable loops (CDR1, CDR2, and CDR3) known as "complementarity determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form β-sheets that provide the structural framework for the domain, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space to generate a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds elements of the complement system and also binds to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity.

[0200] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Some examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear antibodies, single chain variable fragments (scFv), and multispecific antibodies formed with antibody fragments. In some embodiments, the antibody fragment is an antigen-binding fragment.

[0201] For reviews of current antibody engineering and improvement methods, see R. Kontermann and S. Dubel, (2010) Antibody Engineering Vols.1 and 2, Springer Protocols, 2nd Edition and W. Strohl and L. Strohl (2012) Therapeutic antibody engineering: Current and future advances driving the strongest growth area in the pharmaceutical industry, Woodhead Publishing. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and see Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 5-8 and 15.

[0202] The term "antigen" refers to a molecular entity that may be soluble or cell membrane bound, in particular a molecular entity capable of recognition by the adaptive immune system, including but not limited to antibodies or TCRs, or engineered molecules including but not limited to transgenic TCRs, chimeric antigen receptors (CARs), scFvs or multimers thereof, Fab fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule capable of binding to a structure with high affinity.

[0203] "Antigen binding domain" as used herein, in the context of CARs, refers to the region of a CAR that specifically binds to an antigen (and is thereby capable of targeting an antigen-bearing cell). A CAR may comprise one or more antigen binding domains. Generally, the targeting region of a CAR is extracellular. An antigen binding domain may comprise an antibody or an antigen-binding fragment thereof. An antigen binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a diabody. Any molecule that specifically binds to a given antigen may be used as an antigen binding domain, such as an affibody or a ligand binding domain from a naturally occurring receptor. In many cases, the antigen binding domain is an scFv. Typically, in an scFv, the variable portions of immunoglobulin heavy and light chains are fused by a flexible linker to form the scFv. Such linkers can be, for example, (GGGG 4 S) 3 In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as the species in which the CAR will be used. For example, when it is planned to be used therapeutically in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise a human or humanized antibody or an antigen-binding fragment thereof. Human or humanized antibodies or fragments thereof can be produced by various methods well known in the art.

[0204] As used herein, the term "binding affinity" refers to the strength with which one molecule binds to another molecule at a site on the molecule. When a particular molecule binds to or specifically associates with another particular molecule, the two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association and dissociation constants for a pair of molecules, but measuring or determining these constants is not important for the methods herein. Rather, affinity, as used herein to describe the interactions between molecules of the described methods, is generally the apparent affinity observed in empirical studies (unless otherwise specified), which can be used to compare the relative strength with which one molecule (e.g., an antibody or other specific binding partner) will bind to two other molecules (e.g., two versions or variants of a peptide). The concepts of binding affinity, association constant, and dissociation constant are well known.

[0205] The term "cancer" is medically known as malignant neoplasm. Cancer is a broad group of diseases that involve uncontrolled cell growth. In cancer, cells (cancerous cells) divide and grow uncontrollably to form malignant tumors that invade nearby parts of the body. Cancer can also spread to distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known types of cancer that occur in humans.

[0206] The term "chemotherapy" refers to the treatment of cancer (cancerous cells) with one or more cytotoxic anti-neoplastic drugs ("chemotherapeutic agents" or "chemotherapeutic drugs") as part of a standardized regimen. Chemotherapy may be given with a curative intent, or it may be aimed at extending life or alleviating symptoms. It is often used in conjunction with other cancer treatments, such as radiation therapy, surgery, and / or hyperthermia therapy. Traditional chemotherapy drugs work by killing rapidly dividing cells, which is one of the main characteristics of most cancer cells. This means that chemotherapy also harms cells that divide rapidly under normal circumstances, such as cells of the bone marrow, digestive tract, and hair follicles. This results in the most common side effects of chemotherapy, such as myelosuppression (reduced production of blood cells, and therefore also immune suppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss).

[0207] The term "chimeric antigen receptor", abbreviated "CAR", refers to an engineered receptor that grafts antigen specificity onto a cell, such as a T cell or NK cell. The CAR disclosed herein comprises an antigen-binding domain, also known as an antigen-targeting region (typically a single chain variable region composed of an antibody heavy and light chain variable region), an extracellular spacer / linker domain or hinge region, a transmembrane domain, and at least one intracellular signaling domain; which may optionally include other elements, such as at least one co-stimulatory domain. The extracellular domain may also comprise a signal peptide. When the antigen-specific region binds to the corresponding antigen, the signaling domain mediates effector cell function in the host cell.

[0208] The term "combined immunotherapy" refers to the concerted application of two therapeutic approaches, for example, therapeutic approaches known in the art for the treatment of diseases such as cancer. The term "combined immunotherapy" can also refer to the concerted application of an immunotherapy, such as treatment with an antigen-recognizing receptor, with another therapy, such as transplantation of hematopoietic cells, for example, hematopoietic cells that are resistant to recognition by an antigen-recognizing receptor. Expression of an antigen on a cell means that the antigen is sufficiently present on the cell surface of the cell so that the antigen-recognizing receptor can detect, bind and / or recognize it.

[0209] "Co-stimulatory signaling region" (equivalently, costimulatory or "co-stim" domain) refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligand, that is necessary for an immune effector cell to respond efficiently. Examples of costimulatory molecules are discussed above and are known in the art. Small oligopeptide or polypeptide linkers, typically 2-10 amino acids in length, can form the link between elements of the intracellular signaling domain. A prominent linker is a glycine-serine doublet.

[0210] The term "cytokine-induced memory-like," or equivalently, "CIML," in the context of NK cells, means having a "memory" or "memory-like" phenotype and is generated using a priming agent.

[0211] The term "cytotoxicity," as used herein in relation to memory NK cells, refers to the ability of the cells to target and kill diseased cells.

[0212] "Disease cell" refers to a state that deviates from the normal or healthy state for a cell, tissue or organism, which may result from the effects of a pathogen, a toxic agent, irradiation, or internal cellular deregulation. "Disease cell" may also refer to a cell infected with a pathogenic virus. Additionally, the term "disease cell" may refer to a malignant or neoplastic cell that may constitute or give rise to cancer in an individual.

[0213] The terms "engineered cells" and "genetically modified cells" as used herein can be used interchangeably. These terms refer to cells that carry and / or express an exogenous gene or nucleic acid sequence, or that have been genetically modified (e.g., a gene that has been deleted or knocked out) to deviate from its native form or function, which in turn modifies the genotype or phenotype of the cell or its progeny. Cells can be modified by recombinant methods well known in the art to stably or transiently express peptides or proteins not naturally expressed in such cells. Methods for genetically modifying cells include, but are not limited to, transfection, electroporation, nucleofection, transduction using retroviral vectors, lentiviral vectors, non-integrating retroviral or lentiviral vectors, transposons, designer nucleases, including zinc finger nucleases, TALENs, or CRISPR / Cas.

[0214] The term "enrich" as used herein in reference to NK cells means to enrich, purify, or isolate for further analysis or use. Enriched and purified cell populations contain a majority of the desired cells and a negligible proportion of other cells.

[0215] The term "fold selective" as used herein means that the affinity for one target is at least x times higher than its affinity for another target, where x is at least 2, and may be greater, e.g., 10, 20, 50, 100, or 1000. In preferred embodiments, the fold selectivity is therapeutically meaningful, i.e., sufficient to allow cells expressing one target to be killed and cells bearing the other target to survive.

[0216] The term "genetic modification" or "genetically modified" refers to the alteration of the nucleic acid content, including but not limited to, the genomic DNA of a cell. This includes but is not limited to the alteration of a cellular genomic DNA sequence by the introduction of an exchange or deletion of a single nucleotide or fragment of a nucleic acid sequence. The term also refers to any introduction of a nucleic acid into a cell, regardless of whether it results in a direct or indirect alteration of the cellular genomic DNA sequence.

[0217] The term "hematopoietic cells" refers to a population of cells of the hematopoietic system that have hematopoietic potential, including, but not limited to, hematopoietic stem cells and / or hematopoietic progenitor cells (i.e., capable of proliferating and at least partially reconstituting different blood cell types, including erythroid cells, lymphocytes, and myeloid cells). The term "hematopoietic cells," as used herein, also includes cells that differentiate from hematopoietic stem cells and / or hematopoietic progenitor cells to form blood cells (i.e., blood cell types, including erythroid cells, lymphocytes, and myeloid cells).

[0218] Donor hematopoietic cells that are resistant to antigen recognition by an antigen recognition receptor mean that the cells are not as readily susceptible to detection, binding and / or recognition by an antigen recognition receptor specific for the antigen, or that the cells are not killed during immunotherapy because detection, binding and / or recognition is impaired.

[0219] The term "immune cell" or "immune effector cell" refers to a cell that performs a specific effector function, which may be part of the immune system, such as αβT cells, NK cells (including memory NK, ML-NK, and CIML-NK), NKT cells (including iNKT cells), B cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, γδT cells, mesenchymal stem cells or mesenchymal stromal cells (MSCs), monocytes, and macrophages. Preferred immune cells are cells with cytotoxic effector function, such as αβT cells, NK cells (including memory NK, ML-NK, and CIML-NK), NKT cells (including iNKT cells), ILCs, CIK cells, LAK cells, or γδT cells. "Effector function" refers to the specialized function of a cell, for example, in NK cells, the effector function may be cytolytic activity or helper activity, including secretion of cytokines.

[0220] The term "immunotherapy" is a medical term defined as "treatment of disease by induction, enhancement, or suppression of immune response." Immunotherapies designed to elicit or amplify immune response are classified as activating immunotherapies, while immunotherapies that reduce or suppress are classified as suppressing immunotherapies. Cancer immunotherapy as an activating immunotherapy attempts to reject and destroy tumors by stimulating the immune system. Adoptive cell transfer attacks cancer cells using a cell-based cytotoxic response. Immune cells such as T cells with natural or engineered reactivity against the patient's cancer are generated in vitro and then transferred back into the cancer patient.

[0221] As used herein, the term "individual" refers to an animal. Preferentially, the individual is a mammal, such as a mouse, a rat, a cow, a pig, a goat, a chicken, a dog, a monkey or a human. More preferentially, the individual is a human. The individual may be an individual (patient) suffering from a disease, such as cancer, but the subject may also be a healthy subject.

[0222] The "intracellular signaling domain" of a CAR (equivalently, the cytoplasmic signaling domain or effector domain; these are part of the intracellular domain or endodomain) is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to the specialized function of a cell; for example, in NK cells, the effector function can be cytolytic activity or helper activity, including secretion of cytokines. The intracellular signaling domain refers to the part of a protein that transmits the effector function signal and directs the cell expressing the CAR to perform a specialized function.

[0223] The intracellular signaling domain can include any complete or truncated portion of the intracellular signaling domain of a given protein that is sufficient to transmit an effector function signal. Notable examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of receptors and co-receptors that work together to initiate signal transduction following antigen receptor engagement.

[0224] In general, CAR activation of immune effector cells can be mediated by two classes of cytoplasmic signaling sequences, first, those that initiate antigen-dependent primary activation through the CAR (primary cytoplasmic signaling sequences) and second, those that serve to provide a secondary or costimulatory signal in an antigen-independent manner (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can include a primary cytoplasmic signaling domain and, optionally, a secondary cytoplasmic signaling domain (i.e., a costimulatory or "co-stim" domain).

[0225] Primary cytoplasmic signaling sequences that act in a stimulatory manner may include ITAMs (immunoreceptor tyrosine-based activation motif signaling motifs). Examples of primary cytoplasmic signaling sequences with ITAMs that are often used in CARs are disclosed herein and known in the art.

[0226] The term "malignant" or "malignant tumor" refers to a cell, group of cells, or tissue that may constitute a neoplasm, originate from a neoplasm, or be the origin of new neoplastic cells. The term is used to refer to a neoplastic cell in contrast to a normal or healthy cell of the tissue. A malignant tumor contrasts with a non-cancerous benign tumor in that a malignant tumor is not self-limited in its growth, has the ability to invade adjacent tissues, and may have the ability to spread to distant tissues. A benign tumor does not have any of these characteristics. Malignant tumors are characterized by anaplasia, invasiveness, and metastasis, as well as genomic instability. The term "pre-malignant cell" refers to a cell or tissue that is not yet malignant, but is on the verge of becoming malignant.

[0227] The term "memory" or "memory-like", in the context of NK cells, means having an activated phenotype that has improved cytotoxicity and is long-lived / persistent compared to the general population of NK cells, and typically exhibits increased cell surface expression of CD69, CD25, and NKG2A, and maintained expression of CD16, compared to the general population of NK cells.

[0228] The term "monoclonal antibody" (mAb), as applied to antibodies described in this disclosure, refers to a compound that is derived from a single copy or clone from any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. mAbs of this disclosure can exist in a homogenous or substantially homogenous population.

[0229] The term "persistence," as used herein, refers to the ability of a cell, particularly a cell that has been adoptively transferred into a subject, to continue to survive.

[0230] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0231] The term "prime" in the context of NK cells means to stimulate or activate with a priming agent to a memory / memory-like phenotype. A "priming agent" refers to a combination of stimulatory cytokines, e.g., one or more of IL-12, IL-23, IL-27, and IL-35; one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and one or more of IL-18, IL-1a, IL-1b, IL-36a, IL-36b, and IL-36g, or one or more "priming fusion proteins" comprising a functional fragment of such a cytokine, or one or more multi-chain complexes thereof. Examples of such proteins are disclosed herein.

[0232] In general, the term "receptor" refers to a biomolecule that may be soluble or attached to a cell surface membrane and specifically binds to a defined structure that may be attached to a cell surface membrane or soluble. Receptors include, but are not limited to, antibodies and antibody-like structures, adhesion molecules, transgenic or naturally occurring TCRs or CARs. Specifically, the term "antigen-recognizing receptor" as used herein may be a membrane-bound or soluble receptor, such as a natural TCR, a transgenic TCR, a CAR, an scFv or multimer thereof, an Fab fragment or multimer thereof, an antibody or multimer thereof, a bispecific T cell enhancer (BiTE), a diabody, or any other molecule that can perform specific binding with high affinity.

[0233] The term "reducing side effects" refers to reducing the severity of any complication, undesirable or pathological outcome of antigen-recognition receptor-directed immunotherapy, such as toxicity directed at non-target cells expressing the antigen. "Reducing side effects" also refers to measures to reduce or avoid pain, harm or risk of death to a patient during antigen-recognition receptor-directed immunotherapy.

[0234] As used herein, the term "sequence identity" refers to the percentage of identical nucleotides or amino acid residues at corresponding positions of two or more sequences when the sequences are aligned for maximum sequence matching, i.e., taking into account gaps and insertions. Identity can be easily calculated by known methods. Methods for determining identity are designed to give the maximum match between the sequences being examined. Moreover, methods for determining identity are codified into publicly available computer programs. Alignment of sequences for comparison can be performed, for example, by the Smith & Waterman local homology algorithm, by homology alignment algorithms, by similarity search methods, or by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package available from Accelrys, Inc., San Diego, California, USA), or by visual inspection. For an overview, see Altschul, SF et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm.

[0235] "Signal peptide," as used herein, in the context of a CAR, refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example to certain organelles (such as the endoplasmic reticulum) and / or to the cell surface.

[0236] The term "spacer" or "hinge" as used herein in the context of CAR refers to a hydrophilic region between the antigen-binding domain and the transmembrane domain. The CARs disclosed herein may include an extracellular spacer domain, but such a spacer may also be omitted. Spacers may include an Fc fragment or fragment thereof of an antibody, a hinge region or fragment thereof of an antibody, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A prominent example of a spacer is the CD8α hinge.

[0237] The terms "specifically bind" or "specific for" or "specifically recognize" in the context of an antigen recognition receptor refer to an antigen-binding domain of the antigen recognition receptor that recognizes and binds to a specific polymorphic variant of an antigen, but does not substantially recognize or bind other variants.

[0238] The term "side effects" refers to any complication, undesirable or pathological outcome of immunotherapy due to antigen recognition receptors that occurs in addition to the desired therapeutic outcome. The term "side effects" refers preferentially to on-target off-tumor toxicity that may occur during immunotherapy when the target antigen is present on non-target cells that express the antigen, but are not diseased cells as described herein. A side effect of immunotherapy may be the development of graft-versus-host disease.

[0239] The term "target" or "target antigen" refers to any cell surface protein, glycoprotein, glycolipid, or any other structure present on the surface of a target cell. The term also refers to any other structure present on a target cell, particularly a structure that can be recognized by the adaptive immune system, including but not limited to antibodies or TCRs, or engineered molecules, including but not limited to transgenic TCRs, CARs, scFvs or multimers thereof, Fab fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule capable of binding to the structure with high affinity.

[0240] The term "target cell," as used herein, refers to a cell that is recognized by an antigen recognition receptor that is or will be applied to an individual.

[0241] The term "therapeutically effective amount" means an amount that provides a therapeutic benefit.

[0242] The "transmembrane domain" of the CAR can be derived from any desired natural or synthetic source of such a domain. When the source is natural, this domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8α, CD28, NKG2D, or others disclosed herein or known in the art. When the key signaling and antigen recognition modules are on two (or more) polypeptides, then the CAR can have two (or more) transmembrane domains. Splitting the key signaling and antigen recognition modules allows for small molecule-dependent titratable and reversible control of CAR cell expression due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR (Wu et al, 2015, Science 350: 293-303).

[0243] As used herein, the term "transplantation" refers to the administration of a population of donor cells, such as hematopoietic cells or CAR-bearing immune effector cells, to a subject.

[0244] The term "treatment" as used herein means reducing the frequency or severity of at least one sign or symptom of a disease. EXAMPLES

[0245] Working Example The following examples further illustrate the invention.

[0246] Example 1. In vitro culture and activity of expansion only, expansion with priming, and expansion followed by priming Materials and Methods: NK cells were isolated from whole blood using CD3 depletion and CD56 positive selection. Selected NK cells were then cultured in NK MACS medium + supplements + 10% HI-HAB in 96-well plates and primed / expanded under the following conditions (where 1x7t15-21s and ATF1 are at 200nM and 100nM, respectively, and 1x18t15-12s is at 250nM; all dilutions are calculated as indicated from these values). a) Expansion only: +7t15-21s and ATF1 at the indicated concentrations for either 2, 6, or 10 days at 37 degrees, 5% CO2. Every 2 days thereafter, 7t15-21s and ATF1 were replenished with fresh medium to the indicated concentrations. b) Priming and simultaneous expansion: +18t15-12s, 7t15-21s and ATF1 at the indicated concentrations at 37 degrees, 5% CO2 for either 2, 6 or 10 days, followed by replenishment of 18t15-12s, 7t15-21s and ATF1 with fresh medium to the indicated concentrations every 2 days. c) Priming after expansion: +7t15-21s and ATF1 at the indicated concentrations at 37 degrees, 5% CO2 for either 2, 6, or 10 days. Every 2 days thereafter, 7t15-21s and ATF1 were replenished with fresh medium to the indicated concentrations. On days 2, 6, or 10 as indicated, 18t15-12s was added overnight at the indicated concentrations.

[0247] To determine the phenotype of the NK cells generated by the above process, NK cells were harvested at appropriate time points, washed, and assessed for purity and / or receptor expression by staining with a flow panel including anti-CD56, anti-CD3, Live / Dead Yellow, anti-NKG2A, anti-CD69, anti-CD25, and anti-CD16. The following clones were used: Anti-CD45 (HI30 clone) ·Anti-CD56 (CMSSB clone) Anti-CD3 (SK7 clone) ·Live / Dead Yellow(Thermo Fisher) Anti-NKG2A (REA110 clone) Anti-CD69 (FN50 clone) Anti-CD25 (CD25-4E3 clone) Anti-CD16 (eBioCD16 clone) An Attune NXt flow cytometer was used. Data were then analyzed in Flowjo v10.7 by gating on viable CD56+CD3- cells and determining the median fluorescence intensity of each of the above-mentioned markers. Increased expression of CD69, CD25, and NKG2A, and sustained expression of CD16, are indicative of a CIML-NK cell phenotype.

[0248] To determine the killing activity of NK cells generated by the above process, at the appropriate time points, cultured NK cells were harvested, washed, then resuspended in NK MACS medium containing 10% human AB serum (Gibco) and added to 96-well plates with 10,000 luciferase-expressing K562 (K562-Luc) human tumor cells (ATCC) at the indicated effector-to-target (E:T) ratios for 24-48 h with or without IL-2 (Miltenyi), after which luciferase activity (viable K562 cells) was determined by luciferase readout (Promega). Data not shown.

[0249] Results: This example demonstrates the in vitro activity and flow cytometry phenotype of NK cells generated by the process described above.

[0250] The results are shown in Tables 6-11, which show the cumulative fold change in surface protein expression, cell size, and median fluorescence intensity for individual genes.

[0251] [Table 10]

[0252] [Table 11]

[0253] [Table 12]

[0254] [Table 13]

[0255] [Table 14]

[0256] [Table 15]

[0257] Alternatively, lymphoid progenitor cells such as iPSC cells or cord blood NK cells may be cultured in a suitable medium and differentiated into a form capable of priming and / or expanding NK cells.

[0258] Example 2. In vitro expansion and priming with or without priming Materials and Methods: Purified NK cells were treated with various concentrations of expansion agents 7t15-21s and ATF1 every 2 days. On days 6 and 14, cells expanded with 200nM and 100nM 7t15-21s and ATF1 were activated with 250nM 18t15-12s and continued expansion with 200nM and 100nM 7t15-21s and ATF1. After days 6, 13 and 17, NKs were then added at the indicated ratios to plates of K562-Luc2 cells (ATCC) in RPMI + 10% heat inactivated FBS. Plates were then incubated at 37°C and 5% CO2 for 24 hours. Killing of K562 cells was measured by luciferase readout. The lower the EC:50, the better the killing was considered.

[0259] Results: This example demonstrates the in vitro activity and flow cytometry phenotype of NK cells generated by the above process on a large scale.

[0260] The results are shown in Figures 1 to 4.

[0261] Example 3. In vitro culture and activity of expansion only, expansion with priming, expansion followed by priming, and expansion followed by priming and expansion Materials and Methods: NK cells were isolated from whole blood using CD3 depletion and CD56 positive selection. Selected NK cells were then cultured in NK MACS medium + supplements + 10% HI-HAB in 96-well plates and primed / expanded with the following conditions (where 1x7t15-21s and ATF1 are 200 nM and 100 nM, respectively, and x / 4 is 50 nM and 25 nM, respectively): a) Expansion only: +1x or x / 4 7t15-21s and ATF1 for 4 days at 37 degrees, 5% CO2. On day 6 of culture and every 2 days thereafter, 7t15-21s and ATF1 were replenished with fresh medium to 1x or x / 4. b) Priming and simultaneous expansion: 250 nM / 1×, 250 nM / × / 4, 62.5 nM / 1×, or 62.5 nM / × / 4 + 18t15-12s, 7t15-21s and ATF1 for 6 days at 37°C, 5% CO2. On day 6 of culture and every 2 days thereafter, 18t15-12s, 7t15-21s and ATF1 were replenished with fresh medium to the indicated concentrations. c) Priming after expansion: +1x or x / 4 7t15-21s and ATF1 for 4 days at 37 degrees, 5% CO2. On day 6 of culture and every 2 days thereafter, 7t15-21s and ATF1 were replenished with fresh medium to 1x or x / 4. On day 6 or 14 as indicated, 18t15-12s was added at 250nM or 62.5nM for 3 hours. d) Priming and expansion 3 hours after expansion: +1× or × / 4 7t15-21s and ATF1 for 4 days at 37 degrees, 5% CO2. On day 6 of culture and every 2 days thereafter, 7t15-21s and ATF1 were replenished with fresh medium to 1× or × / 4. On day 6 or 14, 18t15-12s, 7t15-21s and ATF1 were added at 250nM / 1×, 250nM / × / 4, 62.5 / 1×, or 62.5 / × / 4 for 3 hours. e) Priming and expansion 48 hours after expansion: +1× or × / 4 7t15-21s and ATF1 for 4 days at 37 degrees, 5% CO2. On day 6 of culture and every 2 days thereafter, 7t15-21s and ATF1 were replenished with fresh medium to 1× or × / 4. On day 6 or 14, 18t15-12s, 7t15-21s and ATF1 were added at 250nM / 1×, 250nM / × / 4, 62.5nM / 1×, or 62.5nM / × / 4 for 48 hours.

[0262] To determine the phenotype of the NK cells generated by the above process, NK cells were harvested at appropriate time points, washed, and assessed for purity and / or receptor expression by staining with a flow panel including anti-CD56, anti-CD3, Live / Dead Yellow, anti-NKG2A, anti-CD69, anti-CD25, and anti-CD16. The following clones were used: Anti-CD45 (HI30 clone) ·Anti-CD56 (CMSSB clone) Anti-CD3 (SK7 clone) ·Live / Dead Yellow(Thermo Fisher) Anti-NKG2A (REA110 clone) Anti-CD69 (FN50 clone) Anti-CD25 (CD25-4E3 clone) Anti-CD16 (eBioCD16 clone) An Attune NXt flow cytometer was used. Data were then analyzed in Flowjo v10.7 by gating on viable CD56+CD3- cells and determining the median fluorescence intensity of each of the above-mentioned markers. Increased expression of CD69, CD25, and NKG2A, and sustained expression of CD16, are indicative of a CIML-NK cell phenotype.

[0263] To determine the killing activity of NK cells generated by the above process, at appropriate time points, cultured NK cells were harvested, washed, then resuspended in NK MACS medium containing 10% human AB serum (Gibco) and added to 96-well plates with 10,000 luciferase-expressing K562 (K562-Luc) human tumor cells (ATCC) at the indicated effector-to-target (E:T) ratios for 24-48 hours with or without IL-2 (Miltenyi), after which luciferase activity (viable K562 cells) was determined by luciferase readout (Promega).

[0264] Results: This example demonstrates the in vitro activity and flow cytometry phenotype of NK cells generated by the process described above.

[0265] The results are shown in Tables 12-17, which show the cumulative fold change in NK cell counts, median fluorescence intensity of individual surface protein expression, and K562-Luc killing.

[0266] [Table 16]

[0267] [Table 17]

[0268] [Table 18]

[0269] [Table 19]

[0270] [Table 20]

[0271] [Table 21]

[0272] Example 4. Large-scale in vitro culture and activity of expansion only and expansion followed by priming Materials and Methods: NK cells were isolated from frozen leukopaks using CD3 depletion and CD56 positive selection on a MACS prodigy. Selected NK cells were then cultured in St. Gobain bags in NK MACS medium + supplements + 10% HI-HAB + 25nM 7t15-21s + 50nM ATF1 at an initial cell concentration of 0.25e6 / mL at 37°C and 5% CO. 2 Cells were cultured at 37°C for 6 days. On day 6 of culture and every 2 days thereafter, cells were counted, diluted to a concentration of 0.25e6 / mL, and supplemented with 7t15-21s and ATF1 to the appropriate concentrations in the final medium volume. On day 14, cells were either frozen (expansion only) or concentrated to 50e6 / mL and 18t15-12 was added to a final concentration of 250 nM (prime after expansion). Cells primed in this manner were incubated at 37°C, 5% CO2 for various times. After addition of 18t15-12s for the indicated length of time (30 min, 1 h, 2 h, 3 h, 5 h or overnight), cells were harvested, washed twice with HBSS(- / -), 0.5% HSA, and resuspended in freezing buffer (90% human serum, 10% DMSO). Cells were frozen at either 2e6 cells / mL or 20e6 cells / mL using a control rate freezer and then transferred to vapor phase liquid nitrogen. The cells were then thawed, washed, counted, and utilized in downstream assays to measure function.

[0273] To determine the phenotype of the NK cells generated by the above process, NK cells were harvested at appropriate time points, washed, and assessed for purity and / or receptor expression by staining with a flow panel including anti-CD56, anti-CD3, Live / Dead Yellow, anti-NKG2A, anti-CD69, anti-CD25, and anti-CD16. The following clones were used: Anti-CD45 (HI30 clone) ·Anti-CD56 (CMSSB clone) Anti-CD3 (SK7 clone) ·Live / Dead Yellow(Thermo Fisher) Anti-NKG2A (REA110 clone) Anti-CD69 (FN50 clone) Anti-CD25 (CD25-4E3 clone) Anti-CD16 (eBioCD16 clone) An Attune NXt flow cytometer was used. Data were then analyzed in Flowjo v10.7 by gating on viable CD56+CD3- cells and determining the median fluorescence intensity of each of the above-mentioned markers. Increased expression of CD69, CD25, and NKG2A, and sustained expression of CD16, are indicative of a CIML NK cell phenotype.

[0274] To determine the killing activity of NK cells generated by the above process, at appropriate time points, cultured NK cells were harvested, washed, then resuspended in NK MACS medium containing 10% human AB serum (Gibco) and added to 96-well plates with 10,000 luciferase-expressing K562 (K562-Luc) human tumor cells (ATCC) at the indicated effector-to-target (E:T) ratios for 24-48 hours with or without IL-2 (Miltenyi), after which luciferase activity (viable K562 cells) was determined by luciferase readout (Promega).

[0275] To determine the cytokine production capacity of NK cells generated by the above process, NK cells were thawed and then resuspended in NK MACS medium containing 10% human AB serum (Gibco) and added to a 96-well plate with 10,000 luciferase-expressing K562 (K562-Luc) human tumor cells (ATCC) at a 1:1 effector-to-target (E:T) ratio for 24 h or alone, after which the supernatants were harvested and IFNg production was determined by IFNg ELISA (R&D Systems).

[0276] Results: This example demonstrates the in vitro activity and flow cytometry phenotype of NK cells generated on a large scale by the process described above.

[0277] The results are shown in Figures 5 to 11.

[0278] Example 5. Large-scale in vitro culture and activity of expansion only, priming followed by expansion, and expansion followed by priming Materials and Methods: NK cells were isolated from frozen leukopaks using CD3 depletion and CD56 positive selection on a MACS prodigy. Selected NK cells were then cultured in St. Gobain bags in NK MACS medium + supplements + 10% HI-HAB + 25nM 7t15-21s + 50nM ATF1 at an initial cell concentration of 0.25e6 / mL for 6 days at 37°C, 5% CO2. On day 6 of culture and every 2 days thereafter, cells were counted, diluted to a concentration of 0.25e6 / mL and 7t15-21s and ATF1 were supplemented to the appropriate concentration in the final medium volume. On day 14, cells were concentrated to various densities (2e6, 5e6, 10e6, 25e6, 35e6 or 50e6 / mL) and 18t15-12 was added to a final concentration of 250nM. Cells were incubated at 37°C, 5% CO2. After addition of 18t15-12 for the indicated length of time (3 hours or overnight), cells were harvested, washed twice with HBSS(- / -), 0.5% HSA, and resuspended in freezing buffer (90% human serum, 10% DMSO). Cells were frozen at either 2e6 cells / mL or 20e6 cells / mL using a control rate freezer and then transferred to vapor phase liquid nitrogen. Cells were then thawed, washed, counted, and utilized in downstream assays to measure function.

[0279] To determine the phenotype of the NK cells generated by the above process, NK cells were harvested at appropriate time points, washed, and assessed for purity and / or receptor expression by staining with a flow panel including anti-CD56, anti-CD3, Live / Dead Yellow, anti-NKG2A, anti-CD69, anti-CD25, and anti-CD16. The following clones were used: Anti-CD45 (HI30 clone) ·Anti-CD56 (CMSSB clone) Anti-CD3 (SK7 clone) ·Live / Dead Yellow(Thermo Fisher) Anti-NKG2A (REA110 clone) Anti-CD69 (FN50 clone) Anti-CD25 (CD25-4E3 clone) Anti-CD16 (eBioCD16 clone) An Attune NXt flow cytometer was used. Data were then analyzed in Flowjo v10.7 by gating on viable CD56+CD3- cells and determining the median fluorescence intensity of each of the above-mentioned markers. Increased expression of CD69, CD25, and NKG2A, and sustained expression of CD16, are indicative of a CIML-NK cell phenotype.

[0280] To determine the killing activity of NK cells generated by the above process, at appropriate time points, cultured NK cells were harvested, washed, then resuspended in NK MACS medium containing 10% human AB serum (Gibco) and added to 96-well plates with 10,000 luciferase-expressing K562 (K562-Luc) human tumor cells (ATCC) at the indicated effector-to-target (E:T) ratios for 24-48 hours with or without IL-2 (Miltenyi), after which luciferase activity (viable K562 cells) was determined by luciferase readout (Promega).

[0281] To determine the cytokine production capacity of K cells generated by the above process, NK cells were thawed and then resuspended in NK MACS medium containing 10% human AB serum (Gibco) and added to a 96-well plate with 10,000 luciferase-expressing K562 (K562-Luc) human tumor cells (ATCC) at a 1:1 effector-to-target (E:T) ratio for 24 h or alone, after which the supernatants were harvested and IFNg production was determined by IFNg ELISA (R&D Systems).

[0282] To determine the in vivo persistence of NK cells generated by the above process, NK cells were thawed and resuspended in HBSS at 20e6 / mL. 2e6-5e6 cells (e.g., in 100uL) were injected intravenously into immunodeficient NSG mice (Jackson Laboratories, Bar Harbor Maine). Mice were supported by administering human IL-2 (Miltenyi Biotec, 50,000IU) every 2 days, and blood was collected on day 7 to measure NK cell counts by staining with a flow panel consisting of: ·Anti-CD56 (CMSSB clone), Anti-CD3 (SK7 clone), ·Live / Dead Yellow(Thermo Fisher), Anti-mouse CD45 (clone 30-F11), and Anti-human CD45 (HI30 clone) After fixation, red blood cells were lysed. Cells were then analyzed for viable huCD45+ mouse CD45-CD3- cell counts using an Attune NXt flow cytometer.

[0283] Results: This example demonstrates the in vitro activity and flow cytometry phenotype of NK cells generated on a large scale by the process described above.

[0284] The results are shown in Figures 12 to 16.

[0285] Example 6. In vitro culture and activity of expansion only and expansion followed by priming Materials and Methods: NK cells were isolated from whole blood using CD3 depletion and CD56 positive selection. The selected NK cells were then cultured in tissue culture treated flasks and then transferred to NK MACS medium + supplements + 10% HI-HAB in cell culture bags and expanded under the following conditions: a) Expansion only: 50 nM 7t15-21s and 25 nM ATF1, 37 degrees, 5% CO2 for 4 days. On day 5 of culture and every 2-3 days thereafter, 7t15-21s and ATF1 were supplemented to 50 nM and 25 nM, respectively, and cells were diluted to the appropriate concentration with fresh medium. On day 14, cells were frozen in 90% HAB, 10% DMSO. b) Priming after expansion: 50nM 7t15-21s and 25nM ATF1, 37 degrees, 5% CO2 for 4 days. On day 5 of culture and every 2-3 days thereafter, 7t15-21s and ATF1 were replenished to 50nM and 25nM, respectively, and cells were diluted to the appropriate concentrations with fresh medium. On day 14, 18t15-12s was added at 250nM for 3 hours. Cells were then frozen in 90% HAB, 10% DMSO.

[0286] To determine the phenotype of the NK cells generated by the above process, the frozen cells were thawed and assessed for receptor expression by staining with a flow panel including purity and / or activation markers, e.g., anti-CD56, anti-CD3, Live / Dead Yellow, anti-NKG2A, anti-CD69, anti-CD25, and anti-CD16. The following clones were used: Anti-CD45 (HI30 clone) ·Anti-CD56 (CMSSB clone) Anti-CD3 (SK7 clone) ·Live / Dead Yellow(Thermo Fisher) Anti-NKG2A (REA110 clone) Anti-CD69 (FN50 clone) Anti-CD25 (CD25-4E3 clone) Anti-CD16 (eBioCD16 clone) An Attune NXt flow cytometer was used. Data was then analyzed in Flowjo v10.7 by gating on viable CD56+CD3- cells and determining the median fluorescence intensity of each of the above markers. Increased expression of CD69, CD25, and NKG2A, and sustained expression of CD16, are indicative of a CIML-NK cell phenotype. Results are shown in Tables 18-22 below.

[0287] [Table 22]

[0288] [Table 23]

[0289] [Table 24]

[0290] [Table 25]

[0291] [Table 26]

[0292] To determine the killing activity of NK cells generated by the above process, at the appropriate time points, cultured NK cells were harvested, washed, then resuspended in NK MACS medium containing 10% human AB serum (Gibco) and added to 96-well plates with 10,000 luciferase-expressing K562 (K562-Luc) human tumor cells (ATCC) at the indicated effector-to-target (E:T) ratios for 24-48 hours with or without IL-2 (Miltenyi), after which luciferase activity (viable K562 cells) was determined by luciferase readout (Promega). The results are shown in Figures 17-18.

[0293] Results: This example demonstrates the in vitro activity and flow cytometry phenotype of NK cells generated by the process described above.

[0294] Example 7. In vivo killing activity of CIML-NK cells To determine the killing efficacy in vivo, NSG mice are implanted with K562-Luc (ATCC) tumor cells. At the end of NK cell culture, cells are harvested, washed, and tumor-bearing animals are injected intravenously with 2-10e6 NK cells, while some control mice are left uninjected. Mice are supported with q2d administration of human IL-2 (50,000IU), and tumor growth is measured weekly by injecting mice with luciferin and reading luciferase in a capable instrument.

[0295] Example 8. Clinical Trial Protocol NK cells as disclosed above may be thawed if cryopreserved and infused into patients in a suitable medium for the treatment of diseases such as cancer. For example, exemplary methods for testing NK cells for safety and efficacy in acute myeloid leukemia and myelodysplastic syndrome are disclosed in clinical trial protocols NCT04354025, NCT03068819, NCT01898793, NCT02782546, and NCT04893915. These protocols involve memory NK cells that have been primed with either a cocktail of IL-12, IL-15, and IL-18 or a priming fusion protein complex, and then optionally expanded. Similar clinical trials may be performed using memory NK cells that have been expanded and then primed, or expanded and primed simultaneously.

[0296] [Table 27]

[0297] [Table 28]

[0298] [Table 29]

[0299] [Table 30]

[0300] [Table 31]

[0301] [Table 32]

[0302] [Table 33]

[0303] [Table 34]

[0304] [Table 35]

[0305] [Table 36]

[0306] [Table 37]

[0307] [Table 38]

[0308] Memory NK cells expanded and primed, or simultaneously expanded and primed, according to the methods disclosed herein are expected to be effective in treating AML, MDS, and other diseases, for example, as shown in the clinical trial protocols described above.

[0309] The detailed description set forth above is provided to assist those skilled in the art in practicing the present invention. However, the present invention described and claimed herein is not limited in scope by the specific embodiments disclosed herein, since those embodiments are intended as illustrations of some aspects of the present invention. Any equivalent embodiments are intended to be within the scope of the present invention. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, without departing from the spirit or scope of the inventive findings. Such modifications are also intended to be included within the scope of the appended claims.

Claims

1. sequentially, a) expanding the purified NK cells; and b) priming the NK cells to produce a population of purified cytokine-induced memory-like natural killer cells (CIML NK cells).

2. The memory NK cells according to claim 1, wherein the NK cells are concentrated from fresh or frozen leukapheresis products or blood donors.

3. The memory NK cells according to claim 1, wherein the NK cells differentiate from lymphocyte progenitor cells.

4. The memory NK cells according to claim 1, wherein the NK cells are purified by negative or positive selection, or a combination thereof.

5. The NK cells are · one or more of IL-12, IL-23, IL-27, and IL-35; · one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and · one or more of IL-18, IL-1a, IL-1b, IL-36a, IL-36b, and IL-36g; or a functional fragment thereof, and / or a fusion protein containing the functional fragment, or any combination of the foregoing The memory NK cells according to claim 1, which are primed by exposure thereto.

6. The memory NK cells according to claim 5, wherein the NK cells are primed by exposure to 18t15-12s.

7. The memory NK cells according to claim 5, wherein the NK cells are primed by exposure to IL-12, IL-15, and IL-18.

8. The memory NK cells according to claim 1, wherein the NK cells are expanded by exposure to 7t15-21s and ATF1.

9. a) at least one extracellular ligand-binding domain that targets an antigen on a target cell; b) a hinge domain; c) a transmembrane domain; d) optionally, one or more co-stimulatory domains; and e) a cytoplasmic signaling domain The memory NK cells according to claim 1, further comprising at least one chimeric antigen receptor (CAR) containing the same.

10. a) purifying a concentrated NK cell population; b) expanding the NK cells; and c) priming the NK cells A method for generating memory NK cells comprising the steps of:

11. The NK cells are · one or more of IL-12, IL-23, IL-27, and IL-35; - One or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and - One or more of IL-18, IL-1a, IL-1b, IL-36a, IL-36b, and IL-36g; Or a functional fragment thereof, and / or a fusion protein comprising the functional fragment thereof, or any combination of the foregoing The method according to claim 10, which is primed by exposure to.

12. The method according to claim 11, wherein the NK cells are primed by exposure to 18t15-12s.

13. The method according to claim 11, wherein the NK cells are primed by exposure to IL-12, IL-15, and IL-18.

14. The method according to claim 10, wherein the NK cells are expanded by exposure to 7t15-21s and ATF1.

15. A method for treating a proliferative malignancy, comprising administering to a patient in need thereof memory NK cells as produced by the method according to claim 1.

16. The method according to claim 15, wherein the cells are administered to the patient fresh.

17. The method according to claim 15, wherein the proliferative malignancy is cancer.

18. The method according to claim 17, wherein the cancer is a hematological cancer.

19. The method according to claim 18, wherein the hematological cancer is T cell lymphoma.

20. The method according to claim 19, wherein the T cell lymphoma is selected from T cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T cell lymphoma (PTCL), T cell chronic lymphocytic leukemia (T-CLL), and Sézary syndrome.

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