Engineered natural killer cells with enhanced antitumor memory response

Abstract

Embodiments of this disclosure include methods and compositions in which engineered NK cells are artificially modified to overexpress a member of the CCAAT / enhancer-binding protein (C / EBP) family. Such engineered NK cells exhibit improved antitumor immunomemory-like properties, enhanced long-term cytotoxic activity, and improved metabolic fitness in vivo and in vitro.

Description

[Technical Field]

[0001] Government support clause This invention was made with government support granted by the National Institutes of Health (NIH) under the code CA127001. The government has certain rights to this invention.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 492,056, filed on 24 March 2023, which is incorporated herein by reference in its entirety.

[0003] Sequence List This application includes a sequence listing filed in ST26 format, which is incorporated into this application in its entirety by reference. The ST26 copy, created on 17 March 2024, is named MDAC_1355WO_Sequence_Listing.xml and has a size of 87,687 bytes.

[0004] I.Technical field This disclosure relates at least to the fields of immunology, cell biology, molecular biology, and medicine (including at least cancer medicine). [Background technology]

[0005] Natural killer (NK) cells have been studied as potential anti-tumor effectors, but several barriers exist that limit their therapeutic application, such as their lack of a significant memory response compared to other immune cells.

[0006] Glioblastoma (GBM) is the most common and invasive primary brain tumor, presenting significant therapeutic challenges. Current treatments include surgery, radiation therapy, and chemotherapy. 1 However, the effect is limited, and the median survival time is only 18-21 months. 2 Glioblastoma stem cell-like cells (GSCs) play a crucial role in tumor development and recurrence and are resistant to conventional chemotherapy and radiotherapy. 3Natural killer (NK) cells have the innate ability to recognize and kill GSCs, which could be a promising immunotherapy strategy against this disease. 4、5 This could be a promising immunotherapy strategy against this disease.

[0007] Cytokines can be used to further enhance the efficacy and in vivo persistence of NK cells against cancer. So far, IL-15 has been shown to promote the cytotoxic activity, proliferation, and persistence of NK cells, which has been the main focus of bridging and clinical studies. Many preclinical studies and initial clinical trials involving patients with lymphoid malignancies support the overall safety and promising activity of systemic administration of secreted IL-15 and NK cells engineered to express chimeric antigen receptors (CARs). 6、7 Furthermore, there are also several studies that have examined the safety and efficacy of locoregional administration of CAR-T cells and NK cells in GBM. However, the safety and activity of locoregionally administered IL-15 NK cells in the context of GBM have not been investigated. IL-21 is another attractive cytokine in cancer immunotherapy and is known to induce metabolic reprogramming and mitochondrial biogenesis in T cells. 8-12 IL-21 also promotes the proliferation, maturation, and metabolic fitness of NK cells. Recombinant IL-21 has been investigated in several clinical trials for metastatic cancer, but despite an acceptable safety profile, its short half-life and the need for repeated dosing have limited its clinical application. 13-15 IL-21 also promotes the proliferation, maturation, and metabolic fitness of NK cells. Recombinant IL-21 has been investigated in several clinical trials for metastatic cancer, but despite an acceptable safety profile, its short half-life and the need for repeated dosing have limited its clinical application. 16 IL-21 also promotes the proliferation, maturation, and metabolic fitness of NK cells. 17-19 Recombinant IL-21 has been investigated in several clinical trials for metastatic cancer, but despite an acceptable safety profile, its short half-life and the need for repeated dosing have limited its clinical application. 20、21 [[ID=十九]]

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present disclosure meets the needs of the art by improving cancer treatment, such as brain tumors, through the use of immunotherapy involving engineered NK cells.

Means for Solving the Problems

[0009] Overview Using multiple patient-derived GSCs and orthotopic in vivo models, the inventors demonstrated that locally administered IL-15 NK cells (NK cells engineered to express secreted IL-15; see Figure 1) were highly toxic and ineffective in controlling GBM tumors. In contrast, similarly administered IL-21 NK cells (NK cells engineered to express secreted IL-21; see Figure 1) were safe and demonstrated long-term antitumor activity. IL-21 NK cells exhibited a specific epigenetic and transcriptional signature, with transcription factors (TFs) of the CCAAT / enhancer-binding protein (C / EBP, CEBP) family emerging as key regulators of the IL-21 NK cell response to GBM. Deletion of CEBPD, the most differentially expressed CEBPTF member in IL-21 NK cells, impaired their potency and long-term antitumor response, while overexpression of CEBPD in NK cells enhanced functional capacity and metabolic suitability. The data demonstrated that STAT3 functions as a major signaling pathway in CEBPD-mediated gene expression regulation. These findings reveal that C / EBP transcription factors, particularly CEBPD, are important transcriptional and epigenetic coordinators of the NK cell response to cancer, supporting the use of IL-21 to arm NK cells as an immunotherapeutic approach for the treatment of GBM.

[0010] Embodiments of this disclosure include at least methods and compositions for treating individuals with a disease (e.g., cancer) using adoptive cell therapy. In one embodiment, provided herein are engineered natural killer (NK) cells modified to overexpress proteins of the CCAAT / enhancer-binding protein (CEBP) transcription factor family. In one embodiment, CEBP transcription factor expression is compared to unengineered NK cells and / or NK cells engineered to express IL-15. In one embodiment, the CEBP proteins are CEBPA (CEBP-alpha, CEBPα), CEBPB (CEBP-beta, CEBPβ), CEBPG (CEBP-gamma, CEBPγ), CEBPD (CEBP-delta, CEBPδ), CEBPE (CEBP-epsilon, CEBPε), and / or CEBP homolog protein (CHOP). In one embodiment, the CEBP protein is CEBPD and / or CEBPB. In one embodiment, the engineered NK cells overexpress transgenic CEBPB and / or CEBPD proteins. In one embodiment, NK cells transgenically express activators of transcription and / or translation of the CEBP protein, or are treated with them. In another embodiment, NK cells transgenically express inhibitors of transcription and / or translation of the CEBP protein, or are inhibited thereof.

[0011] In one embodiment, provided herein are engineered NK cells overexpressing the CEBP protein, the CEBP protein containing at least 80%, 85%, 90%, 95%, 99%, or 100% identical sequences to SEQ ID NO: 19. In one embodiment, the CEBP protein is encoded by a sequence containing at least 80%, 85%, 90%, 95%, 99%, or 100% identical sequences to SEQ ID NO: 20 or 21. In one embodiment, the CEBP protein contains at least 80%, 85%, 90%, 95%, 99%, or 100% identical sequences to any of SEQ ID NO: 23, 25, or 27. In one embodiment, the CEBP protein is encoded by a sequence containing at least 80%, 85%, 90%, 95%, 99%, or 100% identical sequences to any of SEQ ID NO: 24, 26, or 28.

[0012] In one embodiment, engineered NK cells overexpressing CEBP have enhanced mitochondrial fitness and / or memory-like features compared to unengineered NK cells and / or NK cells engineered to express IL-15. In one embodiment, enhanced mitochondrial fitness includes increased basal and / or maximum oxygen consumption rate (OCR) and / or decreased glycolysis (e.g., glycolysis is measured by extracellular oxidation rate (ECAR)) compared to unengineered NK cells and / or NK cells engineered to express IL-15.

[0013] In one embodiment, engineered NK cells overexpressing CEBP exhibit enhanced antitumor cytotoxicity compared to unengineered NK cells and / or NK cells engineered to express IL-15. In another embodiment, NK cells exhibit enhanced antitumor memory compared to unengineered NK cells and / or NK cells engineered to express IL-15. In yet another embodiment, NK cells show higher expression of functional markers, lower expression of inhibitory markers, higher expression of survival genes, lower expression of exhaustion genes, and / or upregulation of activating receptors and markers compared to unengineered NK cells and / or NK cells engineered to express IL-15. In one embodiment, functional markers include granzyme A (GrA), granzyme B (GrB), perforin, and / or Zap70. In yet another embodiment, inhibitory markers include LAG3 and / or KLRG1. In one embodiment, the activation receptor and markers include NKp30, CD25, DNAM, Ki67, CD3ζ, T-bet, and / or FCεRG. In one embodiment, the survival gene includes KLRD1, ITGA1, and / or GZMK. In one embodiment, the exhaustion gene includes DUSP2, CISH, and / or BAX. In one embodiment, NK cells have high expression levels of the cytotoxicity markers GrA, GrB, perforin, TRAIL, and / or CD95, and / or the activation markers / receptors CD25, CD69, DNAM, NKG2D, NKp44, and / or NKp46, compared to unengineered NK cells and / or NK cells engineered to express IL-15. In one embodiment, NK cells have high expression levels of transcription factors important for NK cell maturation, NK cell immune function, NK cell cytotoxicity, IFN-γ response, memory formation, and / or AP-1 complex members, compared to unengineered NK cells and / or NK cells engineered to express IL-15.In one embodiment, the transcription factors include CEBPD, CEBPB, ETS1, IRF1, TBX21, EOMES, IRF9, STAT1, ETS1, JUN, JUNB, JUND, FOS, and / or FOSL1.

[0014] Also provided herein are engineered CEBP-overexpressing NK cells that express one or more interleukins (ILs) through further modification. In one embodiment, the ILs are artificially ligated IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, and / or IL-12 p35 and p40 subunits. In one embodiment, the ILs are IL-21 and / or IL-15. In one embodiment, the ILs are secretory, conjugated, or membrane-bound. In one embodiment, the NK cells described herein enhance tumor cell apoptosis, apoptosis, and / or cytotoxicity. In one embodiment, the NK cells have elevated levels and / or activity of ERK1 / 2, NFκB, IFNG, TNFSF10, FASLG, and / or Nfat.

[0015] In some embodiments, the NK cells described herein are derived from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, NK cell lines, or a combination thereof. In some embodiments, the NK cells are primary NK cells and are not derived from stem cells and / or induced pluripotent stem cells (iPSCs). In some embodiments, the NK cells form complexes with one or more monospecific, bispecific, and / or multispecific antibodies. In some embodiments, the NK cells express one or more antibodies. In some embodiments, the NK cells are further modified to express one or more additional heterologous proteins selected from the group consisting of antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof. In some embodiments, the engineered receptor is an engineered antigen receptor. In some embodiments, the target antigen is a cancer antigen. In some embodiments, the NK cells contain a suicide gene. In some embodiments, the NK cells further contain one or more engineered mutations in an endogenous gene. In some embodiments, the endogenous gene is TGFBR2, CISH, GR, and / or CD38. In some embodiments, the NK cells are pre-activated with one or more cytokines. In one embodiment, one or more cytokines include IL-2.

[0016] Further provided herein are compositions comprising the manipulated NK cells described herein. In some embodiments, the composition comprises pharmaceutically acceptable excipients. In some embodiments, the composition is contained within a delivery device.

[0017] This specification also provides methods for treating diseases in individuals, which may include the step of administering to an individual a therapeutically effective amount of any of the manipulated NK cells or compositions described herein. In some embodiments, the disease is an autoimmune disease, an infection and / or cancer. In some embodiments, the disease is cancer. In some embodiments, the cancer is cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, cervix, or hematological cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the glioblastoma is a mesenchymal, neural, classical, or preneurial subtype of TCGA. In some embodiments, the glioblastoma is a mesenchymal or preneurial subtype. In some embodiments, the glioblastoma has an MGMT unmethylated, methylated, or indeterminate state. In some embodiments, the glioblastoma is primary or recurrent.

[0018] In some embodiments, the engineered NK cells used in the methods described herein exhibit enhanced in vivo antitumor function compared to unengineered NK cells and / or NK cells engineered to express IL-15. In some embodiments, the enhanced in vivo antitumor function includes long-term tumor eradication, a significant extension of the subject's survival time, and / or a reduction in toxicity or weight loss.

[0019] This specification also provides a method for conferring immunological memory against cancer to a subject, which includes administering to an individual a therapeutically effective amount of the manipulated NK cells and / or compositions described herein.

[0020] This specification also provides isolated nucleic acids encoding CEBP proteins fused with heterologous transcriptional regulators. In some embodiments, the CEBP protein contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the CEBP protein is encoded by a sequence that contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 20 or 21. In some embodiments, the heterologous transcriptional regulator is a promoter.

[0021] In some embodiments, methods for treating glioblastoma are also provided herein, which include administering a therapeutically effective amount of NK cells engineered to constitutively express secreted IL-21 to an individual. In some embodiments, the method for treating glioblastoma includes administering NK cells by intracranial injection. In some embodiments, administration is performed by intratumoral injection. In some embodiments, the NK cells engineered to constitutively and / or autonomously express secreted IL-21 include and / or a transgenic polynucleotide sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NOs: 31-32. In some embodiments, the method for treating glioblastoma confers immunological memory to glioblastoma to the subject. In some embodiments, the method for treating glioblastoma confers immunological memory to glioblastoma stem cells to the subject. In one embodiment, NK cells are engineered to stably secrete IL-21 at a rate that reaches an extracellular concentration of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 pg / mL or higher. In one embodiment, NK cells are engineered to stably secrete IL-21 at a rate that reaches an extracellular concentration of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 pg / mL or higher within 1, 2, or 3 days of culture. In one embodiment, NK cells are engineered to stably secrete IL-21 at a rate that reaches extracellular concentrations of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 or higher within 3, 4, or 5 days of culture.In one embodiment, NK cells are engineered to stably secrete IL-21 at a rate that reaches extracellular concentrations of approximately 200–800 pg / mL, 250–750 pg / mL, 300–700 pg / mL, or 350–650 pg / mL, or any value in between. In another embodiment, NK cells are engineered to stably secrete IL-21 at a rate that reaches extracellular concentrations of approximately 200–800 pg / mL, 250–750 pg / mL, 300–700 pg / mL, or 350–650 pg / mL, or any value in between, and maintain this concentration while the culture medium is changed daily, every two days, every three days, every four days, or every five days.

[0022] Specific embodiments of the present disclosure and related inventions can be characterized through the following enumerated embodiments.

[0023] Embodiment 1 is a modified natural killer (NK) cell that is overexpressing CCAAT / enhancer-binding protein (CEBP) transcription factor family proteins.

[0024] Embodiment 2 is the engineered NK cell of Embodiment 1, wherein the expression of the CEBP transcription factor is compared to that of unengineered NK cells and / or NK cells engineered to express IL-15.

[0025] Embodiment 3 is the engineered NK cells of Embodiment 1, wherein the CEBP protein is CEBPD (CEBP-delta, CEBPδ), CEBPA (CEBP-alpha, CEBPα), CEBPB (CEBP-beta, CEBPβ), CEBPG (CEBP-gamma, CEBPγ), CEBPE (CEBP-epsilon, CEBPε), and / or CEBP homolog protein (CHOP).

[0026] Embodiment 4 is an engineered NK cell of Embodiment 1 or 3, wherein the CEBP protein is CEBPD and / or CEBPB.

[0027] Embodiment 5 is an engineered NK cell in any of Embodiments 1 to 4, wherein the NK cell overexpresses the transgenic CEBPB and / or CEBPD proteins.

[0028] Embodiment 6 is a modified NK cell according to any of Embodiments 1 to 5, wherein the NK cell transgenically expresses or is treated with activators of transcription and / or translation of the CEBP protein.

[0029] Embodiment 7 is a modified NK cell in any of Embodiments 1 to 6, wherein the NK cell transgenically expresses or is inhibited by a transcription and / or translation inhibitor of the CEBP protein.

[0030] Embodiment 8 is an engineered NK cell according to any of Embodiments 1 to 7, wherein the CEBP protein contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 19.

[0031] Embodiment 9 is an engineered NK cell in any of Embodiments 1 to 8, wherein the CEBP protein is encoded by a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 20 or 21.

[0032] Embodiment 10 is an engineered NK cell according to any of Embodiments 1 to 9, wherein the CEBP protein contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any of SEQ ID NOs: 23, 25, or 27.

[0033] Embodiment 11 is an engineered NK cell according to any of Embodiments 1 to 10, wherein the CEBP protein is encoded by a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any of SEQ ID NOs: 24, 26, or 28.

[0034] Embodiment 12 is an engineered NK cell from any of Embodiments 1 to 11, having enhanced mitochondrial aptitude and / or memory-like features compared to an unengineered NK cell and / or an NK cell engineered to express IL-15.

[0035] Embodiment 13 is an engineered NK cell of Embodiment 12, wherein the enhanced mitochondrial fitness includes increased basal and / or maximal oxygen consumption rate (OCR) and / or decreased glycolysis (for example, when glycolysis is measured by extracellular oxidation rate (ECAR)) compared to an unengineered NK cell and / or an NK cell engineered to express IL-15.

[0036] Embodiment 14 is an engineered NK cell from any of Embodiments 1 to 13, which has enhanced antitumor cytotoxicity compared to unengineered NK cells and / or NK cells engineered to express IL-15.

[0037] Embodiment 15 is an engineered NK cell from any of Embodiments 1 to 14, which has enhanced antitumor memory compared to unengineered NK cells and / or NK cells engineered to express IL-15.

[0038] Embodiment 16 is an engineered NK cell from any of Embodiments 1 to 15, having high expression of functional markers, low expression of repression markers, high expression of survival genes, low expression of exhaustion genes, and / or upregulation of activating receptors and markers, compared to unengineered NK cells and / or NK cells engineered to express IL-15.

[0039] Embodiment 17 is the engineered NK cells of Embodiment 16, wherein the functional markers include granzyme A (GrA), granzyme B (GrB), perforin, and / or Zap70.

[0040] Embodiment 18 is the engineered NK cells of Embodiment 16, wherein the inhibitory markers include LAG3 and / or KLRG1.

[0041] Embodiment 19 is an engineered NK cell of Embodiment 16, wherein the activating receptor and markers include NKp30, CD25, DNAM, Ki67, CD3ζ, T-bet, and / or FCεRG.

[0042] Embodiment 20 is the engineered NK cells of Embodiment 16, wherein the survival genes include KLRD1, ITGA1, and / or GZMK.

[0043] Embodiment 21 is the engineered NK cells of Embodiment 16, wherein the exhaustion genes include DUSP2, CISH, and / or BAX.

[0044] Embodiment 22 is an engineered NK cell from any of Embodiments 1 to 21, comprising high expression of the cytotoxic markers GrA, GrB, perforin, TRAIL and / or CD95, and / or high expression of the activating markers / receptors CD25, CD69, DNAM, NKG2D, NKp44 and / or NKp46, compared to an unengineered NK cell and / or an NK cell engineered to express IL-15.

[0045] Embodiment 23 is an engineered NK cell from any of Embodiments 1 to 22, which exhibits higher levels of NK cell maturation, NK cell immune function, NK cell cytotoxicity, IFN-γ response, memory formation, and / or high expression levels of transcription factors important to AP-1 complex members, compared to unengineered NK cells and / or NK cells engineered to express IL-15.

[0046] Embodiment 24 is the engineered NK cells of Embodiment 23, wherein the transcription factors include CEBPD, CEBPB, ETS1, IRF1, TBX21, EOMES, IRF9, STAT1, ETS1, JUN, JUNB, JUND, FOS, and / or FOSL1.

[0047] Embodiment 25 is an engineered NK cell from any of Embodiments 1 to 24, wherein the cell is engineered to provide one or more interleukins (ILs).

[0048] Embodiment 26 is an engineered NK cell of Embodiment 25, in which IL is artificially linked to IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, and / or the p35 and p40 subunits of IL-12.

[0049] Embodiment 27 is an engineered NK cell from Embodiment 26, wherein the IL is IL-21 and / or IL-15.

[0050] Embodiment 28 is an engineered NK cell of Embodiment 26 or 27, in which the NK cell acquires tumor cell apoptosis, apoptosis, and / or enhanced cytotoxicity.

[0051] Embodiment 29 is an engineered NK cell from any of Embodiments 26-28, wherein the NK cell has increased levels and / or activity of ERK1 / 2, NFκB, IFNG, TNFSF10, FASLG, and / or Nfat.

[0052] Embodiment 30 is an engineered NK cell according to any of Embodiments 25 to 29, wherein the IL is secreted, bound, or membrane-bound within the cell.

[0053] Embodiment 31 is an engineered NK cell from any of Embodiments 1 to 30, wherein the NK cell is derived from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, an NK cell line, or a combination thereof.

[0054] Embodiment 32 is an engineered NK cell from any of Embodiments 1 to 31, in which the NK cell is a primary NK cell and is not derived from stem cells and / or induced pluripotent stem cells (iPSCs).

[0055] Embodiment 33 is an engineered NK cell according to any of Embodiments 1 to 32, wherein the NK cell forms a complex with one or more monospecific, bispecific, and / or multispecific antibodies.

[0056] Embodiment 34 is the manipulated NK cells of Embodiment 33, wherein the NK cells express one or more antibodies.

[0057] Embodiment 35 is an engineered NK cell according to any of Embodiments 1 to 34, wherein the NK cell is further modified to express one or more additional heterologous proteins selected from antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof.

[0058] Embodiment 36 is the engineered NK cell of Embodiment 35, wherein the engineered receptor is an engineered antigen receptor.

[0059] Embodiment 37 is an engineered NK cell from any of embodiments 33 to 36, wherein the target antigen is a cancer antigen.

[0060] Embodiment 38 is an NK cell that has been manipulated in any of Embodiments 1 to 37, wherein the NK cell contains a suicide gene.

[0061] Embodiment 39 is an engineered NK cell from any of Embodiments 1 to 38, wherein the NK cell further contains an engineered mutation in one or more endogenous genes.

[0062] Embodiment 40 is the engineered NK cell of Embodiment 39, wherein the endogenous genes are TGFBR2, CISH, GR, and / or CD38.

[0063] Embodiment 41 is an engineered NK cell according to any of Embodiments 1 to 40, wherein the NK cell is pre-activated with one or more cytokines.

[0064] Embodiment 42 is an engineered NK cell according to Embodiment 41, wherein one or more cytokines include IL-2.

[0065] Embodiment 43 is a composition comprising manipulated NK cells as described in Embodiments 1 to 42.

[0066] Embodiment 44 is the composition of Embodiment 43, further comprising pharmaceutically acceptable excipients.

[0067] Embodiment 45 is the composition of Embodiment 43 or 44, wherein the composition is included in a delivery device.

[0068] Embodiment 46 is a method for treating a disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of manipulated NK cells or a composition according to any of the preceding embodiments.

[0069] Embodiment 47 is the method of Embodiment 46, wherein the disease is an autoimmune disease, an infectious disease, and / or cancer.

[0070] Embodiment 48 is the method of Embodiment 46 or 47, wherein the disease is cancer.

[0071] Embodiment 49 is the method of Embodiment 48, wherein the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, cervix, or hematological.

[0072] Embodiment 50 is the method of Embodiment 48 or 49, wherein the cancer is glioblastoma.

[0073] Apparatus 51 is the method of Apparatus 50, wherein the glioblastoma is a TCGA mesenchymal, neural, classical, or preneurial subtype.

[0074] Embodiment 52 is the method of Embodiment 51, wherein the glioblastoma is a mesenchymal or anterior nerve subtype.

[0075] Embodiment 53 is the method of Embodiment 50 or 51, wherein the glioblastoma is in an MGMT-unmethylated, methylated, or indeterminate state.

[0076] Apparatus 54 is any of the methods in Apparatus 50 to 53, wherein the glioblastoma is primary or recurrent.

[0077] Embodiment 55 is any of embodiments 48 to 54, wherein engineered NK cells exhibit enhanced antitumor function in vivo compared to unengineered NK cells and / or NK cells engineered to express IL-15.

[0078] Embodiment 56 is the method of Embodiment 55, wherein the enhanced in vivo antitumor function includes long-term tumor eradication, a significant extension of the subject's survival time, and / or a reduction in toxicity or weight loss.

[0079] Embodiment 57 is any of the methods in Embodiments 50 to 56, wherein the administration is by intracranial injection.

[0080] Embodiment 58 is any of the methods in Embodiments 50 to 57, wherein the administration is by intratumor injection.

[0081] Embodiment 59 is any of the embodiments 50 to 58, wherein the method provides immunological memory against glioblastoma.

[0082] Embodiment 60 is any of the methods of Embodiments 50 to 59, wherein the method provides immunological memory to glioblastoma stem cells.

[0083] Embodiment 61 is a method for providing an immunological memory against cancer to a subject, comprising administering to an individual a therapeutically effective amount of manipulated NK cells from any of Embodiments 1 to 42 or a composition from any of Embodiments 43 to 45.

[0084] Embodiment 62 is an isolated nucleic acid encoding a CEBP protein fused with a heterologous transcriptional regulator.

[0085] Embodiment 63 is an isolated nucleic acid of Embodiment 62, wherein the CEBP protein contains a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 19.

[0086] Embodiment 64 is an isolated nucleic acid of Embodiment 62 or 63, wherein the CEBP protein is encoded by a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 20 or 21.

[0087] Embodiment 65 is an isolated nucleic acid according to any of Embodiments 62 to 64, wherein a heterogeneous transcriptional regulatory element is the promoter.

[0088] Embodiment 66 is a method for treating glioblastoma, comprising administering to an individual a therapeutically effective dose of NK cells engineered to autonomously and / or constitutively express secreted IL-21.

[0089] Embodiment 67 is the method of Embodiment 66, wherein the administration is by intracranial injection.

[0090] Embodiment 68 is the method of Embodiment 66 or 67, wherein the administration is by intratumor injection.

[0091] Embodiment 69 is any of embodiments 66 to 68, wherein NK cells engineered to constitutively and / or autonomously express secreted IL-21 include a transgenic polynucleotide sequence encoding and / or containing at least 80%, 85%, 90%, 95%, or 100% identical sequences of SEQ ID NOs. 31-32.

[0092] Embodiment 70 is any of the methods according to embodiments 66 to 69, wherein the method provides immunological memory against glioblastoma.

[0093] Embodiment 71 is any of the methods of Embodiments 66 to 70, wherein the method provides immunological memory to glioblastoma stem cells.

[0094] Embodiment 72 is any of the methods of Embodiments 66 to 71, wherein NK cells are manipulated to stably secrete IL-21 at a rate suitable for reaching an extracellular concentration of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 pg / mL or higher.

[0095] Embodiment 73 is any of the methods of Embodiments 66 to 72, wherein NK cells are manipulated to stably secrete IL-21 at a rate suitable for reaching an extracellular concentration of approximately 200-800 pg / mL, 250-750 pg / mL, 300-700 pg / mL, or 350-650 pg / mL.

[0096] Apparatus 74 is any of the methods in Apparatus 66 to 73, wherein the glioblastoma is of the TCGA mesenchymal, neural, classical, or preneurial subtype.

[0097] Apparatus 75 is one of the methods of Apparatus 66 to 74, wherein the glioblastoma is of the mesenchymal or anterior nerve subtype.

[0098] Embodiment 76 is any of the methods of Embodiments 66 to 75, wherein the glioblastoma is in an MGMT-unmethylated, methylated, or indeterminate state.

[0099] Apparatus 77 is any of the methods in Apparatus 66 to 76, wherein the glioblastoma is primary or recurrent.

[0100] Embodiment 78 is any of embodiments 66 to 77, wherein the manipulated NK cells exhibit enhanced antitumor function in vivo compared to unmanipulated NK cells and / or NK cells manipulated to express IL-15.

[0101] Embodiment 79 is the method of Embodiment 78, wherein the enhanced in vivo antitumor function includes long-term tumor eradication, a significant extension of subject survival, and / or reduction of toxicity or weight loss.

[0102] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples illustrate specific aspects of the invention and are for illustrative purposes only, for various changes and modifications will become apparent to those skilled in the art within the spirit and scope of the invention.

[0103] The following drawings constitute part of this specification and are included to further illustrate specific aspects of the invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0104] [Figure 1-1]Figure 1A-1I-Cytokine arming improved the antitumor activity of NK cells against glioblastoma stem cells (GSCs). (Figure 1A) Schematic diagrams of two retroviral vectors used to transduce NK cells to secrete IL-15 or IL-21. (Figure 1B) NK cells were proliferated for 5 days with uAPC feeder cells and IL-2, and then transduced with retroviral vectors encoding IL-15 or IL-21 (SEQ ID NO: 29 or 31). Transduction efficiency was determined by evaluating IgG expression by flow cytometry using antibodies against human IgG1 (donor n=5-6). (Figure 1C) Total cytokine concentrations (pg / mL; ELISA) of IL-15 (donor n=3) and IL-21 (donor n=6) in the culture supernatant 5 days after transduction of untransduced (NT) NK (NTNK), IL-15-armed NK (IL-15NK), or IL-21-armed NK (IL-21NK) cells. Error bars indicate standard deviation. (Figure 1D) Percentage over time of GSC20 killing by NTNK, IL-15NK, or IL-21NK cells, as measured by a real-time killing assay (E:T ratio 1:1). Asterisks indicate statistical significance. IL-15NK vs NTNK = ***; IL-21NK vs NTNK = ***. (Figure 1E-1F) K562 killing by NTNK, IL-15NK, or IL-21NK cells cultured alone (Figure 1E) or co-cultured with GSC20 for 48 hours (Figure 1F) at an E:T ratio of 1:1. NK cells were purified and their killing ability against the K562 target was evaluated by a real-time killing assay (donor n=6). IL-15NK vs NTNK = ***; IL-21NK vs NTNK = ***. (Figure 1G-1I) 3D killing assay of GSC20 and GSC272 spheroids using NTNK, IL-15NK, or IL-21NK cells (donor n=3). The total integrated intensity of the red objects (y-axis, RCU × μm² / image) shows the growth signal of GSC272 (Figure 1G) or GSC20 (Figure 1H) spheroids over time (x-axis). IL-21NK and IL-15NK cells induced significant levels (P=***) of GSC272 and GSC20 apoptosis compared to NTNK controls.(Figure 1I) Image showing the time course (y axis) of co-culture of NTNK, IL-15NK, or IL-21NK cells with GSC272, or control GSC272 cells (x axis). Time course monitoring of the red signal in the GSC272 spheroid 3D killing assay showed that IL-21NK cells induced significant levels of GSC272 apoptosis within 24 hours of co-culture. The image shows the red signal obtained from live imaging of the GSC272 spheroid 3D killing assay. Statistical significance was determined by two-way ANOVA and Dunnett multiple comparison correction. ***p≦0.001. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above.

[0105] [Figure 2-1]Figures 2A-2C show that P-IL-21 armed NK cells exhibited long-term cytotoxic activity against GSCs and showed higher metabolic compatibility than IL-15 armed or unarmed NK cells. Cytokine-armed NK cells or un-armed (NT) controls were co-cultured with m-cherry-introduced GSC20 (red) at an E:T ratio of 1:1. Every 2-3 days, new GSCs were added to the co-culture without adding new NK cells. The red (GSC) signal was tracked by real-time imaging. (Figures 2A-2C) After IL-15NK, IL-21NK, and NTNK cells (n=3 donors each) were co-cultured alone or with GSC20 for 48 hours, NK cell surface and intracellular marker expression was measured by mass cytometry. (Figure 2A) tSNE plot showing cluster distribution. (Figure 2B) tSNE plot analysis showing cluster distribution, expression, and percentage. The color scale and circle size indicate the expression level and cluster size in each group. (Figure 2C) Comparative heatmap showing NK cell marker expression at the cluster level. Clustering of heatmap columns was identified by FlowSOM analysis. The color scale shows the expression level for each marker, with red indicating high expression and blue indicating low expression (n=3 donors). (Figures 2D, 2F, and 2G) Graphs showing the change in the number of red objects per image over time. This correlated with the percentage of surviving cancer cells after (Figure 2D) GSC20 rechallenge (n=3 donors), (Figure 2F) GSC272 rechallenge (n=3 donors), and (Figure 2G) GSC267 rechallenge (n=3 donors), respectively. The data showed that IL-21NK cells maintained high cytotoxicity against GSC20, GSC272, and GSC267 cells over five rechallenge events, which was not observed in NTNK or IL-15NK. Asterisks indicate statistical differences between groups. Error bars indicate standard deviation. (Figure 2E) Summary of the number of red objects per image after GSC20 rechallenge in two independent experiments (n=6 donors). Error bars indicate the standard deviation. (Figure 2H) Heatmap showing the results of multiplex ELISA measurements of different cytokine concentrations in the supernatant collected from co-cultures of NK cells and GSCs in the experiment shown in Figure 2D.(Figures 2I-2J) Multifunctional scores (Figure 2I) and multifunctional strength indices (Figure 2J) of NTNK, IL-15NK, and IL-21NK cells in response to GSC20 (n=4 donors). Error bars indicate standard deviation. (Figure 2K) Representative measurements of oxygen consumption rate (OCR) after addition of oligomycin (Oligo), FCCP, rotenone, and antamycin A (R / A). Quantified basal respiration (Figure 2L) and maximal respiration (Figure 2M) of purified NTNK, IL-15NK, and IL-21NK cells (n=3 donors) after co-culture with GSC20 for 48 hours. (Figure 2N) Representative measurements of extracellular oxidation rate (ECAR) after addition of glucose, Oligo, and 2-deoxy-D-glucose (2-DG). Quantified basal glycolysis (Figure 2O) and glycolytic activity (Figure 2P) of purified NTNK, IL-15NK, and IL-21NK cells (n=3 donors) after co-culture with GSC20 for 48 hours. Error bars indicate the standard error of the mean. Statistical significance was determined by two-way ANOVA and Bonferroni correction. *p≦0.05, **p≦0.01, ***p≦0.001. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above. [Figure 2-7] Same as above. [Figure 2-8] Same as above.

[0106] [Figure 3-1]Figure 3A-G: IL-21 armed NK cells showed significantly superior in vivo antitumor activity compared to the control group in a rectal PDX mouse model of patient-derived GSCs. (Figure 3A) Schematic diagram showing the injection timeline and representative bioluminescence imaging (BLI) images showing the in vivo tumor state. BLI was used to monitor the proliferation of FFluc-labeled GSC20 tumor cells (0.5 × 10⁶) injected intracranially (IC) into NSG (NOD scid gamma) mice. Tumors were allowed to grow for 7 days, after which mice were treated with (0.1 × 10⁶) NTNK, IL-15NK, or IL-21NK cells by intratumor (IT) injection (n=3-5 mice in each group). BLI images showed that IL-21NK cells effectively eliminated the FFluc-labeled GSC20 tumor cell signal and could maintain this elimination state for at least 176 days after tumor cell inoculation, which was significantly longer than the control group. (Figure 3B) This figure shows the quantitative analysis of individual (thin lines) and average (thick lines) luminance (BLI) data (p / sec / cm2 / sr) obtained from the mice in Figure 3A. Error bars indicate the standard error of the mean. Statistical significance was determined by unpaired t-tests. **p≦0.01, ns=no significant difference. (Figure 3C) Kaplan-Meier plots showing mouse survival rates in each group (n=3-5 mice per group). Animals treated with IL-21NK cells showed significantly better survival by log-rank test compared to the GSC20 monotherapy group (p=0.0042), the IL-15NK group (p=0.0027), or the NTNK control group (p=0.0042). *p≦0.05, **p≦0.01, ns=no significant difference. (Figure 3D) This graph shows the change in body weight (%) of mice in the different groups described in Figure 3C over time. IL-21NK cells maintained or slightly increased body weight throughout the experimental period and showed significantly higher body weight than the control group. Statistical significance was determined by unpaired t-tests. **p≦0.01, ***p≦0.001, ns=no significant difference. (Figure 3E) Schematic diagram showing GSC20 in vivo rechallenge. IL-21NK-treated mice showing no signs of tumor were reinjected with 0.25×10⁶GSC20 400 days after tumor inoculation (n=4 mice). The same injection was given to the NSG control group in a new cohort.Brain tissue was collected 17 days after rechallenge (test group n=4, control group n=5). Flow cytometry was used to detect NK cells (CD56+CD3-CD16+ cells) in brain tissue from the IL-21 NK cell treatment group (3 out of 4 mice with available tissue) and the tumor-only control group (n=5) mice after GSC20 rechallenge (Figure 3F), and CD16 staining by immunohistochemistry (IHC) was performed (Figure 3G). Arrows indicate CD16+ NK cells infiltrating the brain. Representative IHC images of brain sections from two mice (#1, #2) treated with IL-21 NK cells and those that underwent GSC20 rechallenge (left panel), as well as from the tumor-only control (upper right panel) and the tonsil (positive control; lower right panel). Magnification is 10x. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0107] [Figure 4-1]Figures 4A-4J show the epigenetic and transcriptome profiles of IL-21NK cells, characterized by changes over time after GSC co-culture. (Figure 4A) Schematic diagram of characterization experiments using scRNA-seq and scATAC-seq, and UMAP plots of scATAC-seq data showing cluster-level epigenetic evolution of IL-15NK (top) and IL-21NK (bottom) cells from baseline (day 0) to days 3 and 9 after GSC co-culture. (Figure 4B) Fish plots showing the proportion of different scATAC-seq clusters appearing in IL-15NK and IL-21NK cells from day 0 (baseline) to days 3 and 9 after GSC co-culture. (Figure 4C) Transcription factor enrichment at specific peaks in cluster 6 (mainly derived from IL-21NK cells on day 9). (Figure 4D) Volcano plot showing transcription factor enrichment of IL-15NK and IL-21NK cell-specific peaks on day 3 (left) and day 9 (right) after GSC20 co-culture. Red dots indicate transcription factors (TFs) with motifs strongly enriched in product-specific peaks. (Figure 4E) IL-21 motif enrichment on day 9. Motifs of members of the CCAAT / enhancer-binding protein (C / EBP) family are included in the top 20 enriched TFs found, and the binding motifs of selected members are shown. (Figure 4F) UMAP plot of scRNA-seq data showing transcriptome clusters and their evolution in IL-15NK (top) and IL-21NK (bottom) cells at baseline (day 0) and on days 3 and 9 after GSC20 co-culture. (Figure 4G) Fish plot showing the cluster appearance rate of IL-15NK and IL-21NK cells in scRNA-seq data from day 0 (baseline) to days 3 and 9 after GSC20 co-culture. (Figure 4H) Volcano plot showing significantly upregulated genes in IL-15NK cell cluster 3 (left) and IL-21NK cell cluster 4 (right) on day 9 after co-culture with GSC20. Red dots indicate TFs with high gene expression levels.(Figure 4I) Venn diagram showing the overlap between DEGs in cluster 4 determined by scRNA-seq and genes associated with chromatin open regions in cluster 6 determined by scATAC-seq. These two clusters were specific to IL-21NK cells in multiple retries against GSCs. (Figure 4J) Genome coverage plots derived from scATAC and scRNA profiling. After co-culture with GBM cancer cells for 9 days, the chromatin accessibility peak of the CEBPD coding region in IL-21NK cells was higher than in IL-15NK cells. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above.

[0108] [Figure 5-1]Figures 5A-5J show that different regulon activities were observed in IL-21NK cells and IL-15NK cells after GSC co-culture. (Figure 5A) Venn diagram showing overlapping regulons between IL-21NK and IL-15NK cells at each time point (baseline, day 3, day 9). (Figure 5B) Heatmap showing regulon activity of IL-21NK and IL-15NK cells at baseline (day 0) and at days 3 and 9 after GSC co-culture. The color bars show the scaled regulon activity score (AUC) by pySCENIC. (Figures 5C-5D) Violin plots showing scaled gene-level chromatin accessibility estimated by pySCENIC from gene expression profiles for target genes of CEBPB (Figure 5C) and CEBPD (Figure 5D). There were 106 target genes for CEBPD and 85 target genes for CEBPB. (Figures 5E-5F) Top enriched Hallmark pathways of target genes for CEBPB (Figure 5E) and CEBPD (Figure 5F) as estimated by pySCENIC. (Figure 5G) UMAP showing cluster distribution of each NK cell product at baseline (day 0) and at days 3 and 9 after challenge and / or rechallenge with GSC20, based on scRNA-seq data. (Figure 5H) UMAP plot showing CEBPD expression levels across the entire cell population. Highest levels were observed in cluster 4 (IL-21 cluster). (Figure 5I) UMAP plot showing gene set scores of CEBPD regulons at the transcriptome level. (Figure 5J) Notable downstream target genes regulated by CEBPB and CEBPD, identified by pySCENIC. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0109] [Figure 6-1]Figure 6A~6K-CEBPD was required for the potent and sustained antitumor activity and metabolic compatibility of IL-21NK cells. NT, Cas9IL-21 control, and CEBPD-KOIL-21NK cells were co-cultured with m-cherry-transformed GSC20 (red) at an E:T ratio of 1:1. After 2-3 days, fresh GSC20 was added to the co-culture without adding new NK cells or agitation (arrow). The red (tumor) signal was tracked with real-time imaging. (Figure 6A) Graph showing the number of red objects per image in real-time killing analysis (n=4 donors). Comparison of NTNK, IL-21Cas9NK, and CEBPD-KOIL-21NK cells. (Figure 6B) Representative measurements of oxygen consumption rate (OCR) after addition of Oligo, FCCP, rotenone, and antamycin A (R / A). (Figure 6C) Quantification of basal respiration and (Figure 6D) maximal respiration. (Figure 6E) Purified NT, IL-21, and CEBPD-KOIL-21NK cells after 48 hours of co-culture with GSC20 (n=3 donors). Error bars indicate the mean standard error. Graph showing the number of red objects per image in real-time killing analysis of NTNK, CEBPD-KI (knock-in; also called CEBPD-OE (overexpression)) NTNK cells and IL-21NK cells (n=4 donors). Arrows indicate the addition of GSC, i.e., rechallenge. (Figure 6F) Representative measurements of oxygen consumption rate (OCR) after Oligo, FCCP, and R / A addition. (Figure 6G) Quantification of basal respiration and (Figure 6H) maximal respiration. Purified NTNK, CEBPD-KI (Knock-in) NTNK, and IL-21NK cells after 48 hours of co-culture with GSC20 (n=4 donors). Error bars indicate the mean standard error. (Figure 6I) Schematic diagram showing the timeline of the in vivo experiment, and representative BLI images to monitor the proliferation of FFluc-labeled GSC272 tumor cells when NSG mice were injected with GSC272 alone, or with GSC272 and NTNK, CEBPD-KINTNK, IL-21-Cas9 control, or CEBPD-KOIL-21NK cells. (Figure 6J) Mean radiance (BLI) data (p / sec / cm2 / sr) for each treatment condition. Statistical significance was determined by two-way ANOVA and Bonferroni correction for multiple comparisons between NK treatment groups. ***p≦0.001, ns = no significant difference.(Figure 6K) Kaplan-Meier plots showing mouse survival rates in each group (n=5 mice). The data showed that NSG mice administered with CEBPD-KINTNK cells lived significantly longer than mice administered with NTNK cells or tumor cells alone, and that CEBPD is required for IL-21-mediated survival. Error bars indicate the standard error of the mean. Statistical significance was determined based on the log-rank test. *p≦0.05, **p≦0.01, ***p≦0.001, ns=no significant difference. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0110] [Figure 7-1]Figures 7A-7L-cytokine-armed NK cells proliferate and long-term cytotoxic activity against GSCs. (Figure 7A) Doubling of IL-15NK, IL-21NK, and NTNK cells cultured in the presence of uAPC feeder cells and IL-2 (200 IU / mL) at a ratio of uAPC:NK=2:1 (time course, n=4 donors). (Figure 7B) Killing rate of GSC8-11 cells by NTNK, IL-15NK, and IL-21NK measured by real-time killing assay (%) (n=3). Asterisks indicate statistical differences between groups (IL-15NK vs NTNK; IL-21NK vs NTNK). Error bars are standard deviations. Statistical significance was determined by two-way ANOVA (Dunnet corrected). ***p≦0.001. (Figure 7C) Time course graph of the number of red objects per image. This correlates with the percentage of surviving cancer cells after each GSC8-11 rechallenge (n=3 donors). Cytokine-armed NK cells and mCherry-introduced GSC8-11 (red) were co-cultured at E:T=1:1. Fresh GSCs were added every 2-3 days without adding new NK cells. The red (tumor) signal was tracked with real-time imaging. Asterisks indicate differences between groups. Error bars indicate standard deviation. Statistical significance was measured using two-way ANOVA (Bonferroni corrected). ***p≦0.001. (Figure 7D) Absolute number of NK cells (over time) after 1:1 co-culture with GSC20. Error bars indicate standard deviation. (Figure 7E) Bar graph of the percentage of viable NK cells (%) based on Annexin V-negative and Live / Dead-negative gates. Error bars indicate standard deviation. Statistical significance was measured using two-way ANOVA (Bonferroni corrected). (Figure 7F) NTNK cells and GSCs after IL-21 priming were co-cultured at E:T=1:1. Fresh GSCs were added every 2-3 days (no NK cells were added). The red (tumor) signal was tracked in real time. Asterisks indicate differences between groups. Error bars indicate standard deviations. Statistical significance was measured using two-way ANOVA (Bonferroni corrected). ***p≦0.001. (Figures 7G~7K) Changes in IL-21 (Figure 7G), TNF-α (Figure 7H), IFN-γ (Figure 7I), Granzyme B (Figure 7J), and Perforin (Figure 7K) concentrations in the supernatant of NK cells co-cultured with GSC20 in a 1:1 ratio. Fresh GSC cells were added every 2-3 days (without NK cells). Supernatants were collected at each time point and measured by multiplex ELISA.Asterisks indicate group differences (IL-15NK vs NTNK; IL-21NK vs NTNK). Error bars indicate standard deviation. Statistical significance was calculated using two-way ANOVA (Bonferroni corrected). **p≦0.01, ***p≦0.001. (Figure 7L) Multifunctional heatmap of NT, IL-15, and IL-21NK relative to GSC20. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above.

[0111] [Figure 8] Figures 8A-8B - IL-21-armed NK cells showed long-term cytotoxic activity against GSCs. Cytokine-armed NK cells and m-cherry-introduced GSC20 (red) were co-cultured at E:T=1:1. Fresh GSCs were added every 2-3 days (NK cells were not added). The GSC20 killing rate (%) was calculated from the overlap ratio (counts per image) of red (GSC) signals between red and green (dead cell) signals. Signals were tracked in real time. (Figure 8A) GSC20 killing rates (%) by NTNK, IL-15NK, and IL-21NK after each tumor rechallenge (n=3 donors). (Figure 8B) Bar graphs of GSC20 killing rates (%) after each rechallenge in two independent experiments (n=6 donors). Asterisks indicate differences between groups (IL-21NK vs NTNK; IL-21NK vs IL-15NK). Error bars indicate standard deviation. Statistical significance was determined by two-way ANOVA (Bonferroni correction). ***p≦0.001.

[0112] [Figure 9-1]Figures 9A-9I-IL-15NK cells showed increased proliferation and toxicity in an intrarectal (orthotopic) mouse model of GSC. GSC20 was orthotopically transplanted into NSG mice, and 0.5 × 10⁶ IL-15NK or IL-21NK were administered intratumorally (IT). (Figure 9A) Bioluminescence imaging (BLI) was used to monitor the growth of FFluc-labeled GSC20 tumors over time in NSG mice treated with IL-15NK or IL-21NK. (Figure 9B) Mean radiance (BLI) data (p / sec / cm² / sr). Statistical significance was determined by an unpaired t-test at day 14. *p ≤ 0.05. (Figure 9C) Percentage change in body weight (%) graph as an indicator of toxicity between groups. Statistical significance was determined using an unpaired t-test at day 22. *p ≤ 0.05. (Figure 9D) Kaplan-Meier plot showing the survival of IL-15NK (n=3) versus IL-21NK (n=4) treated mice. Based on the log-rank test, IL-15NK treated mice died significantly earlier than IL-21NK treated mice (p=0.01; **p≦0.01). Statistical significance was also assessed by an unpaired t-test on day 22. (Figure 9E) Immunohistochemical staining of human Granzyme B (GrB; left), Iba1 (center), and glial fibrillary acidic protein (GFAP; right) in the brains of mice that died after IL-15NK treatment. Shows marked NK cell proliferation, microgliosis, and astrocytosis. Magnification 20×. Tonsils were used as a positive control for GrB, and mouse brains were used as controls for Iba1 and GFAP. (Figure 9F) Representative GrB immunostaining images of brain sections obtained from NSG mice treated with 0.1 × 10⁶ IL-15NK (n=4), IL-21NK (n=4), or NTNK (n=3) after orthotopic transplantation of GSC20. NSG mice with GSC20 transplantation only (n=3) were used as negative controls. Magnification 10×. The inset of the IL-15NK panel shows NK cells infiltrating the brain (arrows, 20×, brown signal). Staining images from two mice for each treatment condition are shown. (Figure 9G) Number of GrB-positive cells ( / mm²) in brain sections of NTNK, IL-15NK, and IL-21NK treated mice. Error bars indicate standard deviation. Statistical significance was determined using two-way ANOVA (Bonferroni corrected). ***p≦0.001; ns = no significant difference.(Figure 9H) Representative images of mouse GFAP immunostaining of NSG mouse brain sections transplanted with GSC20 and treated with IL-15NK (n=4), IL-21NK (n=4), and NTNK (n=3). Images were obtained at 20× magnification. (Figure 9I) Brain pathology based on human GrB-positive cell (NK cell indicator) infiltration and gliosis score. The presence or absence of GrB-positive cells and glial cell increase (gliosis) in the frontal lobe were evaluated. Gliosis was semi-quantitatively scored on the following scale: 0 = no increase, 1 = minimal (1-10% increase in cell density), 2 = mild (11-30% increase), 3 = moderate (31-50% increase), 4 = severe (>50% increase). [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above.

[0113] [Figure 10-1]Figures 10A-10F - The route of administration affected the degree of efficacy and toxicity caused by IL-15NK cells. (Figure 10A) Mice were orthotopically transplanted with GSC262 and administered IL-15NK cells via tail vein (IV) or intratumor (IT). ITNTNK cell-treated mice were used as controls. Kaplan-Meier plots for each group (n=5 mice) showed that the ITIL-15NK-treated group died significantly earlier than the IV group (log-rank test, p=0.0027; **p≦0.01). (Figure 10B) Graphs showing the percentage change in body weight (%) as an indicator of toxicity for each group described. Statistical significance was determined by two-way ANOVA (Bonferroni corrected). ns = no significant difference. (Figure 10C) Flow cytometry panel showing massive NK cell infiltration in brain tissue of IL-15NK-treated mice administered via IT (top) and IV (bottom). (Figure 10D) Percentage of NK cells (CD56+CD3-) on hCD45+ cells in brain tissue of IL-15NK-treated mice administered via IT (n=4 mice) and IV (n=4 mice). Error bars indicate standard deviation. Statistical significance was determined by t-test. ***p≦0.001. (Figure 10E) GSC20 was orthotopically transplanted into mice, and IL-21NK cells were either administered via tail vein (IV) or left untreated (control). No significant difference was observed between groups in the Kaplan-Meier plots for each group (n=5 mice) (log-rank test). (Figure 10F) Mean radiance (BLI) data (p / sec / cm2 / sr). Error bars indicate standard error of the mean. Statistical significance was determined by paired t-test at each time point. ns = no significant difference. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0114] [Figure 11-1]Figure 11A~11H-IL-21NK cells showed excellent in vivo antitumor activity. (Figure 11A) Schematic timeline of the in vivo experiment. NSG mice were administered GSC8-11 alone, or GSC8-11+NTNK, or GSC8-11+IL-21NK cells, and the proliferation of FFluc-labeled GSC8-11 tumor cells was monitored using bioluminescence imaging (BLI). (Figure 11B) Mean radiance (BLI) data (p / sec / cm2 / sr). Error bars indicate standard error (sem). Statistical significance was determined using paired t-tests at each time point. *p≦0.05. (Figure 11C) Kaplan-Meier plots showing the survival of each group (n=5 animals). Animals treated with IL-21NK had significantly better survival than tumor controls (p=0.0163). On the other hand, there was no significant difference between NTNK treatment and tumor alone (p=0.135, log-rank test). *p≦0.05, ns=no significant difference. (Figure 11D) Time course graph of percentage change in mouse body weight in the GSC8-11 related group. (Figure 11E) Schematic diagram of another experimental timeline. NSG mice were administered GSC267 alone, or GSC267+NTNK, or GSC267+IL-21NK cells, and the proliferation of FFluc-labeled GSC267 tumors was monitored by BLI. (Figure 11F) Mean radiance (BLI) data (p / sec / cm2 / sr). Statistical significance was determined using paired t-tests at each time point. *p≦0.05. (Figure 11G) Kaplan-Meier plots showing the survival of each group (n=3-4 mice). The IL-21NK treatment group had significantly better survival than the tumor control (p=0.0091). On the other hand, there was no significant difference between NTNK and tumor controls (p=0.762, log-rank test). (Figure 11H) Graph of the percentage change in mouse body weight in the GSC267-related group. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above.

[0115] [Figure 12-1]Figures 12A-12D - NK cells appeared after GSC rechallenge in IL-21NK-treated mice. (Figure 12A) Ki-67 immunostaining images of brain sections obtained 17 days after treatment with IL-21NK in NSG mice (n=4) that were subsequently rechallenged with GSC20 (0.25 × 10⁶) 400 days prior, and naive mice (n=5, tumor-only control) that received GSC20 without IL-21NK treatment. Mouse lymph nodes were used as positive controls. Staining images from 4 mice in each group are shown. Images were obtained at 10x magnification. (Figure 12B) Quantification of Ki-67 positive and negative tumor cells in brain sections of the IL-21NK-treated group (rechallenge, n=4) and the GSC-only control group (n=5). This shows that there were fewer proliferative and non-proliferative tumor cells in the IL-21NK-treated group. Error bars indicate standard deviation. Statistical significance was determined using an unpaired t-test. *p≦0.05. (Figure 12C) Flow cytometry gating strategy for identifying NK cells in mouse brain tissue after rechallenge. GSC20 was rechallenged 400 days after IL-21NK treatment and sacrificed 17 days later. The upper panel shows representative FACS plots (n=4). Naive NSG mice (n=5) were administered GSC20 and used as tumor controls (lower panel). (Figure 12D) Quantification of human CD16-positive cells / mm2 in brain sections of the IL-21NK rechallenge group (n=4) and the GSC-alone group (n=5). Error bars indicate standard deviation. Statistical significance was determined using an unpaired t-test. **p≦0.01. [Figure 12-2] Same as above.

[0116] [Figure 13-1] Figures 13A-13B - IL-21NK cells may have remained in the brain. (Figure 13A) Human Granzyme B (GrB) immunostaining images of liver, spleen, and lung sections from mice (n=4) treated with IL-21NK and rechallenged with GSC20 400 days prior. Human tonsils were used as a positive control. Images were obtained at 10x and 20x magnification. (Figure 13B) Flow cytometry panel showing the absence of human CD45+ cells in the lungs, spleen, and bone marrow of IL-21NK-treated mice. Umbilical cord blood NK cells were used as a positive control. [Figure 13-2] Same as above.

[0117] [Figure 14] Figure 14 - Single-cell RNA sequencing profiling of IL-15NK and IL-21NK after GSC20 rechallenge. (Figure 14) Heatmap showing gene expression at the cluster level 9 days after rechallenge. Genes highlighted in red are particularly noteworthy genes that were upregulated in cluster 4 (e.g., CEBPD, CLIC3, EOMES, BIRC3, NFKBIA).

[0118] [Figure 15] Figures 15A-15B: CEBPD expression and regulon scores at the RNA level. (Figure 15A) Violin plot showing CEBPD scRNA-seq expression levels. (Figure 15B) CEBPD regulon scores for each cluster.

[0119] [Figure 16-1]Figures 16A-16F show that IL-21NK cells expressed CEBPD in vivo and in vitro. CEBPD expression in NK cells by qPCR (Figure 16A) and mean fluorescence intensity (MFI) and percentage of CD56+ and CEBPD+ cells by flow cytometry after 21 days of in vitro rechallenge with GSC20 cells (Figures 16B-16D). Error bars indicate standard deviation. Statistical significance was determined by two-way ANOVA (Bonferroni corrected). *p≦0.05, ***p≦0.001; ns = no significant difference. (Figure 16E) Flow panel of brain tissue obtained after transplanting GSC272 into NSG mice and treating with IL-15NK, IL-21NK, and NTNK. Shows CEBPD expression in IL-21NK and NTNK. Gating based on CEBPDKONK cells was used as a control. (Figure 16F) Histogram showing the proportion of CD56+CEBPD+ cells on the CD56+CD3- gate in flow cytometry of pooled brain tissue from three GSC models (GSC20, GSC8-11, GSC272, n=3-8 individuals per group). Error bars indicate standard deviation. Statistical significance was determined by one-way ANOVA (Bonferroni corrected). ns = no significant difference; *p≦0.05, ***p≦0.001. [Figure 16-2] Same as above. [Figure 16-3] Same as above.

[0120] [Figure 17-1] Figures 17A-17C - Enrichment analysis of IL-21 vs IL-15 WT responses. (Figure 17A) Higher canonical pathways differentially activated for IL-15 NK cells compared to IL-21. (Figure 17B) Predicted activity and expression of antitumor-related functions and molecules involved in NK cell signaling pathways. (Figure 17C) Network analysis of regulatory factors differentially activated or suppressed in IL-15 and IL-21 responses. Significant enrichment shown has a Z score <-2 or >2 and a BH-corrected p-value (Q-value) <0.05. Log2fold-change (Log2FC) expression values ​​from corresponding DEGs were overlaid from bulk RNA-seq, and network regulatory factors were cross-validated in both bulk and single-cell ATAC-seq. [Figure 17-2] Same as above.

[0121] [Figure 18-1] Figures 18A-18D: CRISPR / Cas9-mediated deletion of the CEBPD gene in IL-21NK cells. (Figure 18A) The CRISPR-targeted CEBPD region was PCR-amplified using genomic DNA obtained from NTNK, IL-21Cas9 control, and IL-21CEBPD-KONK cells as templates, with forward and reverse primers (SEQ ID NOs: 55, 56), and then size-separated by agarose gel electrophoresis. PCR products from two umbilical cord blood donors are shown. (Figure 18B) CEBPD expression in GSC20 and NK cells after 30 days of rechallenge (flow cytometry, MFI = mean fluorescence intensity). (Figure 18C) Time course of doubling growth in NTNK, IL-21Cas9NK, and IL-21CEBPD-KONK cells in culture with IL-2 and UAPCs. (Figure 18D) Time course of viability in culture of NTNK, IL-21Cas9NK, and IL-21CEBPD-KONK. Error bars indicate standard deviation. Statistical significance was determined by two-way ANOVA (Bonferroni corrected). ns = no significant difference; ***p ≤ 0.001. [Figure 18-2] Same as above.

[0122] [Figure 19-1]Figures 19A-19C: Deletion of CEBPD impaired the long-term cytotoxic activity of IL-21NK cells against GSCs. NTNK, IL-21Cas9NK, and IL-21CEBPD-KONK cells were co-cultured with m-cherry-transformed GSC20 (red) in an E:T ratio of 1:1. GSC20 monoculture was used as a control. Fresh GSC20 cells were added every 2-3 days (no NK cells were added). The red (tumor) signal was tracked with real-time imaging. (Figure 19A) Representative image of GSCs on day 9 of NK rechallenge. This shows that CEBPD-KO reduced the cytotoxic activity of IL-21NK cells against GSC20. Scale bar = 400 μm. (Figure 19B) Multiplexed ELISA bar graphs (n=4 donors) showing cytokine and chemokine production by NTNK (left), IL-21Cas9NK (center), and IL-21CEBPD-KONK (right) after 5 GSC20 rechallenges. Error bars indicate standard deviation. Statistical significance was determined by two-way ANOVA (Bonferroni corrected). ns = no significant difference; *p≦0.05, **p≦0.01, ***p≦0.001. (Figure 19C) Heatmap showing normalized enrichment scores (NES) of transcriptional changes (x-axis) for three interesting comparisons in the selected Hallmark pathway (y-axis). Colors indicate the direction of enrichment. Red at the top of the scale indicates pathway upregulation in CEBPDKO or overexpression (OE) NK cells, and blue at the bottom of the scale indicates upregulation in CEBPDWTNK cells. Asterisks indicate significant pathways with FDR-corrected p-values ​​< 0.05. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0123] [Figure 20]Figures 20A-20B - CEBPD knock-in (KI) efficiency. (Figure 20A) Flow panel showing CEBPD expression (OE = overexpression, or KI = knock-in) in NK cells after introduction with a retroviral vector encoding full-length CEBPD (SEQ ID NO: 19). NTNK (WT) was used as a negative control. (Figure 20B) Histogram showing CEBPD-KINK transduction efficiency 5 days after introduction (n=5 donors). Error bars indicate standard deviation. Statistical significance was determined by unpaired t-test. ***p≦0.001.

[0124] [Figure 21] Figures 21A-21C show that CEBPD-KI reduced mitochondrial-derived reactive oxygen species (ROS) production. Figure 21A shows a representative flow cytometry plot and Figure 21B shows a histogram illustrating the proportion of MitoSOX™ production (CD56+Mitosox+) in NTNK, CEBPD-KINTNK, IL-21NK, and IL-21CEBPD-KONK cells. Error bars indicate standard deviation. Statistical significance was determined by one-way ANOVA with Bonferroni correction and multiple comparisons. ns = no significant difference; *p≦0.05, **p≦0.01. Figure 21C shows that the CRISPR target STAT3 region was PCR amplified using forward primers (SEQ ID NO: 62) and reverse primers (SEQ ID NO: 63) from NK cell genomic DNA derived from two umbilical cord blood donors, and the PCR products were size-separated by agarose gel electrophoresis.

[0125] [Figure 22]Figures 22A-22B - C / EBP family and AP-1 complex gene expression in tumor-infiltrating (TI) NK cells derived from GBM patients. (Figure 22A) UMAP of NK cells obtained from peripheral blood mononuclear cells (PBMCs) of healthy controls (HC-NK) and GBM tumor-infiltrating NK cells (TI-NK) (n=10 patients). HC-NKPBMC samples were downloaded from the 10xGenomics database and contain approximately 3,000 cells. (Figure 22B) Violin plots comparing gene expression of the C / EBP family (top: CEBPA, CEBPB, CEBPD, CEBPE, CEBPG) and AP-1 complex (bottom: JUND, JUNB, FOS, FOSL1, FOSL2) between healthy control PBMCs (HC-NK) and surgical samples of highly malignant gliomas (TI-NK) from 10 patients. P values ​​are shown on each plot. Significance was calculated using an unpaired t-test.

[0126] [Figure 23-1] Figures 23A-23S - Cytokine administration improved the antitumor activity of NK cells against GSCs. (Figures 23A-23S) 3D killing assay of GSC272 spheroids using NTNK, IL-15NK, and IL-21NK cells (7 days). Images were acquired every 2 hours, with representative images shown every 4 hours for the first 24 hours, and every 12 hours thereafter. Red signals indicate proliferation of GSC272 spheroids, and green signals indicate apoptosis (caspase 3 / 7). Scale bar = 600 μm. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above. [Figure 23-5] Same as above. [Figure 23-6] Same as above. [Figure 23-7] Same as above. [Figure 23-8] Same as above. [Figure 23-9] Same as above. [Figure 23-10] Same as above.

[0127] [Figure 24-1]Figures 24A-24H show that pSTAT3 functions as a mediator of CEBPD expression in IL-21NK cells, and CEBPD was validated as a target gene. (Figure 24A) Relative expression levels of CEBPD in IL-21Cas9NK control cells compared to IL-21STAT3KONK cells by qPCR (n=3 donors). Error bars indicate standard deviation. Statistical significance was determined by unpaired t-tests. *p≦0.05. (Figure 24B) After chromatin immunoprecipitation (ChIP) of pSTAT3, CEBPD was analyzed by IgG normalization using qPCR (n=3 donors). CEBPD-BS (CEBPD binding site): Primer for the STAT3 binding site in the promoter region (SEQ ID NOs: 57-58). CEBPD-NC (CEBPD negative control): Primer designed for the region where the STAT3 binding site is absent. Error bars indicate standard deviation. Statistical significance was determined by two-way ANOVA with Bonferroni correction for multiple comparisons. ***p≦0.001. (Figure 24C) Barcode enrichment plot for CEBPD downstream genes; all genes in the dataset are ranked on the x-axis based on enrichment score (y-axis), and the rank of each gene in the signature is displayed on the x-axis. Significance was assessed by rank-based tests, and the enrichment score (ES) reflected the degree of overexpression at both ends of the gene set list using Kolmogorov-Smirnov similarity statistics. Next, the statistical significance of the ES was determined by permutation tests based on phenotypic labels, a null distribution was generated for comparison, and the dependence of the gene set on these labels was confirmed (n=4 donors). (Figure 24D) Heatmap showing mean log2 fold change in the two comparisons (left: NT CEBPDOE vs NT, right: IL-21 CEBPD KO vs IL-21). The list consists of genes in the CEBPD downstream signaling pathway that have been hierarchically clustered (n=4 donors). (Figure 24E) CEBPD occupancy of CEBPD-specific regulon target genes (KLF2, BNIP3L, IRF1, ETS1) in IL-21NK vs NTNK (n=2 donors). Profile plot of CEBPDChIP-seq normalized tag around the promoter region (-300 to +100 bp) of the transcription start site (TSS).(Figure 24F) Box plot of normalized tag density of CEBPDChIP-seq at target gene sites. Statistical significance was determined by unpaired two-sided t-tests between NT and IL-21NK. (Figure 24G) Relative expression levels of KLF2 (left) and BNIP3L (right) by qPCR in IL-21Cas9NK control and IL-21CEBPDKONK cells (n=4 donors). Error bars indicate standard deviation. Statistical significance was determined by unpaired t-tests. *p≦0.05, ***p≦0.001. (Figure 24H) Schematic diagram illustrating how IL-21 induces CEBPD, which in turn regulates downstream target genes (e.g., KLF2, BNIP3L) in NK cells. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above. [Figure 24-5] Same as above.

[0128] [Figure 25-1]Figures 25A-25J - Injection of IL-15NK cells with low transduction efficiency (low IL-15 production) into the brain increased NK infiltration and gliosis. GSC20 was transplanted into orthotopic NSG mice and treated with NT, low transduction IL-15NK cells, and IL-21NK cells. After 2 weeks, brain tissue was collected and analyzed by flow cytometry and IHC staining. (Figure 25A) NK cells were grown for 5 days in the presence of uAPC feeder cells and IL-2, and then transduced with a low dose of retroviral vector encoding IL-15. (Figure 25B) Flow cytometry plot showing a large amount of NK infiltration in the brain of the low IL-15 transduction / expression NK cell group compared to the NT and IL-21NK treatment groups. (Figure 25C) Absolute number of hCD45+CD56+CD3-(NK cells) in brain tissue (n=4-5 mice per group). Error bars indicate standard deviation. Statistical significance was determined by one-way ANOVA with Bonferroni correction and multiple comparisons. *p≦0.05; ns = no significant difference. (Figure 25D) Representative images of immunohistochemical staining for human Granzyme B, GFAP, and Iba1 in brain sections of NSG mice transplanted with orthotopically transplanted GSC20 cells treated with low-introduction IL-15NK (n=3 mice), IL-21NK (n=3 mice), and NTNK (n=3 mice). NSG mice transplanted with GSC20 alone (n=3) were used as a control. (Figure 25E) Number of Granzyme B-positive cells / mm2 in brain sections of NTNK, IL-15NK, and IL-21NK treated mice (n=3 mice in each group). Error bars indicate standard deviation. Statistical significance was determined by one-way ANOVA with Bonferroni correction and multiple comparisons. ***p≦0.001. (Figure 25F) Number of GFAP-positive cells / mm2 in brain sections taken from NSG mice transplanted with GSC20 cells on orthotopic and treated with IL-15NK, IL-21NK, or NTNK cells (n=3 mice per group). Statistical significance was determined by one-way ANOVA with Bonferroni correction for multiple comparisons. *p≦0.05. (Figure 25G) Number of Iba1-positive cells / mm2 in brain sections taken from NSG mice transplanted with GSC20 cells on orthotopic and treated with IL-15NK, IL-21NK, or NTNK cells (n=3 mice per group).(Figures 25H, 25I) Multiplex immunofluorescence images of mouse brain tissue treated with low-dose IL-15NK and IL-21NK two weeks after GSC injection (Iba1: light blue, luciferase: green, Granzyme B: yellow, Caspase-3: red, Ki-67: orange, GFAP: purple, DAPI: dark blue). Images were obtained at 4x and 20x magnification. The white arrow in Figure 25H indicates NK (Granzyme B) in contact with microglia (Iba1). (Figure 25J) Representative immunofluorescence image of green tumor cell signal (GSC20 luciferase) in mouse brain treated with low-dose IL-15NK and IL-21NK. GSC20-only transplanted mice were used as a control. Images were obtained at 2x magnification. [Figure 25-2] Same as above. [Figure 25-3] Same as above. [Figure 25-4] Same as above. [Figure 25-5] Same as above. [Figure 25-6] Same as above. [Modes for carrying out the invention]

[0129] Detailed explanation In accordance with long-standing patent law practice, in this specification, when used in conjunction with the word "comprising" to include the claims, the words "a" and "an" mean "one or more." Some embodiments of this disclosure may consist of, or essentially consist of, one or more elements, method steps, and / or methods of this disclosure. Any method or composition described herein may be carried out in relation to any other method or composition described herein, and different embodiments may be combined.

[0130] Throughout this specification, unless otherwise specified in the context, the terms “comprise,” “comprises,” and “comprising” are understood to mean including the steps or elements or groups of steps or elements described, but not to mean excluding other steps or elements or groups of steps or elements. “Consists of” means including and being limited to what follows the phrase “consists of.” Thus, the expression “consists of” indicates that the enumerated elements are required or essential, and other elements may not be present. “Essentially consists of” means including the elements enumerated after the phrase, and being limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the enumerated elements. Thus, the expression “essentially consists of” indicates that the enumerated elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the enumerated elements.

[0131] Throughout this specification, any reference to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “certain embodiment,” “another embodiment,” or “further embodiment,” or any combination thereof, means that any particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, although the aforementioned terms appear in various places throughout this specification, they do not necessarily all refer to the same embodiment. Furthermore, any particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0132] As used herein, the terms “or” and “and / or” are used to describe multiple components in combination or mutually exclusive. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is particularly intended that x, y, or z may be excluded from embodiments.

[0133] Throughout this application, the term “about” is used in accordance with its plain and common meaning in the fields of cell biology and molecular biology, indicating that the value includes the standard deviation of error in the apparatus or method employed to determine that value.

[0134] As used herein, the terms “CD3 receptor complex” or “CD3 coreceptor complex” refer to the protein complex that acts as a T cell coreceptor in nature and is composed of a CD3ζ chain, a CD3γ chain, a CD3δ chain, and two CD3ε chains (although alternatives use only one CD3ε chain).

[0135] As used herein, the term “engineered” refers to human-made entities, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, the engineered entities are synthetic and comprise elements that do not exist in nature or are not constructed in the manner utilized herein. In specific embodiments, vectors are engineered by recombinant nucleic acid technology, and cells are engineered by transfection or transduction of the engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cell because the heterologous protein and / or the transcript encoding it are recombinant or synthetic, or because the cell does not naturally express the protein, or to overexpress heterologous proteins that could naturally be expressed by the cell.

[0136] The phrase "pharmaceutically or pharmacologically acceptable" means, as appropriate, molecular entities and compositions that, when administered to animals such as humans, do not cause adverse reactions, allergic reactions, or other adverse reactions. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients will be known to those skilled in the art in light of this disclosure. Furthermore, it will be understood that when administered to animals (e.g., humans), preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA's Office of Biological Standards.

[0137] As used herein, “pharmaceutically acceptable carriers” include any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline, sodium chloride, ringer's dextrose, and other parenteral vehicles), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as etyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, body fluids, and nutritional supplements, of which such materials and combinations thereof will be known to those skilled in the art. The pH and precise concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0138] As used herein, the term “subject” generally refers to an individual having or suspected of having cancer. A subject may be any living organism or animal subject that is the subject of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject may be a patient who has or is suspected of having a disease (sometimes called a medical condition), such as a benign or malignant neoplasm or cancer. A subject may be undergoing or have previously undergone treatment. A subject may be asymptomatic. A subject may be a healthy individual who desires cancer prevention. The term “individual” is used interchangeably, at least in some cases. As used herein, “subject” or “individual” may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. An individual may receive “one or more medical compositions” via the internet. Individuals may include all ages of human or non-human animals, and therefore include both adults and adolescents (i.e., children) and infants, as well as individuals in utero. Since this term does not imply a need for medical treatment, individuals may participate in experiments voluntarily or involuntarily, whether clinical or in support of basic scientific research.

[0139] As used herein, “treatment” or “procedure” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally include either the alleviation or improvement of one or more symptoms of a disease or condition, or the delay of the progression of a disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of a disease or condition, or any symptoms associated therewith.

[0140] I. Embodiments of Disclosure Natural killer (NK) cells are innate immune cells with potent antitumor activity. While NK cells have been reported to exhibit adaptive memory-like responses to previously encountered viruses, the factors that facilitate memory against cancer remain unexplained and unclear. This specification presents data and analysis results demonstrating that NK cells engineered to express IL-21 (IL-21 NK) acquire memory-like characteristics and possess the ability to respond to glioblastoma (GBM) rechallenge in vitro and in vivo. Furthermore, using a single-cell multi-omics platform, it is shown that IL-21 NK cells possess a unique signature in chromatin accessibility, particularly revealing that CCAAT / enhancer-binding proteins (C / EBP, or CEBP), including CEBPD, function as key transcription factors regulating the memory response of NK cells. CEBPD deletion resulted in a loss of IL-21 NK cell efficacy, while CEBPD overexpression in NK cells enhanced their long-term cytotoxic activity, metabolic fitness, and in vivo anti-GBM effects. The results presented herein indicate that IL-21 epigenetically reprograms NK cells to acquire antitumor immunological memory. Furthermore, the results presented herein demonstrate that this antitumor immunological memory is highly dependent on members of the C / EBP family, particularly CEBPD, and that CEBPD overexpression is sufficient to reproduce IL-21-mediated phenotypes such as long-term cytotoxic activity, metabolic fitness, and anti-cancer (e.g., anti-GBM) effects. Accordingly, in some embodiments, compositions and methods are provided herein that include NK cells engineered to overexpress one or more CEBP family protein members.

[0141] Until very recently, NK cells were thought to lack the memory properties characteristic of adaptive immune cells such as T cells and B cells. However, in recent years, this conventional theory has been overturned, and memory-like responses of NK cells to haptens and viruses, as well as memory-like responses of NK cells to haptens and viruses after exposure to inflammatory cytokines such as (IL)-12, IL-15, and IL-18, have been reported. While NK subsets exhibiting memory-like characteristics after cytomegalovirus (CMV) infection in mice and humans have been reported, there is no evidence that such subsets exist after tumor attack, and the molecular mechanisms controlling the memory response to cancer remain unclear.

[0142] The efficacy and in vivo persistence of NK cells against cancer can be enhanced by cytokine armament, and many translational and clinical studies have focused on IL-15. IL-15 is released from numerous cell types, including macrophages and dendritic cells, and promotes the cytotoxic activity and proliferation of NK cells. Multiple preclinical and early clinical trials support the overall safety and promising activity of IL-15-armed NK cells.

[0143] Another attractive cytokine for cancer immunotherapy is IL-21, which induces metabolic reprogramming and mitochondrial regeneration in T cells and activates CD4 + IL-21 is produced by multiple immune cells, including T cells and natural killer T (NKT) cells. IL-21 also promotes NK cell maturation and metabolic adaptability, and modulates NK cell function. Recombinant IL-21 has been studied in several metastatic cancer clinical trials and has shown an acceptable safety profile; however, its short half-life and the need for repeated administration have significantly limited its clinical application. Therefore, these data indicate that IL-15 and IL-21 play crucial roles in regulating diverse NK cell functions and support cytokine manipulation as a strategy to enhance the antitumor effects of NK cells.

[0144] As described herein, multiple direct-link models of patient-derived glioblastoma stem cells (GSCs) were used to compare the antitumor activity of engineered IL-21-equipped NK cells (IL-21NK) and engineered IL-15-equipped NK cells (IL-15NK). The data presented herein showed that engineered IL-21NK cells exhibited superior antitumor activity with memory-like features compared to IL-15NK cells and / or unequipped NK cells, and were associated with distinctly different transcriptional and epigenetic signatures. Furthermore, the data presented herein were used to identify the C / EBP family transcription factor (TF) and activating protein 1 (AP-1) complex as key regulators of NK cell memory. Surprisingly, deletion of CEBPD, which the inventors found to be the most differentially expressed TF in IL-21NK cells, impaired the potency and restorative response of IL-21NK cells, while overexpression of CEBPD in wild-type NK cells enhanced the functional capacity and metabolic fitness of NK cells. Finally, as shown herein, the inventors confirmed the expression of CEBPD in tumor-infiltrating NK cells derived from GBM patients. These data identify the CEBP protein family, particularly CEBPD, as important transcriptional and epigenetic regulators of NK cell memory against cancer. Furthermore, these data support the idea that NK cell armament by IL-21 (cytokine armament) and / or NK cell armament by overexpression of CEBP family proteins represents a novel immunotherapeutic approach to cancer treatment, including GBM.

[0145] This disclosure relates, in particular, to NK cells that have been modified to enhance their function as immunotherapies compared to unmodified NK cells. This modification allows NK cells to have greater versatility and functionality when used alone or in combination with other therapeutic agents.

[0146] II. Compositions Disclosed This disclosure relates to compositions and methods comprising engineered NK cells modified to overexpress and / or upregulate the activity of at least one or more members of the CCAAT / enhancer-binding protein (CEBP) family. In some embodiments, engineered NK cells may be transgenically modified to overexpress nucleic acids encoding members of the CEBP protein family. In some embodiments, engineered NK cells transgenicly express and / or be affected by activators of transcription and / or translation of CEBP proteins. In some embodiments, engineered NK cells transgenicly express and / or be affected by inhibitors of transcription and / or translation of CEBP proteins.

[0147] A. Modification of NK cells In certain embodiments, the compositions disclosed herein include engineered NK cells that have been artificially modified to overexpress some or all of the CEBP protein.

[0148] In one embodiment, engineered natural killer (NK) cells are modified to overexpress CEBP proteins compared to unengineered NK cells and / or NK cells engineered to express IL-15. In one embodiment, the CEBP proteins are CEBPA (CEBP-alpha, CEBPα), CEBPB (CEBP-beta, CEBPβ), CEBPG (CEBP-gamma, CEBPγ), CEBPD (CEBP-delta, CEBPδ), CEBPE (CEBP-epsilon, CEBPε), and / or CEBP homolog protein (CHOP). In one embodiment, the CEBP protein is CEBPD. In one embodiment, the CEBP protein is CEBPB. In one embodiment, the CEBP proteins are CEBPD and CEBPB. In one embodiment, engineered natural killer cells are modified to express heterologous CEBP transcription factor family proteins (e.g., overexpressing CEBP transcription factor family proteins that are not normally expressed, or that are naturally expressed at relatively low levels).

[0149] If a portion of the CEBP protein is utilized, that portion may consist of at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids, and may include consecutive and / or discontinuous amino acids. In certain embodiments, the CEBP protein may contain at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the amino acids of the wild-type CEBP protein.

[0150] In one embodiment, the CEBP protein and / or the transcript encoding the CEBP protein are overexpressed at least 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, or more than 10 times, and within any range thereof, compared to control cells. In one embodiment, the CEBP protein and / or the transcript encoding the CEBP protein are overexpressed at least 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, or more than 10 times, and within any range thereof, compared to unmanipulated cells. In one embodiment, the CEBP protein and / or the transcript encoding the CEBP protein are overexpressed at least 1.0-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3.0-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4.0-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5.0-fold, 5.25-fold, 5.5-fold, 5.75-fold, 6.0-fold, 6.25-fold, 6.5-fold, 6.75-fold, 7.0-fold, 7.25-fold, 7.5-fold, 7.75-fold, 8.0-fold, 8.25-fold, 8.5-fold, 8.75-fold, 9.0-fold, 9.25-fold, 9.5-fold, 9.75-fold, 10.0-fold, or more than 10-fold, or within any range thereof, compared to IL-15 expressing cells.

[0151] In certain cases, any of the sequences included herein may be used to modify NK cells, while in other cases, sequences homologous to them may be used. For example, related sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences included herein may be used in this disclosure.

[0152] In specific cases, one or more vectors used for transduction of NK cells may or may not be multicistronic, as they can ultimately produce two or more distinct polypeptides. If one or more multicistronic vectors are employed, they may utilize one or more internal ribosome entry sites (IRESs) and / or one or more 2A self-cleaving peptide sites. If one or more 2A sequences are utilized, the GSG is an optional linker.

[0153] T2A (GSG) EGRGSLLTCGDVEENPGP (Sequence ID 1)

[0154] P2A (GSG) ATNFSLLKQAGDVEENPGP (Sequence ID 2)

[0155] E2A (GSG) QCTNYALLKLAGDVESNPGP (Sequence ID 3)

[0156] F2A (GSG) VKQTLNFDLLKLAGDVESNPGP(Sequence ID 24)

[0157] In situations where multiple protein components are expressed from a multicistronic vector, the 5'-to-3' order on the polynucleotide vector can be any order; however, in other embodiments, they may be present on the vector in a specific order. A multicistronic vector can express multiple heterologous proteins. In certain embodiments, a multicistronic vector may include one or more marker proteins (e.g., fluorescent tags) and one or more functional proteins (e.g., CEBP proteins, cytokines, engineered antigen receptors).

[0158] In certain embodiments, the engineered natural killer (NK) cells described herein are modified to acquire, or have the ability to acquire, memory (e.g., immunological memory) and / or memory-like phenotypes toward a target (e.g., an antigen, cell, cancer cell, virus, amoeba, parasite, etc.). In certain embodiments, the engineered NK cells acquire memory and / or memory-like phenotypes directed toward the target, and as a result, the NK cells have at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 days after exposure to the target. , 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st , 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th Day, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, 144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th,180 days, 181 days, 182 days, 183 days, 184 days, 185 days, 186 days, 187 days, 188 days, 189 days, 190 days, 191 days, 192 days, 193 days, 194 days, 195 days, 196 days, 197 days, 198 days, 199 days, 200 days, 201 days, 202 days, 203 days, 204 days, 205th, 206th, 207th, 208th, 209th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 218th, 219th, 220th, 221st, 222nd, 223rd, 224th, 225th, 226th, 227th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 255th, 256th, 257th, 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 277th, 278th, 279th, 280 days, 281 days, 282 days, 283 days, 284 days, 285 days, 286 days, 287 days, 288 days, 289 days, 290 days, 291 days, 292 days, 293 days, 294 days, 295 days, 296 days, 297 days, 298 days, 299 days, 300 days, 301 days, 302 days, 303 days, 304 days, 305th, 306th, 307th, 308th, 309th, 310th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 319th, 320th, 321st, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th, 336th, 337th, 338th, 339th, 340th, 341st, 342nd, 343rd, 344th, 345th, 346th, 347th, 348th, 349th, 350th, 351st, 352nd, 353rd, 354th, 355 days, 356 days, 357 days, 358 days, 359 days, 360 days, 361 days, 362 days, 363 days, 364 days, 365 days, 366 days, 367 days, 368 days, 369 days, 370 days, 371 days, 372 days, 373 days, 374 days, 375 days, 376 days, 377 days, 378 days, 379 days,The target can be recognized and activated over a period of 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400 days, or more than 400 days. In certain embodiments, manipulated NK cells remain active for at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 days after target clearance. 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th, 98th, 99th Day, 100th day, 101st day, 102nd day, 103rd day, 104th day, 105th day, 106th day, 107th day, 108th day, 109th day, 110th day, 111th day, 112th day, 113th day, 114th day, 115th day, 116th day, 117th day, 118th day, 119th day, 120th day, 121st day, 122nd day, 123rd day, 124th day, 125th day, 126th day, 127th day, 128th day, 129th day, 130th day, 131st day, 132nd day, 133rd day, 134th day, 135th day, 136th day, 137th day, 138th day, 139th day, 140th day, 141st day, 1 42nd, 143rd, 144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th, 181st, 182nd, 183rd, 184th,185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 194th, 195th, 196th, 197th, 198th, 199th, 200th, 201st, 202nd, 203rd, 204th, 205th, 206th, 207th, 208th, 209th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 218th, 219th, 220th, 221st, 222nd, 223rd, 224th, 225th, 226th, 227th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 255th, 256th, 257th, 258th, 259th, 260 days, 261 days, 262 days, 263 days, 264 days, 265 days, 266 days, 267 days, 268 days, 269 days, 270 days, 271 days, 272 days, 273 days, 274 days, 275 days, 276 days, 277 days, 278 days, 279 days, 280 days, 281 days, 282 days, 283 days, 284 days, 285th, 286th, 287th, 288th, 289th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 306th, 307th, 308th, 309th, 310th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 319th, 320th, 321st, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th, 336th, 337th, 338th, 339th, 340th, 341st, 342nd, 343rd, 344th, 345th, 346th, 347th, 348th, 349th, 350th, 351st, 352nd, 353rd, 354th, 355th, 356th, 357th, 358th, 359th, 360 days, 361 days, 362 days, 363 days, 364 days, 365 days, 366 days, 367 days, 368 days, 369 days, 370 days, 371 days, 372 days, 373 days, 374 days, 375 days, 376 days, 377 days, 378 days, 379 days, 380 days, 381 days, 382 days, 383 days, 384 days,Over a period of 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400 days, or more than 400 days, the system acquires memories and / or memory-like phenotypes that enable the recognition and activation of the target.

[0159] In certain embodiments, manipulated NK cells acquire memory and / or memory-like phenotypes directed towards the target, and as a result, these NK cells remain active for at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, 74th, 75th, 76th Day 77, Day 78, Day 79, Day 80, Day 81, Day 82, Day 83, Day 84, Day 85, Day 86, Day 87, Day 88, Day 89, Day 90, Day 91, Day 92, Day 93, Day 94, Day 95, Day 96, Day 97, Day 98, Day 99, Day 100, Day 101, Day 102, Day 103, Day 104, Day 105, Day 1 06th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, 144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 169th, 170th, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th, 181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 194th, 195th, 196th, 197th, 198th, 199th, 200th, 201st, 202nd, 203rd, 204th, 205th,206th, 207th, 208th, 209th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 218th, 219th, 220th, 221st, 222nd, 223rd, 224th, 225th, 226th, 227th, 228th, 229th, 230th , 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 251st, 252nd, 253rd, 254th, 255 256th, 257th, 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 277th, 278th, 279th, 28 Day 0, Day 281, Day 282, Day 283, Day 284, Day 285, Day 286, Day 287, Day 288, Day 289, Day 290, Day 291, Day 292, Day 293, Day 294, Day 295, Day 296, Day 297, Day 298, Day 299, Day 300, Day 301, Day 302, Day 303, Day 304, Day 3 5th, 306th, 307th, 308th, 309th, 310th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 319th, 320th, 321st, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330 days, 331 days, 332 days, 333 days, 334 days, 335 days, 336 days, 337 days, 338 days, 339 days, 340 days, 341 days, 342 days, 343 days, 344 days, 345 days, 346 days, 347 days, 348 days, 349 days, 350 days, 351 days, 352 days, 353 days, 354 days , over a period of 355 days, 356 days, 357 days, 358 days, 359 days, 360 days, 361 days, 362 days, 363 days, 364 days, 365 days, 366 days, 367 days, 368 days, 369 days, 370 days, 371 days, 372 days, 373 days, 374 days, 375 days, 376 days, 377 days, 378 days, 379 days, 380 days, 381 days, 382 days, 383 days, 384 days, 385 days, 386 days, 387 days, 388 days, 389 days, 390 days, 391 days, 392 days, 393 days, 394 days, 395 days, 396 days, 397 days, 398 days, 399 days, 400 days, or over a period of more than 400 days,It can provide immunity against the target in question.

[0160] In certain embodiments, NK cells engineered to overexpress and / or upregulate the activity of one or more members of the CEBP family can be obtained from any suitable source, including fresh or frozen. In certain embodiments, the NK cells are not obtained from iPSC differentiation. In certain embodiments, NK cells can be obtained from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte apheresis products (PBSCs), NK cell lines (e.g., NK-92), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known to those skilled in the art. Specifically, NK cells can be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, NK cell lines, or mixtures thereof. In certain embodiments, NK cells are isolated from mixed CBs. CBs can be pooled from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more units. The NK cells can be autologous or allogeneic to the recipient. Isolated NK cells may or may not have a haplotype match with the subject. NK cells can be detected by specific cell surface markers, such as CD16 and CD56 in humans. In some cases, the source of NK cells is umbilical cord blood, where NK cells are contained within a heterogeneous mixed cell population, and specific cells expressing CD3 can be removed. Alternatively, NK cells can be induced by isolating CD34+ cells from umbilical cord blood.

[0161] NK cells may be pre-activated by one or more inflammatory cytokines, and may be proliferated or non-proliferated. In some cases, NK cells are pre-activated before or after modification to overexpress and / or upregulate the activity of one or more members of the CEBP family. In certain embodiments, pre-activation of NK cells includes culturing isolated NK cells in the presence of one or more cytokines. NK cells may be stimulated by IL-2, or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, etc.). In certain embodiments, the pre-activating cytokines are selected from IL-12, IL-15, IL-18, and combinations thereof. One or more additional cytokines may be used in the pre-activation step. Pre-activation may be short in duration, for example, 5 to 72 hours, 10 to 50 hours, particularly 10 to 20 hours, for example, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, specifically about 16 hours. Pre-activated cultures may contain IL-12 at concentrations of 0.1–150 ng / mL, e.g., 0.5–50 ng / mL, particularly 1–20 ng / mL, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, specifically about 10 ng / mL. Pre-activated cultures may contain IL-18 and / or IL-15 at concentrations of 10–100 ng / mL, e.g., 40–60 ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, specifically about 50 ng / mL.

[0162] In some cases, NK cells are proliferated before and / or after modification to overexpress and / or upregulate the activity of one or more members of the CEBP family. Pre-activated NK cells may be proliferated in the presence of artificial antigen-presenting cells (aAPCs) and / or feeders / fragments, or NK-activating beads. Pre-activated NK cells may be washed before proliferation, for example, two, three, four, or five times, particularly three times. aAPCs may be engineered to express CD137 ligand and / or membrane-bound cytokines. Membrane-bound cytokines may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, aAPCs are engineered to express CD137 ligand and mIL-21. aAPCs may be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous HLA class I, II, or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58). In particular, aAPC may be K562 cells, for example, K562 cells engineered to express CD137 ligand and mRNA-21. aAPC may be irradiated. In some embodiments, fragments of APC may be used for NK cell proliferation. The operation may be carried out by any method well known to those skilled in the art, for example, by retroviral transduction. Retroviral transduction may be carried out at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after co-culture of NK cells with antigen-presenting cells. In some embodiments, retroviral transduction includes co-transduction of one or more constructs. In some embodiments, retroviral transduction is carried out after co-culture with antigen-presenting cells or about 5 days later. In one embodiment, co-culture with antigen-presenting cells continues after the introduction of NK cells. Growth can last approximately 2 to 30 days, for example 3 to 20 days, particularly 12 to 16 days, for example 12, 13, 14, 15, 16, 17, 18, or 19 days, specifically about 14 days. Pre-activated NK cells and aAPCs may be present in a ratio of approximately 3:1 to 1:3, for example 2:1, 1:1, 1:2, particularly about 1:2.The growth culture may further contain growth-promoting cytokines, such as IL-2. IL-2 may be present at concentrations of approximately 10–500 U / mL, for example, 100–300 U / mL, and particularly around 200 U / mL. IL-2 can be replenished in the growth culture, for example, every 2–3 days. aAPC can be added to the culture at least twice, for example, on day 7 of growth.

[0163] In certain embodiments, NK cells may be transfected or transduction with one or more membrane-bound cytokines, such as IL-21, IL-12, IL-18, IL-23, IL-7, or IL-15, either secreted by the NK cells or bound to the NK cell membrane. In such cases, the membrane-bound cytokines may be bound to the NK cell membrane by specific transmembrane domains, such as the transmembrane domains of CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, and DAP12. In certain embodiments, the cytokines are bound to the NK cell membrane together with polypeptides containing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical sequences to the peptide represented and / or encoded by any of SEQ ID NOs. 5-13. In certain embodiments, the cytokine is fused to the extracellular domain of a polypeptide containing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical sequences to the peptide represented and / or encoded by any of SEQ ID NOs. 13-14. In certain embodiments, the extracellular domain of the polypeptide may be the N-terminus or C-terminus of the cytokine. In certain embodiments, the cytokine comprises a signal peptide fused to the N-terminal domain, the signal peptide containing at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical sequences to the peptide represented and / or encoded by any of SEQ ID NOs. 15-18. Transmembrane domain amino acid sequence derived from SEQ ID NO: 5-CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 5) Transmembrane domain nucleic acid sequence derived from SEQ ID NO: 6-CD28 TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 6) Transmembrane domain amino acid sequence derived from SEQ ID NO: 7-CD19 VSAVTLAYLIFCLCSLVGILHL (SEQ ID NO: 7) Transmembrane domain nucleic acid sequence derived from SEQ ID NO: 8-CD19 GTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATTCTTCATCTT (SEQ ID NO: 8) Transmembrane domain amino acid sequence derived from SEQ ID NO: 9-CD8 IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 9) Transmembrane domain nucleic acid sequence derived from SEQ ID NO: 10-CD8 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTG (SEQ ID NO: 10) Transmembrane domain amino acid sequence derived from SEQ ID NO: 11-CD4 MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 11) Transmembrane domain nucleic acid sequence derived from SEQ ID NO: 12-CD4 ATGGCCCTGATCGTGCTGGGCGGAGTGGCTGGCCTGCTGCTGTTTATCGGCCTGGGCATATTCTTT (SEQ ID NO: 12) Extracellular domain amino acid sequence derived from SEQ ID NO: 13-CD8 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 13) Extracellular domain nucleic acid sequence derived from sequence number 14-CD8 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 14) SEQ ID NO: 15-GMCSF-R signal peptide amino acid sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 15) Sequence ID 16 - GMCSF-R signal peptide nucleic acid sequence ATGCTGCTGCTCGTGACCTCTCTGCTGCTGTGCGAGCTGCCCCACCCTGCCTTTCTGCTGATCCCT (SEQ ID NO: 16) SEQ ID NO: 17 - Exemplary signal peptide amino acid sequence EFGLSWLFLVAILKGVQCSR (SEQ ID NO: 17) SEQ ID NO: 18 - Exemplary signal peptide nucleic acid sequence GAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGA (SEQ ID NO: 18)

[0164] After preparation, the manipulated NK cells (including, in certain embodiments, one or more monospecific, bispecific, or multispecific antibodies in an effective amount) may be administered immediately or stored by cryopreservation. In some cases, when using cryopreserved NK cells, they may have been inactivated with an inactivating agent (e.g., kinase inhibitors, dasatinib, nilotinib, rapamycin, etc.) before cryopreservation. In some embodiments, the cells may be passaged ex vivo as a bulk population for several days, weeks, or months and may be prepared within approximately 1, 2, 3, 4, or 5 days.

[0165] C. Loading into NK cells In certain embodiments, engineered NK cells are loaded with an antibody before use. NK cell loading may be performed by any specific method, such as during culture or immediately before administration, to form a complex with the antibody. The conditions should be appropriate enough for an effective amount of antibody to bind to the NK cell surface. When using a monospecific antibody, the Fc region of the monospecific antibody binds to the NK cell, and the antigen-binding domain of the antibody is free to bind to the target antigen. In certain embodiments using a multispecific antibody, one or more antigen-binding domains of the antibody bind to the NK cell surface via an antigen on the NK cell surface (e.g., CD3, CD32, CD64, KIRs, etc., but not limited to examples), and other antigen-binding domains are free to bind to the target antigen. In certain embodiments using a multispecific antibody, one or more antigen-binding domains of the antibody may bind to one or more target antigens.

[0166] The culture conditions under which NK cells are loaded may or may not be of a specific type with specific parameters. In certain embodiments, loading is performed under a specific temperature in culture, for example, 37°C, but in other embodiments, it may be 36°C or 38°C, or lower or higher. The duration of the loading process is flexible and may range from 1 minute to 24 hours or more. For example, it may be 1 minute to 24 hours, 1 minute to 18 hours, 1 minute to 12 hours, 1 minute to 6 hours, 1 minute to 1 hour, 30 minutes to 24 hours, 30 minutes to 18 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 1 hour, 1 to 24 hours, 1 to 18 hours, 1 to 12 hours, 1 to 6 hours, 6 to 24 hours, 6 to 18 hours, 6 to 12 hours, 12 to 24 hours, 12 to 18 hours, or 18 to 24 hours. In some embodiments, the loading time may be approximately 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours or longer, or within that range. In certain embodiments, the culture medium is a basal medium or a compound medium. In some cases, the culture may contain one or more of the reagents used in the pre-activation and / or growth steps, but in other cases, it may not. In certain embodiments, the culture contains one or more cytokines (e.g., one or more of IL-12, IL-15, IL-2, IL-18). In some cases, the culture contains any type of APC.

[0167] In certain embodiments, an antibody from the composition described herein is brought into contact with an effective amount of NK cells of the disclosure in an effective amount to form a “chimeric antigen receptor-like” complex. Specifically, the antigen-binding domain of the antibody binds to the NK cell via an antigen, which is a cell surface protein. Multiple antibodies may be brought into contact with multiple NK cells to form a large number of cell / antibody complexes. The antibody may be monospecific, bispecific, or multispecific, and in certain cases (such as an engager in the art, i.e., a fusion protein consisting of two single-chain variable regions (scFv) derived from different antibodies), the antibody binds to both the NK cell and the target antigen via its antigen-binding domain. In the case of a monospecific antibody, the antigen-binding domain of the antibody binds to the target antigen (e.g., a cancer antigen), and another part of the antibody (e.g., the Fc region) binds to the NK cell. If the antibody is multispecific, one or more antigen-binding domains of the antibody bind to the NK cell (which may be a naturally expressed antigen or a transgene-introduced antigen, e.g., CD3), and one or more antigen-binding domains further bind to one or more target antigens. Polyspecific antibodies can be, for example, bispecific, triplicate, or quadruplicate. If an antibody is triplicate or quadruplicate, additional antigen-binding domains may be able to bind to other cells, such as stem cells.

[0168] In certain embodiments, the antibody can enhance the response and specificity to various tumors by binding to any NK cell surface antigen (which may or may not be a receptor) on NK cells, such as CD16 (including CD16a or CD16b), CD32, CD56, CD64, type C lectins such as NKG2D and NKG2C, costimulatory molecules such as CS1, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR, and redirecting NK cells to target them. In certain embodiments, the antibody may also bind to a transgenic NK cell surface antigen, such as CD3.

[0169] In some embodiments, the antibody may bind to any suitable antigen (e.g., the antigens described herein). In certain embodiments, the antibody targets CD19. In certain embodiments, the antibody targets CD20. In certain embodiments, the antibody targets CD123. In certain embodiments, the antibody targets EGFR. In certain embodiments, the antibody targets EGFR2.

[0170] In certain embodiments, the preparation of loaded NK cells may be carried out by any suitable means that provide sufficient conditions for the appropriate region of the antibody to bind to the appropriate surface region of the NK cell. In certain cases, the culture medium may be any, specifically Plasmalight A and / or human serum albumin, but may or may not be used. After the complex is formed in culture, it may or may not be washed before administration (e.g., infusion). In another embodiment, the NK cells and antibody are administered separately, and the complex is formed in vivo.

[0171] D. Pre-activation In some embodiments, NK cells are pre-activated before administration to the recipient. The pre-activation step may or may not occur before the proliferation step. In certain embodiments, NK cells are pre-activated with one or more cytokines, and in certain embodiments, NK cells are pre-activated with one or more, e.g., two, three, or more, of IL-12, IL-15, IL-2, and IL-18. If all three IL-12, IL-15, IL-2, and IL-18 are not used, then IL-12 and IL-15 may be used without IL-18, or IL-12 and IL-18 may be used without IL-15, or IL-15 and IL-18 may be used without IL-12. IL-2 may or may not be a substitute for IL-15.

[0172] In certain embodiments, the pre-activating cytokines are IL-12, IL-15, and IL-18. One or more additional cytokines may be used in the pre-activation step. Pre-activation may be carried out for a short period of time, from 5 to 72 hours, for example, 10 to 50 hours, particularly 10 to 20 hours, for example, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, and in some cases particularly about 16 hours. The pre-activation culture may contain IL-18 and / or IL-15 at concentrations of 10 to 100 ng / mL, for example, 40 to 60 ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, specifically at a concentration of about 50 ng / mL. In some cases, the pre-activation culture may contain IL-12 at concentrations of 0.1–150 ng / mL, e.g., 1–20 ng / mL, e.g., 10 ng / mL. In another embodiment, NK cells may be stimulated with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-21, etc.), which may be used in addition to, or as a substitute for, IL-12, IL-15, and IL-18. In such cases, the pre-activation culture may contain IL-12 at concentrations of 0.1–150 ng / mL, e.g., 0.5–50 ng / mL, particularly 1–20 ng / mL, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, specifically about 10 ng / mL.

[0173] E. Proliferation In certain embodiments, NK cells are proliferated to increase their quantity before administration to the target individual. The proliferating cells may be derived from pre-activated NK cells, in which case a pre-activation step is performed before the proliferation step. The NK cell proliferation step may be carried out in any manner appropriate for growing the NK cell population, but in certain cases, specific reagents (e.g., in culture) are used to enhance the proliferation. In some cases, NK cells may not be proliferated. IL-2, IL-15, IL-18, or any combination thereof may be added to the proliferation culture before or during proliferation. In certain embodiments, NK cells may be proliferated in vitro in a flask or in one of several different bioreactor configurations with continuous perfusion of medium / additives.

[0174] In certain cases, NK cells (whether pre-activated or not) may be washed with PBS, plasma light, human serum albumin, culture medium, or a combination thereof before and / or after proliferation. Washing may be performed one, two, three, four, or five times, and in particular three times. In certain embodiments, NK cells are grown in the presence of artificial antigen-presenting cells (aAPCs). In certain embodiments, NK cells are grown in the presence of a fragment of aAPC. aAPCs may be engineered to express CD137 ligand and / or membrane-bound cytokines. Membrane-bound cytokines may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, aAPCs are engineered to express CD137 ligand and mIL-21. aAPCs may be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous HLA class I, II, or CD1d molecules. ICAM-1 (CD54), LFA-3 (CD58), or CD48 may be expressed. In particular, aAPC may be K562 cells, for example, K562 cells engineered to express CD137 ligand and mIL-21. The engineering may be carried out by methods well known to those skilled in the art, such as retroviral transduction, but any viral or non-viral vector may also be used. aAPC may or may not be irradiated. Growth may be carried out for a certain period, for example about 2 to 30 days, for example 3 to 20 days, particularly 12 to 16 days, for example 12, 13, 14, 15, 16, 17, 18, or 19 days, specifically about 14 days. Pre-activated NK cells and aAPC may be present in a ratio of about 3:1 to 1:3, for example 2:1, 1:1, 1:2, specifically about 1:2. The growth culture may further contain one or more cytokines to promote growth, for example IL-2. IL-2 can be present at concentrations of approximately 10–500 U / mL, for example, 100–300 U / mL, and particularly around 200 U / mL. IL-2 can be supplemented in the growth culture, for example, every 2–3 days. aAPC can be added to the culture at least twice, for example, about 7 days after the start of growth. Any cytokines used in the pre-activation and / or growth process may be recombinant human cytokines.

[0175] In some embodiments, after proliferation, NK cells may be used immediately and, for example, conjugated with one or more antibodies, or stored by cryopreservation. In certain cases, the cells may be passaged in vitro as a bulk population for several days, weeks, or months within 1, 2, 3, 4, or 5 days.

[0176] Activated and / or proliferating NK cells may secrete type I cytokines, such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF). These activate both innate and adaptive immune cells and also induce other cytokines and chemokines. Measurement of these cytokines can be used to determine the activation state of NK cells. Furthermore, other methods known to those skilled in the art for determining NK cell activation may also be used to characterize the NK cells of this disclosure.

[0177] Therefore, with respect to the specific pre-activation and proliferation aspects of this disclosure, in certain embodiments, a highly potent cell preparation can be obtained by subjecting NK cells pre-activated with any combination of IL-12, IL-15, and / or IL-18 to proliferation with aAPCs such as K562 cells expressing mIL-21 and CD137 ligand. Thus, the NK cell-based methods of this disclosure are provided for the treatment of various diseases, including immunotherapy for cancer patients. In the exemplary method, isolated NK cells are subjected to approximately 16 hours of pre-activation with a combination of cytokines such as interleukin-12 (IL-12), IL-15, and / or IL-18, and then proliferated with artificial antigen-presenting cells (aAPCs) such as K562 feeder cells expressing membrane-bound IL-21 and CD137 ligand, and / or exogenous IL-2. IL-2, IL-15, or IL-18, or any combination thereof, may be added to the proliferation culture at least twice.

[0178] F. Cryopreservation In certain cases, the NK cells and / or antibodies of this disclosure are stored in a cryopreservation medium composition comprising at least one cryoprotective agent, serum (human or animal serum) or a non-serum substitute (not human or animal serum), and at least one cytokine and / or at least one growth factor. In some cases, the cryoprotective agent is dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethyl starch, or a combination thereof. The non-serum substitute may be any kind, comprising at least platelet lysate and / or blood product lysate (e.g., human serum albumin). In embodiments of compositions utilizing one or more (including two or more) cytokines, the cytokines may be natural, recombinant, or synthetic proteins. At least one of the cytokines may be a Food and Drug Administration (FDA) approved cytokine. Examples of cytokines and growth factors include at least IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferon, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or combinations thereof. In the serum embodiment, the serum may be human serum (including human AB serum) or animal-derived serum such as bovine serum. DMSO and other cryoprotective agents, if used, may constitute 4-10%, 4-6%, 4-8%, 5-10%, 5-8%, 6-10%, 6-8%, 8-10%, etc., of the composition.In embodiments where serum is used, the serum is available in the following concentrations: 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, and 10-60%. %, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 20-99%, 20-95%, 20-90%, 20-85%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 30-99%, 30-95%, 30-90 %, 30-85%, 30-80%, 30-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 40-99%, 40-95%, 40-90%, 40-85%, 40-80%, 40-75%, 40-70%, 40-65%, 40-60%, 40-55%, 40-50%, 40-45%, 50-99%, 50-95%, 50-90%, 50-85%, 50-80 The composition may contain %, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99%. The composition may contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less of serum.In certain embodiments, the composition comprises platelet lysate, which may be at any concentration in the composition, but in certain embodiments, the platelet lysate is 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85% , 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 20-99%, 20-95%, 20-90%, 20-85%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20- 25%, 30-99%, 30-95%, 30-90%, 30-85%, 30-80%, 30-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 40-99%, 40-95%, 40-90%, 40-85%, 40-80%, 40-75%, 40-70%, 40-65%, 40-60%, 40-55%, 40-50%, 40-45%, 50-99%, 50-95%, 50-90% The composition contains 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99%. The composition may contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less of platelet lysates.

[0179] The composition may contain components at specific concentrations, including cytokines and / or growth factors. In specific cases, any cytokine, including, for example, IL-2, IL-21, and / or IL-15, may be present in the composition at specific concentrations. IL-2 may be present at concentrations of, for example, 1-5000, 1-1000, 1-500, 1-100, 100-5000, 100-5000, 500-5000, 500-1000, or 1000-5000 U / mL. In specific cases, IL-2 may be present in the composition at concentrations of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 U / mL, or less. In a particular embodiment, IL-21 is present in the composition at concentrations of 10-3000, 10-2000, 10-1000, 10-500, 10-100, 100-3000, 100-2000, 100-1000, 500-3000, 500-2000, 500-1000, 1000-3000, 1000-2000, or 2000-3000 ng / mL. IL-21 may be present in the composition at concentrations of at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, or 3000 ng / mL, or lower. IL-15 may be present in the composition at concentrations of 1-2000, 1-1000, 1-500, 1-100, 100-2000, 100-1000, 100-500, 500-2000, 500-1000, or 1000-2000 ng / mL. IL-15 may be present in the composition at concentrations of at least 10, 50, 100, 500, 1000, 1500, or 2000 ng / mL, or less.

[0180] A composition as encompassed herein, comprising at least one cryoprotective agent, serum or a non-serum substitute for serum, and at least one cytokine and / or at least one growth factor, may further comprise any number of immune cells and / or stem cells of any kind. In specific embodiments, the cells are NK cells, T cells, B cells, NKT cells derived from mature bone marrow or peripheral blood cells; cell lines such as tumor cell lines (e.g., NK92 or other NK lines) which may be derived from bone marrow, peripheral blood, skin, adipose tissue, or a combination thereof; hematopoietic stem cells, induced pluripotent stem cells, MSCs (cell populations also referred to in the literature as “mesenchymal stem cells” and “mesenchymal stromal cells”), or mixtures thereof. In embodiments utilizing NK cells, the NK cells may be proliferating NK cells or not. Embodiments of the Disclosure also encompass pharmaceutical compositions comprising any composition of the Disclosure and a suitable pharmaceutically acceptable carrier.

[0181] In certain embodiments, cells and / or antibodies are treated with one or more inactivators (e.g., kinase inhibitors, e.g., dasatinib, nilotinib, rapamycin, etc.) before cryopreservation.

[0182] In some embodiments, the techniques described herein include inactivating NK cells, which involves treating NK cells with an effective amount of one or more inactivators under conditions that produce inactivated NK cells. In some embodiments, the inactivator is a kinase inhibitor. In some embodiments, the inactivator is a mechanical target of a rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is rapamycin, everolimus, and / or temsirolimus. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the inactivator is a tyrosine kinase (TK) inhibitor. In some embodiments, TK inhibitors include lorlatinib, brigatinib, ceritinib, alectinib, crizotinib, bosutinib, ponatinib, nilotinib, dasatinib, imatinib, zanubrutinib, acalabrutinib, ibrutinib, capmatinib, pexidartinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdafitinib, nintedanib, gilteritinib, midos These include taurine, tucatinib, neratinib, baricitinib, ruxolitinib, fedratinib, tofacitinib, ripretinib, selumetinib, binimetinib, cobimetinib, trametinib, upadacitinib, avapritinib, serpercatinib, cabozantinib, fostamatinib, lalotrectinib, entrectinib, axitinib, regorafenib, pazopanib, sorafenib, lenvatinib, vandetanib, and / or sunitinib. In some embodiments, the TK inhibitor is a BCR-Abl inhibitor. In some embodiments, the TK inhibitor is bosutinib, ponatinib, nilotinib, dasatinib, and / or imatinib. In some embodiments, the TK inhibitor is dasatinib and / or nilotinib.

[0183] In some embodiments, treatment with the inactivator is performed at any point during the culture of NK cells. In some embodiments, the treatment is performed for approximately 24 hours to approximately 96 hours, approximately 36 hours to approximately 84 hours, or approximately 48 hours to approximately 72 hours. In some embodiments, the treatment is performed for approximately 24 hours, approximately 48 hours, or approximately 72 hours. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 1 to approximately 1000 nM. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 5 to approximately 500 nM. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 20 to approximately 200 nM. In some embodiments, NK cells are treated with an inactivator at a concentration of approximately 30 to approximately 100 nM. In some embodiments, inactivated NK cells have increased expression of one or more C-kit, CCR-5, CD62L, and / or CXCR4, and / or decreased expression of one or more NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS, and / or CD95, compared to activated NK cells. In some embodiments, the techniques described herein include a method for maintaining the viability of a population of cells at least 50% after cryopreservation of the population, the method including the steps of: subjecting the population to an effective amount of one or more inactivators (e.g., tyrosine kinase inhibitors) to inactivate the cells before cryopreservation; cryopreserving the cells; and thawing the population, wherein the viability of the population at thawing is at least 50%. In some cases, at thawing the cells, the viability of the population of cells after cryopreservation of the population is at least 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0184] III. Heterogeneous proteins and mutants In certain embodiments, NK cells are engineered (e.g., heterologously expressed) to overexpress and / or upregulate the activity of one or more members of the CEBP family, and may further be engineered to express one or more other heterologous proteins. In some embodiments, NK cells are designed to express one or more heterologous proteins. The heterologous proteins can enhance the function of NK cells in any manner including, at least, activation, persistence, proliferation, homing, and / or cytotoxic activity.

[0185] The oligonucleotides, polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of this disclosure are, for SEQ ID NOs: 1-63, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any range derived therefrom) of the mutant amino acids. It may contain nucleic acid substitutions, or at least, exactly, or at most, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derived therefrom) similar, identical or homologous, or at least, exactly, or The maximum is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 1, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、It may contain 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1600, 1800, or 2000 or more (or any range thereof) consecutive amino acids or nucleic acids. In certain embodiments, the nucleic acid encoding the peptide or polypeptide is codon-optimized for expression in mammals. In some embodiments, peptides or polypeptides do not exist in nature and / or exist as combinations of multiple peptides or polypeptides.

[0186] The polypeptides of this disclosure are at least, at most, or exactly, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 ,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,11 4, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 ,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 2 08, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 23 9, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,It may include 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, or 422 substitutions (or any range derived therefrom). In certain embodiments, the substitution is effected by alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.,

[0187] According to one theory, polypeptide is used for amino acids contained in any of the following sequence numbers: 1-5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41, with respect to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 5 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 1 60, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 19 1, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 ,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、The amino acid positions 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, or 529 may include one or more substitutions. Here, each substitution is independently selected from amino acids selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. Furthermore, the polypeptide has sequence identity of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derived therefrom) with respect to any of sequence numbers 1-5, 7, 9, 11, 13, 15, 17, 19, 23, 25, 27, 29, 31, 33, 35, 37, 39, or 41.

[0188] According to one theory, a protein or polypeptide is a protein that contains amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 29, 31, 33, 35, 37, 39, or 41 of any of the numbers 1-5, 7, 9, 11, 13, 15, 17, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 1 28, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 1 59, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 19 0, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 ,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、It may contain amino acids 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, or 529.

[0189] According to one theory, a protein or polypeptide is a protein that contains amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 29, 31, 33, 35, 37, 39, or 41 of any of the numbers 1-5, 7, 9, 11, 13, 15, 17, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 1 28, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 1 59, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 19 0, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 ,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、Including 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, or 529, and further including at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66% of any of sequence numbers 1-63. They may have sequence identity of 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range that can be derived from there).

[0190] In one embodiment, a protein, polypeptide, or nucleic acid is a sequence of amino acids or nucleic acids from SEQ ID NOs. 1 to 63, specifically 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 ,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107 ,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 1 70, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 20 1, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232 ,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 3 15, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 36 6, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417 It may contain, at least, or at most, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1600, 1800, or 2000 (or any range that can be derived therefrom) amino acids or nucleic acids or nucleic acids.

[0191] In one embodiment, a polypeptide, protein, or nucleic acid is a sequence of amino acids or nucleic acids from SEQ ID NOs: 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 ,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107 ,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 1 70, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 20 1, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232 ,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 120 Including 0, 1266, 1400, 1600, 1800, or 2000 (or any range that can be derived therefrom), and with at least, at most, or exactly, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,Alternatively, they may be 100% (or to any extent that can be derived from that) similar, identical, or homologous.

[0192] In one embodiment, a nucleic acid molecule or polypeptide is located at positions 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 in any of SEQ ID NOs: 1 to 63. , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 ,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 1 73, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 20 4, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 ,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 3 29, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 36 0, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391 Starting from 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, or 422, and further including at least, at most, or exactly, any of the sequence numbers 1 through 63, from 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 41 It may contain 2, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 1000, 1200, 1266, 1400, 1600, 1800, or 2000 (or a range that can be derived therefrom) consecutive amino acids or nucleic acids.

[0193] A. Transcription regulators In certain embodiments, NK cells are engineered to express one or more transcription factors. In some embodiments, the transcription factors are transcription factors (TFs). In some embodiments, the transcription factors are positive and / or negative regulators of transcription factors.

[0194] 1. CCAAT / enhancer-binding protein (C / EBP) In some embodiments, the transcription factor is a transcription factor belonging to the CEBP (also referred to herein as C / EBP) protein family. In certain embodiments, engineered natural killer (NK) cells are modified to overexpress CEBP protein compared to unengineered NK cells and / or NK cells engineered to express IL-15. In certain embodiments, the CEBP protein is CEBPA (CEBP-alpha, CEBPα), CEBPB (CEBP-beta, CEBPβ), CEBPG (CEBP-gamma, CEBPγ), CEBPD (CEBP-delta, CEBPδ), CEBPE (CEBP-epsilon, CEBPε), and / or CEBP homolog protein (CHOP). In certain embodiments, the CEBP protein is CEBPD. In certain embodiments, the CEBP protein is CEBPB. In certain embodiments, the CEBP protein is CEBPD and CEBPB.

[0195] In certain embodiments, the manipulated NK cells are genetically modified to basically constitute, or constitute, a nucleic acid sequence and / or protein encoding a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs. In certain embodiments, CEBP family members may be expressed as part of a multicistronic construct. In certain embodiments, CEBP family members may be transcriptionally ligated to one or more marker proteins. In certain embodiments, CEBP family members may be operably ligated to exogenous transcriptional regulatory sequences, not limited to promoters, enhancers, 5'UTR, 3'UTR, target sites of RNA interference mechanisms (e.g., targets such as one or more miRNAs, siRNAs, shRNAs, etc.), and / or polyadenylation signals. Sequence ID 19 - CEBP Delta (CEBPD, CEBPδ) amino acid sequence MSAALFSLDGPARGAPWPAEPAPFYEPGRAGKPGRGAEPGALGEPGAAAPAMYDDESAIDFSAYIDSMAAVPTLELCHDELFADLFNSNHKAGGAGPLELLPGGPARPLGPGPAAPRLLKREPDWGDGDAPGSL LPAQVAACAQTVVSLAAAGQPTPPTSPEPPRSSPRQTPAPGPAREKSAGKRGPDRGSPEYRQRRERNNIAVRKSRDKAKRRNQEMQQKLVELSAENEKLHQRVEQLTRDLAGLRQFFKQLPSPPFLPAAGTADCR (SEQ ID NO: 19) Sequence ID 20 - CEBP Delta (CEBPD, CEBPδ) Codon-Optimized Nucleic Acid Sequence ATGTCAGCAGCTCTTTTTTCTCTTGATGGTCCTGCCCGGGGCGCGCCATGGCCCGCTGAGCCAGCTCCCTTTTACGAGCCAGGTAGAGCTGGAAAACCAGGGAGAGGGGCGGAGCCCGGGGCTCTCGGGGAGCCTGGAGCTGCAGCTCCGGCCATGTATGACGATGAATCAGCAATCGATTTCTCAGCGTACATTGATTCCATGGCCGCCGTTCCAACGTTGGAACTCTGTCATGATGAGCTTTTCGCAGACCTCTTTAACTCTAATCACAAAGCTGGCGGCGCTGGTCCACTGGAACTTCTTCCTGGGGGCCCCGCTCGGCCGCTCGGGCCCGGTCCCGCCGCCCCGAGATTGCTGAAAAGGGAACCGGATTGGGGAGATGGTGACGCGCCAGGAAGCTTGCTGCCTGCACAGGTAGCGGCATGCGCCCAGACCGTGGTGAGTCTGGCTGCAGCGGGCCAACCAACCCCTCCAACGTCACCGGAACCACCACGATCATCACCTAGACAAACACCAGCGCCCGGCCCGGCGCGCGAAAAGTCCGCAGGGAAAAGAGGGCCTGACAGAGGCAGTCCGGAATACAGACAACGCCGGGAGAGGAACAATATAGCTGTGAGAAAGAGTCGCGATAAGGCGAAACGGCGGAACCAAGAAATGCAGCAGAAGCTGGTAGAACTCAGCGCAGAGAACGAGAAGCTCCATCAACGGGTTGAGCAACTGACTAGGGACCTCGCGGGCCTGAGACAATTTTTCAAGCAGCTGCCGAGTCCACCCTTTCTTCCCGCAGCGGGGACTGCCGACTGCCGC (SEQ ID NO: 20) Sequence number 21 - CEBP delta (CEBPD, CEBPδ) nucleic acid sequence ATGAGCGCCGCGCTCTTCAGCCTGGACGGCCCGGCGCGCGGCGCGCCCTGGCCTGCGGAGCCTGCGCCCTTCTACGAACCGGGCCGGGCGGGCAAGCCGGGCCGCGGGGCCGAGCCAGGGGCCCTAGGCGAGCCAGGCGCCGCCGCCCCCGCCATGTACGACGACGAGAGCGCCATCGACTTCAGCGCCTACATCGACTCCATGGCCGCCGTGCCCACCCTGGAGCTGTGCCACGACGAGCTCTTCGCCGACCTCTTCAACAGCAATCACAAGGCGGGCGGCGCGGGGCCCCTGGAGCTTCTTCCCGGCGGCCCCGCGCGCCCCTTGGGCCCGGGCCCTGCCGCTCCCCGCCTGCTCAAGCGCGAGCCCGACTGGGGCGACGGCGACGCGCCCGGCTCGCTGTTGCCCGCGCAGGTGGCCGCGTGCGCACAGACCGTGGTGAGCTTGGCGGCCGCAGGGCAGCCCACCCCGCCCACGTCGCCGGAGCCGCCGCGCAGCAGCCCCAGGCAGACCCCCGCGCCCGGCCCCGCCCGGGAGAAGAGCGCCGGCAAGAGGGGCCCGGACCGCGGCAGCCCCGAGTACCGGCAGCGGCGCGAGCGCAACAACATCGCCGTGCGCAAGAGCCGCGACAAGGCCAAGCGGCGCAACCAGGAGATGCAGCAGAAGTTGGTGGAGCTGTCGGCTGAGAACGAGAAGCTGCACCAGCGCGTGGAGCAGCTCACGCGGGACCTGGCCGGCCTCCGGCAGTTCTTCAAGCAGCTGCCCAGCCCGCCCTTCCTGCCGGCCGCCGGGACAGCAGACTGCCGGTAA (SEQ ID NO: 21) Construct containing the sequence number 22 - CEBP delta (CEBPD, CEBPδ) nucleic acid sequence Sequence ID 23 - CEBP beta (CEBPB, CEBPβ) isoform A amino acid sequence MQRLVAWDPACLPLPPPPPAFKSMEVANFYYEADCLAAAYGGKAAPAAPPAARPGPRPPAGELGSIGDHERAIDFSPYLEPLGAPQAPAPATATDTFEAAPPAPAPAPASSGQHHDFLSDLFSDDYGGKNCKKPAEYGYVSLGRLGAAKGALHPGCFAPLHPPPPPPPPPAE LKAEPGFEPADCKRKEEAGAPGGGAGMAAGFPYALRAYLGYQAVPSGSSGSLSTSSSSSPPGTPSPADAKAPPTACYAGAAPAPSQVKSKAKKTVDKHSDEYKIRRERNNIAVRKSRDKAKMRNLETQHKVLELTAENERLQKKVEQLSRELSTLRNLFKQLPEPLLASSGHC (SEQ ID NO: 23) Sequence ID 24-CEBPbeta (CEBPB,CEBPβ) transcription variant 2 (encoding isoform A) nucleic acid sequence Sequence ID 25 - CEBP beta (CEBPB, CEBPβ) isoform B amino acid sequence MEVANFYYEADCLAAAYGGKAAPAAPPAARPGPRPPAGELGSIGDHERAIDFSPYLEPLGAPQAPAPATATDTFEAAPPAPAPASSGQHHDFLSDLFSDDYGGKNCKKPAEYGYVSLGRLGAAKGALHPGCFAPLHPPPPPPPPPAELKAEPGFEPADC KRKEEEAGAPGGGAGMAAGFPYALRAYLGYQAVPSGSSGSLSTSSSSSPPGTPSPADAKAPPTACYAGAAPAPSQVKSKAKKTVDKHSDEYKIRRERNNIAVRKSRDKAKMRNLETQHKVLELTAENERLQKKVEQLSRELSTLRNLFKQLPEPLLASSGHC (SEQ ID NO: 25) Sequence ID 26 - CEBP beta (CEBPB, CEBPβ) transcription variant 1 (encoding isoform B) nucleic acid sequence ATGGAAGTGGCCAACTTCTACTACGAGGCGGACTGCTTGGCTGCTGCGTACGGCGGCAAGGCGGCCCCCGCGGCGCCCCCCGCGGCCAGACCCGGGCCGCGCCCCCCCGCCGGCGAGCTGGGCAGCATCGGCGACCACGAGCGCGCCATCGACTTCAGCCCGTACCTGGAGCCGCTGGGCGCGCCGCAGGCCCCGGCGCCCGCCACGGCCACGGACACCTTCGAGGCGGCTCCGCCCGCGCCCGCCCCCGCGCCCGCCTCCTCCGGGCAGCACCACGACTTCCTCTCCGACCTCTTCTCCGACGACTACGGGGGCAAGAACTGCAAGAAGCCGGCCGAGTACGGCTACGTGAGCCTGGGGCGCCTGGGGGCCGCCAAGGGCGCGCTGCACCCCGGCTGCTTCGCGCCCCTGCACCCACCGCCCCCGCCGCCGCCGCCGCCCGCCGAGCTCAAGGCGGAGCCGGGCTTCGAGCCCGCGGACTGCAAGCGGAAGGAGGAGGCCGGGGCGCCGGGCGGCGGCGCAGGCATGGCGGCGGGCTTCCCGTACGCGCTGCGCGCTTACCTCGGCTACCAGGCGGTGCCGAGCGGCAGCAGCGGGAGCCTCTCCACGTCCTCCTCGTCCAGCCCGCCCGGCACGCCGAGCCCCGCTGACGCCAAGGCGCCCCCGACCGCCTGCTACGCGGGGGCCGCGCCGGCGCCCTCGCAGGTCAAGAGCAAGGCCAAGAAGACCGTGGACAAGCACAGCGACGAGTACAAGATCCGGCGCGAGCGCAACAACATCGCCGTGCGCAAGAGCCGCGACAAGGCCAAGATGCGCAACCTGGAGACGCAGCACAAGGTCCTGGAGCTCACGGCCGAGAACGAGCGGCTGCAGAAGAAGGTGGAGCAGCTGTCGCGCGAGCTCAGCACCCTGCGGAACTTGTTCAAGCAGCTGCCCGAGCCCCTGCTCGCCTCCTCCGGCCACTGCTAG (SEQ ID NO: 26) Sequence ID 27-CEBPbeta (CEBPB,CEBPβ) isoform C amino acid sequence MAAGFPYALRAYLGYQAVPSGSSGSLSTSSSSSPPGTPSPADAKAPPTACYAGAAPAPSQVKSKAKKTVDKHSDEYKIRRERNNIAVRKSRDKAKMRNLETQHKVLELTAENERLQKKVEQLSRELSTLRNLFKQLPEPLLASSGHC (SEQ ID NO: 27) Sequence ID 28-CEBPbeta (CEBPB,CEBPβ) transcription variant 3 (encoding isoform C) nucleic acid sequence ATGGCGGCGGGCTTCCCGTACGCGCTGCGCGCTTACCTCGGCTACCAGGCGGTGCCGAGCGGCAGCAGCGGGAGCCTCTCCACGTCCTCCTCGTCCAGCCCGCCCGGCACGCCGAGCCCCGCTGACGCCAAGGCGCCCCCGACCGCCTGCTACGCGGGGGCCGCGCCGGCGCCCTCGCAGGTCAAGAGCAAGGCCAAGAAGACCGTGGACAAGCACAGCGAC GAGTACAAGATCCGGCGCGAGCGCAACAACATCGCCGTGCGCAAGAGCCGCGACAAGGCCAAGATGCGCAACCTGGAGACGCAGCACAAGGTCCTGGAGCTCACGGCCGAGAACGAGCGGCTGCAGAAGAAGGTGGAGCAGCTGTCGCGAGCTCAGCACCCTGCGGAACTTGTTCAAGCAGCTGCCCGAGCCCCTGCTCGCCTCCTCCGGCCACTGCTAG (SEQ ID NO: 28)

[0196] B. Bispecific antibodies or multispecific antibodies In one embodiment, NK cells are modified to express one or more bispecific or multispecific antibodies, while in other cases, NK cells do not express antibodies, and the antibodies are used in combination with NK cells.

[0197] If NK cells are modified to express antibodies, these antibodies may act as engagers, bridging specific immune effector cells to specific target cells and destroying the target cells. In some embodiments, engineered NK cells are complexed with or used in combination with T cell engagers (BiTEs) and / or bispecific NK engagers (BiKEs). In some embodiments, a BiKE comprises an antibody that binds to a cell surface protein on an NK cell (including, but not limited to, naturally expressed proteins such as NKp30, NKp44, NKp46, CD16, CD32, CD64, KIRs, etc.) and an antibody that binds to a desired target antigen. A BiKE can target NK cells via antibodies that bind to NK cell surface proteins such as CD16, CS1, CD32, CD64, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIRs. In such cases, the BiKE used in the present invention may also target cancer antigens or viral antigens designed to match the medical condition of the recipient individual. For example, the BiKE may be designed to bind to tumor antigens specific to the cancer cells of a target cancer patient.

[0198] In one embodiment, the antibody is blinatumomab. In one embodiment, the antibody is teventafusp. In one embodiment, the antibody is mosnetuzumab. In one embodiment, the antibody is tecristamag. In one embodiment, the antibody is grofitamab. In one embodiment, the antibody is epcolitamab. In one embodiment, the antibody is furotuzumab. In one embodiment, the antibody is APVO436. In one embodiment, the antibody is TNB383B. In one embodiment, the antibody is TNB383B. In a use case of a multispecific antibody, in one embodiment, one or more antigen-binding domains of the antibody can bind to one or more target antigens.

[0199] C. Cytokines In certain embodiments, cells expressing NK cells are engineered to express one or more heterologous cytokines and / or engineered to upregulate normal expression of one or more heterologous cytokines. These cells may or may not be transduced or transfected with one or more cytokines on the same vector as other genes. In certain embodiments, NK cells may be modified to express one or more cytokines, cytokine receptors, chemokines, chemokine receptors, and / or suicide genes.

[0200] One or more cytokines may be co-expressed from the vector, and these may be expressed as polypeptides separate from the components involved in the overexpression and / or upregulation of the activity of one or more CEBP family proteins. In some embodiments, for example, interleukin-15 (IL-15) is tissue-specific and is observed only in serum or systemic levels under pathological conditions. In some embodiments, IL-15 has several desirable properties for adoptive cell therapy. In some embodiments, IL-15 is a homeostatic cytokine that induces the development and proliferation of natural killer cells, promotes the eradication of established tumors by desuppressing the function of tumor resident cells, and further inhibits activation-induced cell death (AICD). In addition to IL-15, other cytokines may be considered in some embodiments. These include, but are not limited to, IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. In one embodiment, NK cells expressing IL-15 enable sustained supportive cytokine signaling useful for post-infusion survival. In one embodiment, engineered NK cells are not transgenically modified to express the cytokine IL-15. In one embodiment, engineered NK cells autonomously and / or constitutively express and secrete IL-21, enabling sustained supportive cytokine signaling useful for post-infusion survival. In one embodiment, engineered NK cells expressing IL-21 have enhanced immunological memory against glioblastoma stem cells compared to cells engineered to express IL-15. In one embodiment, NK cells are engineered to autonomously express IL-21. In one embodiment, NK cells are engineered to constitutively express IL-21. In one embodiment, NK cells are engineered to secrete IL-21.In one embodiment, NK cells stably secrete IL-21, and extracellular concentrations are maintained at 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 900, and 1000. The system is operated to reach a level that can be maintained at concentrations of 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 pg / mL, or above 3000 pg / mL. In one embodiment, NK cells are manipulated to stably secrete IL-21 and thus reach extracellular concentrations of IL-21 equal to or near 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 pg / mL, or any value deriveable in between. In one embodiment, NK cells are manipulated to stably secrete IL-21, and thus to reach extracellular concentrations of IL-21 equal to or within the range of approximately 200–800 pg / mL, 250–750 pg / mL, 300–700 pg / mL, 350–650 pg / mL, or 400–600 pg / mL.

[0201] In some embodiments, cells express one or more exogenously provided engineered receptors, where the engineered receptors include chemokine receptors and / or cytokine receptors. In some embodiments, the cytokine receptor is an IL-15 receptor. In some embodiments, the cytokine receptor is a non-naturally occurring variant of a cytokine receptor. In some embodiments, the cytokine receptor is an IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, or GMCSF receptor, or a combination thereof.

[0202] In specific embodiments, cells express one or more exogenously supplied cytokines. For example, cytokines may be IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. Cytokines can be exogenously supplied to NK cells because they are expressed from intracellular expression vectors. In other cases, intracellular endogenous cytokines are upregulated by manipulations of endogenous cytokine expression control, such as genetic recombination at the cytokine promoter site. When cytokines are supplied to cells on an expression construct, the cytokines may be encoded from the same vector as one or more other components described herein.

[0203] In one embodiment, manipulated NK cells are transgenically modified to express nucleic acid sequences and / or cytokines containing them that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of sequence numbers 29-34.

[0204] In one embodiment, the following specific IL-15 sequence is used (underlined portion indicates the signal peptide sequence, which may or may not be included): SEQ ID NO: 29-IL-15 amino acid sequence (signal peptide underlined) MRISKPHLRSISIQCYLCLLLNSHFLTEA GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 29) Sequence ID 30-IL-15 nucleic acid sequence (signal peptide underlined) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGC AGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 30)

[0205] In one embodiment, the following specific IL-21 sequence is used (underlined portion indicates the signal peptide sequence, which may or may not be included): Sequence ID 31 - Codon-optimized IL-21 amino acid sequence (signal peptide underlined) MRSSPGNMERIVICLMVIFLGTLV HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 31) Sequence ID 32 - Codon-optimized IL-21 nucleic acid sequence (signal peptide underlined) ATGAGGAGCAGTCCAGGCAATATGGAGCGGATAGTAATTTGTCTCATGGTAATATTCCTCGGTACTCTGGTACATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAA CGGATAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT (SEQ ID NO: 32) Amino acid sequence of SEQ ID NO: 33-IL-21 GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 33) Sequence ID 34-IL-21 nucleic acid sequence GGCCAGGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCTGCCCCCGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATC AACGTGTCCATCAAGAAGCTGAAGCGGAAGCCCCCCAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCCCCTAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCCGGACCCACGGCTCTGAGGACAGC (SEQ ID NO: 34)

[0206] In one embodiment, cytokines are expressed as part of a multi-cistronic construct along with one or more marker proteins. In one embodiment, engineered NK cells are transgenically modified to express nucleic acids and / or marker proteins containing them that encode sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs.

[0207] In one embodiment, the manipulated NK cells are transgenically modified to express nucleic acids that contain, essentially consist of, or comprise sequences that are at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of sequence numbers 43–51. Sequence ID 35-IgG Fc amino acid sequence EPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKETTPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 35) Sequence ID 36 - IgG Fc nucleic acid sequence GAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGAAACAACCCCAAATAAAGGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTATCTGGGCACACGTGTTTCACGTTGACAGGTTTGCTTGGGACGCTAGTAACCATGGGCTTGCTGACT (SEQ ID NO: 36) Sequence number 37 - IgG4 amino acid sequence ESKYGPPCPPPAPEFLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSLGK (SEQ ID NO: 37) SEQ ID NO:38-IgG4 nucleic acid sequence GAGTCTAAGTAACGGCCCTCCCTGCCCTCCTTGTCCAGCCCCTGAATTTCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCTGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTT AATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAAGGGCCTGCCCAGCAGCATCG AGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAAACCCCAGGTGTACACACTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAA CTACAAGACCACCCCCCGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCCCGCCTGACCGTGGACAAGAGCAGATGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCCAGAAGTCCCTGTCTCTGAGCCTGGGCAAG (SEQ ID NO: 38) Amino acid sequence of the extracellular domain of CD19 containing the signal peptide SEQ ID NO: 39 MPPPRLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKL YVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHL (SEQ ID NO: 39) Nucleic acid sequence of the extracellular domain of CD19 containing the signal peptide SEQ ID NO: 40 ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCTATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACTATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGGGCATTCTTCATCTT (SEQ ID NO: 40) Sequence No. 41 - CD19 Amino Acid Sequence PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPK LYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK (SEQ ID NO: 41) Sequence ID 42-CD19 nucleic acid sequence CCCGAGGAGCCCCTGGTGGTGAAGGTGGAGGAGGGCGACAACGCCGTGCTGCAGTGCCTGAAGGGCACCAGCGACGGCCCCACCCAGCAGCTGACCTGGAGCAGAGAGAGCCCCCTGAAGCCCTTCCTGAAGCTGAGCCTGGGCCTGCCCGGCCTGGGCATCCACATGAGACCCCTGGCCATCTGGCTGTTCATCTTCAACGTGAGCCAGCAGATGGGCGGCTTCTACCTGTGCCAGCCCGGCCCCCCCAGCGAGAAGGCCTGGCAGCCCGGCTGGACCGTGAACGTGGAGGGCAGCGGCGAGCTGTTCAGATGGAACGTGAGCGACCTGGGCGGCCTGGGCTGCGGCCTGAAGAACAGAAGCAGCGAGGGCCCCAGCAGCCCCAGCGGCAAGCTGATGAGCCCCAAGCTGTACGTGTGGGCCAAGGACAGACCCGAGATCTGGGAGGGCGAGCCCCCCTGCCTGCCCCCCAGAGACAGCCTGAACCAGAGCCTGAGCCAGGACCTGACCATGGCCCCCGGCAGCACCCTGTGGCTGAGCTGCGGCGTGCCCCCCGACAGCGTGAGCAGAGGCCCCCTGAGCTGGACCCACGTGCACCCCAAGGGCCCCAAGAGCCTGCTGAGCCTGGAGCTGAAGGACGACAGACCCGCCAGAGACATGTGGGTGATGGAGACCGGCCTGCTGCTGCCCAGAGCCACCGCCCAGGACGCCGGCAAGTACTACTGCCACAGAGGCAACCTGACCATGAGCTTCCACCTGGAGATCACCGCCAGACCCGTGCTGTGGCACTGGCTGCTGAGAACCGGCGGCTGGAAG (SEQ ID NO: 42) Sequence number 43 - Representative SPCD19IgG1COIL21 vector - Nucleic acid sequence (signal peptide underlined) ATGAGGAGCAGTCCAGGCAATATGGAGCGGATAGTAATTTGTCTCATGGTAATATTCCTCGGTACTCTGGTA CATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAAC GGATAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGTTGA (SEQ ID NO: 43) Sequence ID 44 - Representative IL-15 vector - Nucleic acid sequence (signal peptide underlined) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCC GGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGC AGGTGATCAGCCTGGAAAGCGGCGACGCCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAAACACCAGC (SEQ ID NO: 44) sequence number 45- representative of 19ACLTBC mbIL21Fc vector-nucleic acid sequence Sequence ID 46 - Representative 21AAMWXC IC9SP8mbIL21tmCD8 vector - Nucleic acid sequence Sequence ID 47 - Representative 21AAMWYC IC9SP8mbIL21tm28 vector - Nucleic acid sequence Sequence ID 48 - Representative CD8SPcoIL21CD8TMD vector - Nucleic acid sequence (signal peptide is underlined) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG ATGAGGAGCAGTCCAGGCAATATGGAGCGGATAGTAATTTGTCTCATGGTAATATTCCTCGGTACTCTGGTA Sequence ID 49 - Representative CD8SPcoIL21CD28TMD vector - Nucleic acid sequence (signal peptide is underlined) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG ATGAGGAGCAGTCCAGGCAATATGGAGCGGATAGTAATTTGTCTCATGGTAATATTCCTCGGTACTCTGGTA Sequence ID 50 - Representative CEBPD-GFP vector - nucleic acid sequence Sequence ID 51 - Representative CEBPD-GFP (with IL-15) vector - Nucleic acid sequence

[0208] D. Antigen The manipulated NK cells described herein can be used in combination with monospecific, bispecific, or multispecific antibodies that target one or more specific antigens.

[0209] Some antigens targeted by antibodies are expressed in the context of diseases, conditions, or cell types targeted via adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, and include hematological cancers, lymphomas, leukemias, and / or cancers of the immune system, such as myelomas, including B, T, and myeloid leukemias, lymphomas, and multiple myeloma. In some embodiments, antigens are selectively expressed or overexpressed on disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, antigens are expressed on normal cells and / or on manipulated cells.

[0210] This method allows for the targeting of any suitable antigen. Antigens may, in some cases, be associated with specific cancer cells but not with non-cancer cells. Exemplary antigens include, but are not limited to, infectious agent-derived antigen molecules, auto / autoantigens, tumor / cancer-associated antigens, and neoplastic tumor antigens (Linnemann et al., 2015). In certain embodiments, antigens may include NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigens, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigens, CD319 (CS1), ROR1, folate-binding proteins, HIV-1 envelope glycoprotein gp120, and HIV-1 envelope glycoprotein gp 41, CD5, CD23, CD30, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinase (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinase), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MA GE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-associated protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phospho Inositide 3 kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II,CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulator 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-binding kinase (ILK), transcriptional signaling and activating factors STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-B), Notch receptors (e.g., Notch1-4), NY ESO 1, c-Met, mammalian targeted rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), and their regulatory subunits, PMSA, PR-3, MDM2, mesoserine, renal cell carcinoma-5T4, SM22α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) This includes ETS fusion genes, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesocerian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Examples of antigen sequences can be found, for example, in GenBank. 登録商標In the database: CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number: NC_000023.11), EGFRvIII (accession number: NG_007726.3), MUC1 (accession number: NG_029383.1), HER2 (accession number: NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10) are known in the art.

[0211] Tumor-associated antigens may originate, for example, from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain tumor, bone cancer, gastric cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens such as those disclosed in International Patent Publication WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-exclusive examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Examples of prostate cancer tumor-associated antigens include prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and prostate 6-stage membrane epithelial antigen (STEAP).

[0212] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Furthermore, tumor antigens can also be autopeptide hormones; for example, gonadotrophin hormone-releasing hormone (GnRH), a short peptide with a total length of 10 amino acids, is useful in the treatment of many cancers.

[0213] Antigens may include genes mutated in tumor cells, such as telomerase enzymes, survivorin, mesoserine, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and abnormally expressed intron sequences like N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens containing epitopic regions or epitopic peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and epitope regions or epitope peptides derived from genes transcribed at different levels in tumor cells compared to normal cells, such as carcinoembryonic antigens and unmutated oncophetal proteins with tumor-selective expression, such as α-fetoprotein.

[0214] 2. Suicide gene In certain embodiments, suicide genes are used in conjunction with NK cell therapy to control their use and enable termination of cell therapy at a desired event and / or time. Suicide genes are employed in transdextrins for the purpose of inducing the death of transdextrins when needed. Cells of this disclosure, modified to possess one or more vectors incorporated herein, may contain one or more suicide genes. In some embodiments, the term “suicide gene” as used herein is defined as a gene that, upon administration of a prodrug or other agent, results in the transfer of a gene product to a compound that kills a host cell. In other embodiments, the suicide gene optionally encodes a gene product that is targeted by a drug (such as an antibody) that targets the suicide gene product.

[0215] In some cases, cell therapy may be subject to the use of one or more suicide genes of any type if the individual receiving cell therapy and / or the individual who has received cell therapy is considered to be at risk of exhibiting, or being imminent to exhibit, one or more symptoms of one or more adverse events such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity (for example). The use of suicide genes may be part of a planned protocol for treatment, or it may be used only when deemed necessary. In some cases, cell therapy may be terminated with a drug that targets a suicide gene or its gene product because treatment is no longer necessary.

[0216] The use of suicide genes can be initiated when at least one adverse event occurs in the individual, and this adverse event can be recognized by any means, including during periodic monitoring, whether continuous or not, from the initiation of cell therapy. Adverse events may be detected by laboratory tests and / or examinations. If the individual is experiencing cytokine release syndrome (sometimes called a cytokine storm), the individual may exhibit elevated levels of inflammatory cytokines (one or more) (for example: interferon-γ, granulocyte-macrophage colony-stimulating factor, IL-10, IL-6, and TNF-α), fever, fatigue, hypotension, hypoxia, tachycardia, nausea, capillary leakage, cardiac / renal / hepatic dysfunction, or a combination thereof. If the individual is neurotoxic, the individual may exhibit confusion, delirium, aplasia, and / or seizures. In some cases, the individual may be tested for markers associated with the development and / or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF-α, and / or ferritin.

[0217] Examples of suicide genes include engineered non-secretory (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see PCT / US19 / 62009, the whole of which is incorporated herein by reference), which may be affected by the delivery of antibodies that bind to TNF-α variants. Examples of suicide gene / prodrug combinations that may be used include herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Escherichia coli purine nucleoside phosphorylase is a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine and may be used. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzyme, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0218] F. Knockout or knockdown of endogenous genes In certain embodiments of this disclosure, NK cells may include gene editing of NK cells to remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous genes in the NK cells. In some cases, gene editing is performed in NK cells expressing one or more heterologous transgenes (e.g., CEBPD, CEBPB, IL-21, IL-15, etc.), while in other cases, gene editing is performed in at least some NK cells that do not express heterologous transgenes but will eventually express one or more heterologous transgenes. In certain embodiments, the NK cells to be gene edited are proliferating NK cells.

[0219] In certain embodiments, one or more endogenous genes in NK cells are modified, such as by disruption of expression, which reduces some or all of their expression. In certain cases, one or more genes are knocked down or knocked out using the process of this disclosure. In certain cases, multiple genes are knocked down or knocked out using the same process as the process of this disclosure. The genes edited in NK cells may be any, but in specific embodiments, the genes are those whose gene product inhibits the activity and / or proliferation of NK cells. In certain cases, the genes edited in NK cells enable NK cells to function more effectively in the tumor microenvironment. In specific cases, the genes are one or more of the following: NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, GR, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, β2-microglobulin, HLA, CD73, CREB, CREM, ICER, and CD39. In specific embodiments, the TGFBR2 gene is knocked out or knocked down in NK cells. In specific embodiments, the CISH gene is knocked out or knocked down in NK cells. In specific embodiments, the CD38 gene is knocked out or knocked down in NK cells. In specific embodiments, the glucocorticoid receptor (GR) gene is knocked out or knocked down in NK cells.

[0220] In some embodiments, gene editing is carried out using one or more DNA-binding nucleic acids, such as modifications mediated by RNA-guided endonucleases (RGENs). For example, modifications can be carried out using clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-related (Cas) proteins. Generally, the “CRISPR system” refers collectively to transcripts and other elements involved in directing the expression or activity of CRISPR-related (“Cas”) genes, and includes the sequence encoding the Cas gene, the tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr-mate sequence (including “direct repeats” and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), the guide sequence (also called “spacers” in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus. Methods utilizing the CRISPR system are well known in the art.

[0221] IV. Administration of the therapeutic composition In certain embodiments, engineered NK cells are administered to individuals who require them. In some embodiments, engineered NK cells are administered to individuals who require them in combination with one or more antibodies, in proximity to the antibodies so that they can interact with the antibodies. In some cases, the two components (engineered NK cells and antibodies) are administered to the individual separately, while in other cases, the two components are conjugated ex vivo before administration. In another embodiment, the NK cells express antibodies. In some cases, the two components are not conjugated before administration and are administered simultaneously via any appropriate route of administration, such as co-infusion to the patient.

[0222] In certain embodiments, this disclosure relates to methods of using compositions comprising NK cells and antibodies provided herein for the treatment or prevention of medical diseases or disorders. The methods include administering a therapeutically effective amount of engineered NK cells and antibodies to a subject, thereby treating or preventing a disease in the subject, including reducing the risk of the disease, reducing the severity of the disease, and / or delaying the onset of the disease. In certain embodiments of this disclosure, cancer or infection is treated by the transfer of a composition comprising an NK cell population and corresponding antibodies. In at least some cases, NK cells may enhance the adaptive immune response by reversing the anti-inflammatory tumor microenvironment due to the release of inflammatory cytokines and promoting the differentiation, activation, and / or recruitment of accessory immune cells to malignant tumor sites. In certain embodiments, the providing steps may include co-culturing engineered NK cells with antibody molecules for a specific time (e.g., from about 5 minutes to about 24 hours or more) and storing the NK cells and antibody molecules for a certain period (e.g., about 1, 2, 3, 4, 5 days, or 5 days or more) before injection / administration.

[0223] The cancers for which the therapeutic methods of the present invention are useful include all malignant cell types, such as those found in solid tumors or hematological malignancies. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of the pancreas, colon, cecum, stomach, brain, head, neck, ovaries, kidneys, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological malignancies include tumors of the bone marrow, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, and myeloma. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric cancer or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various head and neck cancers, and melanoma.

[0224] Cancer may, but is not limited to, the following histological types: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; dermal papillary carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyp; adenocarcinoma, familial adenomatous polyposis; solid tumor; malignant carcinoid tumor; lobulated-alveolar adenocarcinoma; papillary adenocarcinoma; chromatocarcinoma; Eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; unencapsulated sclerotic carcinoma; adrenal cortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; keratinized gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary gland; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; sarcoma, malignant; granulosa cell malignant tumors; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; angiosarcoma; malignant melanoma; achromatic melanoma; superficial spreading melanoma; lentigo malignant melanoma; lentigo genitalia; nodular melanoma; malignant melanoma of giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müller's mixed tumor; nephroblastoma; hepatoblastoma Carcinosarcoma; Mesenchymal tumor, malignant; Brenner tumor, malignant; Philodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Embryonic disorders; Embryonic carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesoothelioma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangioexinodermoma, malignant; Lymphangiosarcoma; Osteosarcoma; Soft cortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloepithelial gingivoma; Ameloepithelioma, malignant; Ameloepithelial fibrosarcoma; Pineal glandoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma;Fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermoma; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin; paragranuloma; malignant lymphoma, microlymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin lymphomas; B-cell lymphoma; low-grade / follicular non-Hodgkin lymphoma (NHL); microlymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-necrotic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenström macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythrocyte leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myelosarcoma; pilocytic cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0225] Therapies provided herein may include the combined administration of multiple therapeutic agents, such as a first cancer treatment and a second cancer treatment. These treatments may be administered in any suitable manner known to those skilled in the art. For example, the first and second cancer treatments may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered in separate compositions. In other embodiments, the first and second cancer treatments are contained in the same composition. Aspects of this disclosure relate to compositions and methods comprising therapeutic compositions. Different treatments may be administered in one composition or in multiple compositions, such as two, three, or four. Various combinations of therapeutic agents may be available. Examples of treatments other than those disclosed herein include surgery, chemotherapy, drug therapy, radiotherapy, hormone therapy, immunotherapy (other than those disclosed herein), or combinations thereof.

[0226] The therapeutic agents disclosed herein may be administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, or orbitally, by implantation, by inhalation, intrathecally, intraventricularly, or nasally. In some embodiments, antibiotics are administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, or orbitally, by implantation, by inhalation, intrathecally, intraventricularly, or nasally. The appropriate dosage may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0227] Therapeutic agents may include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount administered, as well as the specific route and prescription, is within the scope of the clinical practitioner's judgment. A unit dose does not need to be administered as a single injection, but may include continuous infusions over a period of time. In some embodiments, a unit dose includes a single-dose dose.

[0228] The dosage, both in terms of the number of treatments and the unit dose, depends on the desired therapeutic effect. The effective dose is understood to refer to the amount required to achieve a particular effect. In practice, in certain embodiments, it is intended that doses ranging from 10 mg / kg to 200 mg / kg may affect the protective capacity of these drugs. Therefore, possible doses include approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derived from there. Furthermore, such doses can be administered multiple times a day and / or over multiple days, weeks, or months.

[0229] In one embodiment, the method includes administering one or more doses of NK cells as described herein, in which case at least one dose is approximately 1 × 10⁶ 6 ~Approx. 1×10 10 This is the range that can be introduced into cells or between them. In one embodiment, at least one dose is about 4 × 10 5 , 8×10 5 , 4×10 6 , 8×10 6 , 4×10 7 , 8×10 7 , 4×10 8 , 8×10 8 , 4×10 9 , 8×10 9 , 4×10 10 , or 8×10 10 or within the range that can be reached in between.

[0230] In one embodiment, the NK cell-containing composition described herein is approximately 1 × 10⁻⁶ 6 ~About 100×10 6 The concentration of cells is or can be in the range between . In one embodiment, the NK cell-containing composition described herein is about 3 × 10 6 ~Approx. 25×10 6 This is the range that can be induced in cells or between cells at that concentration.

[0231] In certain embodiments, an effective amount of the pharmaceutical composition may provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective amount may provide a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range from which it can be derived). In other embodiments, the dose is the following blood levels of the drug derived from the therapeutic agent administered to the subject: approximately, at least, or at most approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, The concentrations may be 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derived therefrom. In certain embodiments, the therapeutic agent administered to a subject is metabolized in the body to become a metabolized therapeutic agent, in which case the blood concentration may refer to the amount of that therapeutic agent. Alternatively, in the range in which the therapeutic agent is not metabolized by the subject, the blood concentration discussed herein may refer to the unmetabolized therapeutic agent.

[0232] The precise amount of therapeutic composition depends on the clinician's judgment and is individual to individual. Factors influencing the dosage include the patient's physical and clinical condition, route of administration, purpose of treatment (symptom relief or cure), and the potency, stability, and toxicity of the specific therapeutic substance or other treatments the patient is receiving.

[0233] Those skilled in the art will understand and recognize that dosage units of μg / kg or mg / kg per body weight can be expressed in comparable concentration units (μg / mL or mM (blood concentration), e.g., 4 μM to 100 μM). They will also understand that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions regarding uptake and concentration measurements are well known, enabling those skilled in the art to convert one concentration measurement to another and to make reasonable comparisons and draw conclusions regarding the doses, efficacy, and results described herein.

[0234] In one embodiment, the manipulated NK cells provided herein are used to treat cancer. In one embodiment, the cancer is glioblastoma. In one embodiment, the glioblastoma has a mesenchymal subtype annotated by TCGA. In one embodiment, the glioblastoma has a preneurial subtype annotated by TCGA. In one embodiment, the glioblastoma has a non-methylated MGMT status. In one embodiment, the glioblastoma has a methylated MGMT status. In one embodiment, the glioblastoma has an uncertain MGMT status. In one embodiment, the glioblastoma is EGFRVIII negative. In one embodiment, the glioblastoma is EGFRVIII positive. In one embodiment, the glioblastoma is primary glioblastoma (primary GBM status). In one embodiment, the glioblastoma is secondary glioblastoma (secondary GBM status). In one embodiment, the glioblastoma is classified as wild-type isocitrate dehydrogenase 1 (IDH-1) and / or wild-type isocitrate dehydrogenase (NADP(+)) 2 (IDH-2). In one embodiment, glioblastoma is classified as mutant isocitrate dehydrogenase 1 (IDH-1) and / or mutant isocitrate dehydrogenase (NADP(+))2 (IDH-2). In one embodiment, glioblastoma is negative for the glioma CpG island methylation phenotype (GCIMP). In one embodiment, glioblastoma is GCIMP positive.

[0235] In some embodiments, the methods provided herein relate to methods for immunizing a subject from cancer by administering the described engineered NK cells. In some embodiments, immunizing a subject from cancer includes immunizing a subject from glioblastoma and includes administering NK cells engineered to express IL-21 by gene transfer. In some embodiments, immunizing a subject from cancer includes immunizing a subject from glioblastoma and includes administering NK cells engineered to overexpress CEBP family proteins by gene transfer. In some embodiments, immunizing a subject from cancer includes immunizing a subject from glioblastoma and includes administering NK cells modified to overexpress CEBPD by gene transfer.

[0236] V. Kit Certain aspects of this disclosure also relate to kits comprising compositions for carrying out the compositions and / or methods of this disclosure. In certain embodiments, the kit comprises fresh or cryopreserved NK cells, which may or may not be pre-activated or amplified. The NK cells may or may not be already engineered to overexpress one or more CEBP proteins compared to unengineered NK cells and / or NK cells engineered to express IL-15. If the NK cells have not yet been engineered to overexpress one or more CEBP proteins, the kit may comprise reagents for transfection or transduction of the corresponding NK cells. These may comprise reagents such as vectors for expressing the components and primers for amplifying the components. In some cases, the NK cells may or may not already express one or more heterologous proteins, and if not, the kit may comprise vectors for expressing the heterologous proteins, primers for amplifying the heterologous proteins, and so on.

[0237] In one embodiment, the kit is for a subject of approximately 1 × 10 6 ~Approx. 1×10 10It is designed to allow the recipient to receive a cell dose, or a range of cell doses that can be induced in between. In one embodiment, the kit is designed to allow the target to receive approximately 4 × 10 5 , 8×10 5 , 4×10 6 , 8×10 6 , 4×10 7 , 8×10 7 , 4×10 8 , 8×10 8 , 4×10 9 , 8×10 9 , 4×10 10 , or 8×10 10 It is designed to allow the patient to receive a range of cell doses that can be achieved in between.

[0238] In one embodiment, the kit is approximately 1 × 10 6 ~About 100×10 6 The kit contains a cell composition dispensed at a cell concentration of approximately 3 × 10 6 ~Approx. 25×10 6 It contains a cell composition dispensed at a cell concentration of [value].

[0239] The kit can be prepared by individually packaging each component or by placing them in tubes, bottles, vials, syringes, or other suitable containers. Individual components may also be provided in concentrated quantities within the kit. In some embodiments, components are provided individually at the same concentration as they would be in solution with the other components. Component concentrations may be 1x, 2x, 5x, 10x, or 20x or higher.

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[0241] VII. Examples The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments are techniques found by the inventors to function well in the implementation of the invention and therefore constitute preferred forms of implementation. However, those skilled in the art should understand that, in light of this disclosure, many modifications can be made to the specific embodiments disclosed to obtain similar or comparable results without departing from the spirit and scope of the invention.

[0242] material and method Unless otherwise specified, the assays, assay materials, and experiments described in the following examples were carried out using the methods described herein.

[0243] Glioblastoma stem cells (GSC) GSCs were obtained from human primary GBM samples following methods described in previous studies. GSCs were cultured in Dulbecco modified Eagle medium:nutrient mixture F-12 (DMEM / F12) supplemented with 20 ng / ml epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) (both PEPROTech®, Cranbury, NJ), B27 supplement (1:50; Invitrogen®, Carlsbad, CA), 100 units of penicillin, and 100 mg / ml streptomycin (Thermo Fisher Scientific®, Waltham, MA), and subcultured every 4-5 days. All GSC cell lines used in this study were prepared at MD Anderson Cancer Center.

[0244] Amplification of NK cells NK cells were purified from umbilical cord blood (CB) using an NK cell isolation kit (Miltenyi Biotec, Inc., San Diego, CA). NK cells were stimulated on day 0 in complete Cell Genix® GMP SCGM stem cell growth medium (Cell Genix® GmbH, Freiburg, Germany) supplemented with recombinant human IL-2 (Proleukin, 200 IU / ml; Chiron, Emeryville, CA) in a 2:1 ratio (feeder cells:NK) with irradiated (100 Gy) K562-based feeder cells (modified to express 4-1BB ligand and CD137 ligand, called universal antigen-presenting cells [uAPC]). For IL-21 priming, NTNK cells were cultured for 2 days in exogenous human IL-21 (3 ng / ml, PEPROTech®, Cranbury, NJ).

[0245] Retrovirus transfection and transduction Transfection of 293T cells and transduction of CB-derived NK cells were performed using retroviral vectors encoding human IL-15 or IL-21. NK cells were transduced on human fibronectin-coated plates (Clontech® Laboratories, Inc., Mountain View, CA) on day +5. Untransfected 293T cells or untransduced (NT) NK cells were used as negative controls. To generate NK cells overexpressing CEBPD (CEBPD OE, also known as CEBPD KI), CEBPD cDNA (SEQ ID NO: 20) was cloned into a pSFG retroviral expression vector to produce the retrovirus.

[0246] Flow cytometry CB-NK cells were stained with Live / Dead-aqua (Thermo Fisher Scientific®) and anti-human CD56 antibody (clone HCD56) and anti-human CD3 antibody (clone SK7, BioLegend®, San Diego, CA). Anti-human IgG1 antibody (Jackson ImmunoResearch) was used to confirm the transfection and transduction efficiency of 293T and NK cells, respectively. Cell viability was evaluated using Annexin V (Thermo Fisher Scientific®) and Live / Dead according to the manufacturer's instructions. For CEBPD staining, cells were first stained with a surface antibody for 20 minutes, washed, fixed / permeabilized, and then stained with anti-human CEBPD antibody (clone C6, Santa Cruz Biotechnology, Dallas, TX) at room temperature for 30 minutes. Mouse tissue cell suspensions were first blocked for 10 minutes with a human Fc receptor inhibitor solution (BD Biosciences, San Jose, CA), and then surface-stained with anti-human CD45 antibody (clone HI30, BioLegend®), anti-mouse CD45 antibody (clone 30-F11, BioLegend®), anti-human CD56 antibody, anti-human CD16 antibody (clone 3G-8, BioLegend®), and anti-human CD3 antibody. All cells were treated with BD LSR Fortessa TM Data was acquired and analyzed using the instrument, and the data was analyzed with FlowJo software (version 10.7.1).

[0247] Suppression Assay NK cells were cultured alone or in a 1:1 ratio with GSCs and incubated at 37°C for 48 hours before functional assays were performed. After co-culture, NK cells were isolated using the human NK cell isolation kit and human CD105 microbeads from Miltenyi Biotec, Inc. (San Diego, CA, USA) according to the manufacturer's instructions.

[0248] Real-time NK cell cytotoxicity assay NK cells were co-cultured in a 1:1 ratio with K562 or GSC target cells labeled with CellTracker® Deep Red Dye (Thermo Fisher Scientific®, Waltham, MA), or with GSC target cells pre-transduced with an mCherry-expressing retroviral vector. In the re-challenge experiment, fresh GSC target cells pre-transduced with mCherry were added to the plate every 2-3 days. Apoptosis was induced by CellEvent TM Detection was performed using Caspase-3 / 7 Green Detection Reagent (Thermo Fisher Scientific®). An IncuCyte® S3 live cell analysis system (Sartorius®, Goettingen, Germany) was used, with a 10x objective lens, measuring 1.75 × 1.29 mm at four locations per well. 2 Frames were acquired from the region at 1-hour or 2-hour intervals over a 24-hour period. Values ​​from four regions in each well were aggregated, and the average of three replicates was calculated. The results were graphed as a percentage of cytotoxicity, calculated by dividing the ratio of overlapping red and green signals (counts per image) by the red signal (counts per image). Tumor cell counts (red signal) were plotted over time to track tumor growth or elimination by NK cells (tumor cell index).

[0249] 3D Real-Time Cell Injury Assay GSC spheroids were prepared by suspending 10,000 single cells in 100 μl of CellGenix® GMP SCGM stem cell growth medium (CellGenix®) and seeding them in a 96-well transparent round-bottom ultra-low adhesion microplate (Corning®, Glendale, AR). After 48 hours, spheroid formation was confirmed by microscopy, and 20,000 NK cells were added to the spheroid-containing wells. Frames were acquired at 2-hour intervals for 6 days using a 10x objective lens. Red signals (tumors) were quantified using the IncuCyte® S3 live cell analysis system (Sartorius®).

[0250] Single-cell secretome assay (IsoPlexis®) IL-15NK, IL-21NK, and NT-NK cells were labeled with a fluorescent dye (IsoPlexis® stain cell membrane 405), co-cultured with GSC20 cells for 2 hours, and then purified by positive selection using CD56+ microbeads (Miltenyi Biotec, Waltham, MA). Total 3 × 10⁶ 4 Individual NK cells are individually coded according to the manufacturer's protocol (IsoPlexis®, Branford, CT). TM The cells were loaded onto a chip, and single-cell 32-plex cytokine sequence profiling was performed using a fully validated cytokine panel with the IsoPlexis® single-cell platform. TM Using software, the percentage of cells secreting multiple cytokines (2, 3, 4, or 5 or more) was measured in each sample. These cells were then classified into effector, stimulant, regulatory, or chemotactic categories, and the polyfunctionality index (PSI) was calculated by multiplying the mean fluorescence intensity (MFI) of the proteins secreted by NK cells by the percentage of multifunctional cells.

[0251] ELISA and multiplex ELISA IL-15 and IL-21 levels were measured using Invitrogen® IL-15 or IL-21 ELISA kits according to manufacturer instructions, collected from the supernatant of cytokine-transduced NK cells 5 days post-transduction. For multiplex ELISA, co-culture supernatants of GSCs and NK cells were collected at different time points, and cytokine and chemokine production was evaluated using Milliplex® Human Cytokine / Chemokine Magnetic bead Premixed 41 Plex Kit (EMD Millipore Corporation, Burlington, MA). Samples were taken from Millipore MAGPIX. TM The device (EMD Millipore) reads the data, and data acquisition is performed using Luminex® xPONENT® software, and Belysa TM The data was analyzed using Analysis Software (EMD Millipore). The data was plotted using Morpheus (https: / / software.broadinstitute.org / morpheus).

[0252] Metabolic assay Extracellular oxidation rate (ECAR) was measured using the Glycolytic Stress Test, and oxygen consumption rate (OCR) was measured using the Mito Stress Test or XF Substrate Oxidation Stress Test with an Agilent® Seahorse® XFe96 Analyzer (Agilent®) according to the manufacturer's instructions. NK cells were assayed alone or after 48 hours of co-culture with GSC20 cells followed by purification. Cells were seeded at 250,000 cells / well in 96-well microplates in the medium for each assay. Data were analyzed using Wave Software (Agilent®).

[0253] Measurement of reactive oxygen species (ROS) To identify reactive oxygen species (ROS) produced by NK cells, cells were stained and analyzed by flow cytometry. MitoSOX was used for mitochondrial ROS staining. TM Red (Cat #M36008, Life Technologies®, Grand Island, NY, USA) was used according to the manufacturer's protocol. Cells were taken from BD LSRFortessa. TM The results were analyzed using an instrument and expressed as the percentage of MitoSOXTM-positive NK cells.

[0254] Mass cytometry The antibody conjugation method is described elsewhere. Table 2 lists the antibodies used to characterize NK cells in this study. [Table 1]

[0255] Mass cytometry - Sample preparation, staining, and acquisition

[0256] NK cells were cultured alone or with GSC20 cells (E:T ratio 1:1) for 48 hours. After washing with cell staining buffer (0.5% BSA / PBS), NK cells were treated with human Fc receptor blocking solution (Trustain FcX™, BioLegend®, San Diego, CA) at room temperature for 10 minutes. Subsequently, they were stained with a CyTOF® antibody mixture against cell surface markers (Table 2). The antibody conjugation method has been previously described. Cells were recorded using a Helios instrument (Fluidigm®) at a rate of 300 events per second with Helios 6.5.358 acquisition software (Fluidigm®). Mass cytometry data were normalized based on the EQTM 4-element signal shift over time using Fluidigm® normalization software 2. Initial data quality control was performed using FlowJo version 10.7. Singlets were selected based on iridium-193 staining and event duration, and calibration beads were excluded by gating. Dead cells were excluded by Pt195 channeling, followed by CD45+ cells, and then the target NK cell population (CD3). - CD56 + ) was selected. For automated clustering, a total of 320,000 cells were proportionally extracted from each sample.

[0257] Data Analysis

[0258] Mass cytometry data were integrated using principal component analysis (PCA) with the "RunPCA" function of the R package Seurat(v3). Dimensionality reduction was performed using the top 20 principal components with the "RunUMAP" function of Seurat(v3). UMAP plots were generated using the R package ggplot2(v3.2.1). The data were analyzed using a combination of automated dimensionality reduction methods (including viSNE) and FlowSOM, and clustering was performed for deep phenotyping of immune cells. Related cell clusters were further subdivided using a custom-built pipeline. CD45 was used for heatmap generation. + CD56 + CD3 -The gated FCS file was exported from FlowJo to R, and the "read.FCS" function of the R package flowCore (v3.10) was used. The mean values ​​of all markers were plotted as a heatmap using the "pheatmap" function of the R package pheatmap (v1.0.12). Markers showing similar expression were clustered hierarchically.

[0259] Xenotransplant mouse models (GBM) To evaluate the antitumor effect of cytokine-transduced NK cells against GSCs in vivo, a human xenograft model of NOD / SCID IL-2Rγnull (NSG) was used (Jackson Laboratories, Bar Harbor, ME). Patient-derived GSC mouse models were used because they exhibited higher invasiveness and migratory ability upon intracranial transplantation compared to conventional glioma cell lines. 24 Patient-derived GSC20, GSC262, GSC267, GSC8-11, or GSC272 cells were each measured in 0.5 × 10⁶ units. 6 One individual was intracranially implanted into the right frontal lobe of a 5-week-old female NSG mouse using a guide screw system (previously reported 24). In the re-challenge experiment, 0.25 × 10⁻¹⁴ 6 Ten GSC20 cells were intracranially transplanted. To enhance the uniformity of engraftment and growth of xenografts, the cells were simultaneously injected into 10 animals using a multiport microinfusion syringe pump (Harvard Apparatus, Holliston, MA). During the procedure, the animals were anesthetized with xylazine / ketamine. For in vivo bioluminescence imaging, GSCs expressed luciferase by lentiviral transduction. Tumor growth rates were monitored weekly using bioluminescence imaging (BLI) (Xenogen-IVIS® 200 Imaging system; Caliper, Waltham, MA). Signal quantification in photons / second (p / s) was performed by calculating the photon flux rate within a standardized region of interest (ROI) using Living Image software (Caliper). Seven days after tumor transplantation, NK cells (0.5 × 10⁶) were measured. 6 or 0.1 × 10 6A suspension of NK cells (in 4 μl) was injected intratumorally via a guide screw. Mice exhibiting neurological symptoms (e.g., hydrocephalus, seizures, ataxia) or in a necrotic state were euthanized. Subsequently, the brain and other tissues were excised and processed for analysis.

[0260] Histopathological analysis

[0261] Brain tissue samples were collected from untreated control mice and mice treated with IL-15 transduction NK cells or IL-21 transduction NK cells. Brain tissue was fixed in 10% neutral buffered formalin and then embedded in paraffin. Formalin-fixed, paraffin-embedded tissue was prepared into 4 μm sections and stained with hematoxylin and eosin using standard methods. The brain was examined for the presence of glioblastoma tumor cells, general gliosis, and NK cell infiltration. Sections without tumors were examined by a board-certified veterinary pathologist using a Leica® DM 2500 optical microscope to confirm evidence of meningoencephalitis. One section was examined from each sample. Representative images were taken using a Leica® DFC495 camera with a 10x objective lens from comparable areas of the cerebral hemispheres.

[0262] Immunohistochemical staining

[0263] Formalin-fixed, paraffin-embedded tissue was typically sectioned to 4 μm thickness and mounted on glass slides. After deparaffinization, Granzyme B (human-specific, rabbit monoclonal antibody clone D6E9W, Cell Signaling Technologies) or CD16 (human-specific, rabbit polyclonal antibody, Thermo Fisher Scientific®) antibody was added to the tissue sections to identify human NK cells. Anti-Ki-67 antibody (rabbit monoclonal antibody clone SP6, Abcam) was used to identify proliferating GSC tumor cells in brain sections. Anti-GFP antibody was used to identify astrocytes, and anti-Iba1 antibody was used to identify microglia. Before staining, antigen retrieval was performed in citrate buffer at 95°C for 20 minutes. The primary antibody was diluted 1:100 and reacted for 60 minutes. The nuclei were counterstained with hematoxylin, and the sections were mounted on coverslips as usual. Slides were observed with a Leica® DM2500 microscope, and images were captured at 4x, 10x, 20x, and 40x magnification using a Leica® DMC6200 camera and Leica® Application Suite (LAS) software. Six antibodies were used for multiplex immunofluorescence: anti-Granzyme B, anti-Iba1 (clone EPR16589, Abcam), anti-GFAP (Dako), anti-Cleaved Caspase-3 (BioCare Medical), anti-luciferase (clone EPR17790, Abcam), and anti-Ki-67. DAPI was used for cell nucleus identification. Slides were scanned with an Aperio AT2, and images were aligned and fused using HALO v.3.6.

[0264] Processing of mouse brain tissue Mononuclear cells were identified using the protocol developed by Pino et al. 24Cells were isolated from mouse brain tissue using a Percoll® (GE HealthCare, Chicago, IL) density gradient. In short, the brain tissue was dissociated and passed through a 70 μm cell strainer (Life Science, Durham, NC) to obtain a homogeneous cell suspension. A 70% isotonic Percoll® solution was then overlaid on the cell suspension, which was suspended in a 30% isotonic Percoll® solution. The cells were centrifuged at 18°C ​​at 500 × g for 30 minutes without using a brake. 2–3 ml from the 70%-30% interface was collected in a clean tube and washed once with PBS 1X. After this treatment, the cells were ready for use in the immunostaining described above.

[0265] Single-cell ATAC sequence IL-15 transduction NK cells and IL-21 transduction NK cells were co-cultured with GSC20 cells (E:T ratio 1:1). Fresh GSC cells were added to the co-culture every 2-3 days. Cells were harvested and cryopreserved on day 3 (1X GSC) and day 9 (3X GSC). NK cells cultured in the absence of GSCs were harvested on day 0 and used as a baseline control.

[0266] The cryopreserved cell suspensions were thawed in a 37°C water bath and their viability was measured. The cells were treated with nuclear lysis buffer (10X Genomics™, San Francisco, CA) to wash the nuclei, and then diluted with nuclear buffer (10X Genomics™). TMThe samples were resuspended in ) and counted in volumes that yielded 5,000–10,000 nuclei. Mononuclear libraries were prepared using 10X Genomics™ Chromium Next GEM ATAC Capture and Library (V1.1) according to the manufacturer's protocol. Nine samples were pooled to a final concentration of 10 nM. The pooled samples were validated by qPCR before sequencing using a NovaSeq 6000 sequencer with an S1, 100-cycle flow cell. Sequencing was performed with 50 cycles for read 1, 8 cycles for i7, 16 cycles for the i5 index, and 49 cycles for read 2. The data is publicly available under accession number GSE227098.

[0267] Upstream analysis of scATAC-seq data For each sample, fragment and peak profiles were obtained using 10X Genomics. TM CellRanger TM The fragments were generated using the pipeline's default parameters. The five columns constituting the fragment file were chromosome, start position, end position, cell barcode, and PCR duplicate count, which were specified as a coordinate-sorted, block gzip-compressed (bgzip) and indexed browser-extended data file. The locations of the two Tn5 integration events that generated the sequenced DNA fragments were indicated in the start and end fields of the fragment file.

[0268] Downstream analysis of scATAC-seq data Downstream analysis was performed using R 4.0 with Signac 63. Since peak calling was performed independently for each sample, a common peak set was created for all samples using Signac's reduce function. Peaks with a width of less than 20 bp or greater than 10,000 bp were removed. The fragment matrix for each sample was adjusted based on the common peak set and generated using the CreateFragmentObject function. Peak profiles from multiple samples were combined into a single unified matrix, and peak coordinates were shared across all samples.

[0269] As a QC step, cells with fewer than 500 fragments and features observed in cells with fewer than 500 fragments were excluded. To perform dimensionality reduction, RunTFIDF was run on the top peaks identified using the FindTopFeatures function (min.cutoff=10). The RunSVD function was used to perform dimensionality reduction in the peak assay, with the dimension set to 30.

[0270] By integrating and clustering single-cell ATAC peak profiles of 7,000–11,000 NK cells (Table 3) across three time points—baseline, day 3, and day 9—using the Signac pipeline, 10 distinct NK cell clusters were identified. [Table 2]

[0271] To identify peaks that differed between samples and clusters, differential accessibility (DA) tests were performed using the FindMarkers function, with minimum.pct set to 5% and the number of fragments as a latent variable. To identify important regulatory sequences specific to samples and clusters, the FindMotifs function was used to search for DNA motifs (i.e., transcription factors: TFs) that were excessively present in the differentially accessible peak set. This calculates the probability that a motif would appear by chance at the observed frequency compared to a background peak set matching the GC content. A higher score for a motif indicates that its TF is abundant in the differential peak. The EnhancedVolcano function was used to visualize the fold-change of TF enrichment between two samples (https: / / github.com / kevinblighe / enhancedvolcano).

[0272] To determine the accessibility levels of CEBPD and CEBPB targets, the GeneActivity function from the Signac package was used to quantify ATAC-seq counts in the 2 kb upstream region and the gene itself in each cell, and gene-level accessibility was calculated.

[0273] Single-cell RNA sequencing IL-15NK cells or IL-21NK cells were co-cultured with GSC20 cells (E:T ratio 1:1). Fresh GSC cells were added to the co-culture every 2-3 days. Cells were harvested and cryopreserved on day 3 (1X GSC) and day 9 (3X GSC). NK cells cultured in the absence of GSCs were harvested on day 0 and used as a baseline control.

[0274] The frozen cell suspension was thawed in a 37°C water bath and then Countessed using Trypan Blue staining solution (0.4%). TM Cell viability was measured using II FL. Cells were suspended in a solution of phosphate-buffered saline (PBS) with 0.4% bovine serum albumin (BSA), loaded at a recommended rate of 500–1000 cells per μl, and 7,000–10,000 cells were collected for single-cell RNA sequencing (scRNA-seq). Single-cell capture, barcoding, and library preparation were performed using 10X Genomics. TM The Single Cell Chromium 3' protocol (CG000183, V3.1) was followed. Ten libraries were pooled to a final concentration of 10 nM, and after confirming the concentration by qPCR, sequencing was performed on a NovaSeq 6000 sequencer using the S2 100-cycle flow cell. Using the MD Anderson ATGC (Advanced Technology Genomics Core) core, read 1 was sequenced in 28 cycles, the i7 index in 8 cycles, and read 2 in 91 cycles. The data is publicly available under accession number GSE227098.

[0275] Downstream analysis was performed using Seurat software 63 in R 4.0. As a QC step, features with fewer than 50 features and features found only in fewer than 100 cells were removed. The resulting single-cell dataset included a total of 52,873 cells across three time points, targeting IL-21NK cells and IL-15NK cells (with and without GSCs) (see Table 3). For dimensionality reduction, RunPCA was performed using the top 2,500 variable genes identified by the FindTopFeatures function. Sample / cluster-to-cluster DEG analysis was performed using the FindMarkers function with minimum.pct set to 5% and gene count as a latent variable.

[0276] The CEBPD regulon score at the gene expression level was calculated by averaging the expression levels of 106 CEBPD target genes estimated by pySCENIC at the single-cell level using the AddModuleScore function.

[0277] Pathways rich in CEBPB and CEBPD target genes were identified by searching the MSigDB Hallmark database and using the Enricher tool. The corrected p-value was 10. -5 Only the higher-scoring paths were retained.

[0278] For genomic coverage derived from scATAC and scRNA profiling, we retained BAM alignment mapping files obtained from cellranger count or cellranger-atac count pipelines that processed scRNA or scATAC sequencing data. Cell barcodes for filtered scRNA or scATAC samples were exported from the Seurat or Signac packages in R, respectively. BAM files of these filtered cells were extracted and generated using the filterbarcodes function of sinto software (https: / / timoast.github.io / sinto / ). The resulting BAM files were converted to bigwig format using the bamCoverage command in deepTools software and visualized in IGV (Integrative Genomics Viewer).

[0279] How to create a pySCENIC diagram To estimate major transcription factors (TFs) and gene regulatory networks from scRNA-seq data, we used the pySCENIC v0.11.2 (Single-Cell Regulatory Network Inference and Clustering) pipeline. In summary, the first step involved using pySCENIC with default parameters on a high-performance computing system to estimate regulatory interactions between a predefined list of TFs and candidate target genes. This was done using the GRNBoost2 algorithm (gradient boosting regression) and Arboreto, which utilizes co-expression patterns from scRNA-seq data. As a result, we obtained an adjacencies matrix linking each TF to a target gene, and importance scores separating highly reliable and weak interactions. Next, candidate modules were generated from these interactions, each consisting of a TF regulator and its target gene list. To distinguish between direct and indirect targets of a given regulator, these co-expression modules were refined by selecting target genes that possess their TF-specific DNA motifs in their promoter regions. This was achieved by cis-regulatory module scoring using RcisTarget, and cisTarget motif enrichment was searched using the whole-genome ranking of all known TF-related motifs registered in the pySCENIC database. This analysis yielded a normalized enrichment score (NES) that quantitatively indicates the enrichment of individual motifs. Only target genes with NES > 4 were used for downstream analysis. Finally, an AUC score was calculated to measure and quantify the relative biological activity of each regulon at the cellular level. The pySCENIC analysis defined 456 regulons with direct target genes showing significant TF motif enrichment, and the AUC regulon activity score for each was also calculated. These scores were overlaid on scRNA-seq data using Seurat 4.1.0, and differential regulons over time between IL-21 NK cell samples and IL-15 NK cell samples were identified using Seurat's FindMarkers function.The notable differential regulons were visualized using the DoHeatmap function on a scaled AUC matrix to facilitate comparisons between samples.

[0280] Gene expression was quantified using RSEM (1.3.3) with the bowtie2 (2.4.2) aligner from a fastq file. Gene counts were read from the RSEM output file using the R package tximport. The data is available as accession number GSE227098. Differential gene expression analysis (DEG) was performed using a 2-way ANOVA-fitted model in NTNK cells, IL-15NK cells, IL-21NK cells, and under conditions of CEBPD presence (CEBPD KI (OE)) or absence (CEBPD KO) (donor n=4), with NTNK cells, IL-15NK cells, IL-21NK cells, or the WT group set as reference levels. Differentially expressed genes (DEGs) were defined by a Log2 multiplier change <0.3 or >0.3 and a p-value <0.05. DEG was performed in R using the OBIF package (version 1.0).

[0281] Enrichment analyses were performed by QIAGEN® IPA certified analysts using Ingenuity Pathway Analysis Software (QIAGEN® Inc.) with core analysis functions applied to expression levels. Unsupervised enrichment was performed for pathways, disease and function, regulators, and networks, and the IL-21 vs IL-15 wild-type (WT) response was evaluated. Significant enrichment was defined as a Z score <-2 or >2 and FDR <0.05. Expression levels from bulk RNA-seq differential analysis were superimposed, and regulatory network analyses were adjusted using regions accessible by bulk and single-cell ATAC-seq as references.

[0282] Differential gene expression analysis (DEG) was performed individually on IL-21-transfected NK cells and non-transfected NK cells, corrected for umbilical cord blood. CEBPD KI(OE), CEBPD KO, and NTNK cells were compared, with NTNK cells used as the reference level. DEGs were defined as FDR-corrected p-values ​​< 0.05. DEG was performed using the DESeq2 package in R (version 4.1.1).

[0283] Pathway enrichment analysis (GSEA) was performed using predefined CEBPD downstream genes. GSEA was executed using the gsea function in the clusterProfiler package in R (version 4.1.1), employing the ranked Wald test statistic for all genes in differential gene expression analysis.

[0284] Bulk ATAC-seq analysis ATAC-seq library preparation was performed using the MDACC Epigenomics Profiling Core, with some modifications to the previously reported protocol 12. In short, 50,000 nuclei isolated from IL-15NK cells, IL-21NK cells, and NTNK cells (including CEBPD KI(OE) and KO conditions, respectively) from four donors were fragmented, and the resulting libraries were purified using SPRISelect™ beads (Beckman Coulter). The libraries were sequenced at 2 × 100 bp using an Illumina™ NovaSeq 6000, yielding at least 50 million high-quality mapping reads per sample. The data are available under accession number GSE227098.

[0285] For each bulk ATAC-seq sample, paired-end reads from the fastq file were aligned to the human genome (GRCh38) using bwa mem mode with duplicate reads removed. The 5' ends of the ATAC-seq reads were shifted to the actual transposase cleavage sites using the DeepTools alignmentSieve module. Peaks were called using paired-end read information with Macs2, and the minimum FDR (q value) cutoff for peak detection was set to 0.05. Macs2 outputs from multiple samples were read using DiffBind, and overlapping peak sets across samples were identified using DiffBind's bUseSummarizeOverlaps function. Transcription factor (TF) activity levels were calculated using Signac's RunChromVAR function, and differential TFs between different groups were identified using the FindMarkers function (min.pct=0.5, logfc.threshold=2).

[0286] Chromatin immunoprecipitation (ChIP) qPCR and ChIP sequencing (ChIP-seq) Chromatin immunoprecipitation (ChIP) was performed at the MD Anderson Cancer Center Epigenomic Profile Core. IL-15NK, IL-21NK, and NTNK cells (donor n=2-3) were crosslinked with 1% formaldehyde at room temperature for 10 minutes, followed by the addition of glycine (final concentration 125 mM) to pause the reaction for 5 minutes. 64 Chromatin was sonicated to an average of 600 bp. Chromatin and binding proteins were immunoprecipitated using anti-CEBPD antibody (Santa Cruz Biotechnology) and IgG antibody. Input DNA and CEBPD ChIP DNA libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB). ChIP-Seq libraries and the corresponding input libraries were sequenced using Illumina® NextSeq® 500 and HiSeq® 3000 with a 50-base single-read protocol.

[0287] In ChIP quantitative PCR (ChIP-qPCR), chromatin was immunoprecipitated at 4°C for 12-16 hours using antibodies that recognize pSTAT1 (Tyr701) or pSTAT3 (Tyr705) (both from Cell Signaling Technology), or control rabbit immunoglobulin G (IgG). After decrosslinking, quantitative real-time PCR was performed on the precipitated DNA using primers corresponding to specific target gene regions. The primers used are as follows: 65 : CEBPD Forward:5'- GGTTTCACCATGTTGACCAG -3'(Sequence ID: 57) CEBPD Reverse:5'- AGAATGGGCTTTGTCATGTG -3'(Sequence ID: 58)

[0288] The target DNA region was detected by SYBR real-time quantitative PCR, and its relative enrichment relative to the input was calculated.

[0289] ChIP-seq analysis Sequence adapters and low-quality bases were trimmed with Trimmomatic 0.38. Cleaned reads were aligned to the reference genome hg38 using bowtie2. Peak signals were visualized with Integrative Genomics Viewer Version 2.16.1.

[0290] CEBPD ChIP-seq peak and tag density profiles From the aligned donor sample files (BAM) for each processing condition, HOMER 66The makeTagDirectory function (-genome hg38 -checkGC) was used to create an integrated tag directory. The annotatePeaks.pl function was used to identify the transcription start sites (TSS) of CEBPD-specific gene targets (tss hg38 -list<gene list> Create an annotated region list for ) and for each sample, the area around TSS (<annotated region list> hg38 -size -300,100 -d<combined tag directories> The normalized tag density profile (per base per peak) was calculated. Using GraphPad Prism 10 software, a box plot showing the sequenced normalized tag density of CEBPD-specific gene targets, and a peak profile plot (5bp bin) showing the sequenced CEBPD occupancy from all donors were created. Genomic visualization of specific CEBPD regulatory gene targets was performed using the sample BAM file with Integrative Genomics Viewer. 67 The analysis was performed on the human genome GRCh38 using (IGV 2.16.0) software.

[0291] qPCR RNA was extracted from NK cells, iscript TM Reverse transcription was performed using a cDNA Synthesis kit (Bio-Rad®, Mylan, Italy). The relative expression level of the target gene was evaluated by RT-qPCR using Applied Biosystems® Fast SYBR® Green qPCR master mix (Thermo Fisher Scientific®). Pre-made primers for CEBPD, KLF2, and BNIP3L were used. TM It was purchased from [source]. The relative expression level was determined by comparison with the housekeeping gene S18.

[0292] CRISPR gene editing We targeted the CEBPD gene in NK cells using pre-designed sgRNAs. The sgRNAs were ordered from SYNTHEGO™ (Gene Knockout Kit v2 - human - CEBPD - 1.5 nmol, Gene Knockout Kit v2 - human - STAT3 - 1.5 nmol), and Cas9 was obtained from IDT. TM I purchased a proprietary Alt-RTM HiFi (cat #1081061) from [manufacturer name].

[0293] The following three types of sgRNAs were used for CEBPD gene mutations:

[0294] sgRNA #1: GCCGUCCAGGCUGAAGAGCG (Sequence ID: 52)

[0295] sgRNA #2: CCCGGUUCGUAGAAGGGCGC (Sequence ID: 53)

[0296] sgRNA #3: CUCUCGUCGUCGUACAUGGC (Sequence ID: 54)

[0297] The following three types of sgRNAs were used for STAT3 gene mutations:

[0298] sgRNA #1: AAUCUUGACUCUCAAUCCAA (Sequence ID: 59)

[0299] sgRNA #2: AGCUGUCACUGUAGAGCUGA (Sequence ID: 60)

[0300] sgRNA #3: AUUUUAGCAGGAUGGCCCAA (Sequence ID: 61)

[0301] The sgRNA derived from the kit was resuspended in nuclease-free TE buffer to a final concentration of 100 μM. The sgRNA was diluted to 30 μM in nuclease-free water. The sgRNA was mixed with Cas9 and T buffer (Neon™ Electroporation Kit, Invitrogen®) in a 3:1 ratio. The ribonucleoprotein complexes (RNPs) were incubated at room temperature (RT) for 10 minutes. During incubation, each T25 flask was filled with culture medium, Universal APC (effector:target = 1:2), and 200 IU / ml of IL-2 (for NK cells only), and placed in a 37°C incubator. Effector cells were harvested by centrifugation and washed twice with PBS in aliquots of 500,000 cells each. For electroporation, the supernatant was removed as much as possible without disturbing the pellet, and the cells were suspended in Resuspension Buffer T. The final concentrations used for electroporation were 1.8 μM sgRNA, 0.62 μM Cas9 nuclease, and 0.45 μM Cas9 electroporation enhancer. Cells were processed using a Neon Transfection System at 1600 V, with a pulse width of 10 ms and 3 pulses, using a 10 μl electroporation tip (Thermo Fisher Scientific®, cat #MPK5000). After electroporation, the cells were transferred to a prepared flask and placed in a 37°C incubator. Knockout efficiency was evaluated by PCR and agarose gel electrophoresis.

[0302] The following primers were used for CEBPD gene amplification: Forward:5'- GACAGCCTCGCTTGGAC -3'(Sequence ID: 55) Reverse: 5'- CAAGCTCACCACGGTCTG -3' (Sequence ID: 56)

[0303] The following primers were used for STAT3 gene amplification: Forward:5'- GGGTGCCCCTTTATCTCCTG -3'(Sequence ID: 62) Reverse: 5'- GGGAGAAAGAAGCAGGGTCC -3' (Sequence number: 63)

[0304] statistical analysis Statistical analysis and plotting were performed using Prism 9.2.0 software (GraphPad, San Diego, CA). Student's t-test was used for significance testing between two groups, one-way ANOVA was used for group comparisons under certain conditions, and two-way ANOVA was used for group comparisons over specific time periods. Dunnett correction was used for comparisons with reference or control groups. Bonferroni correction was also applied to repeated measures. Mean ± standard error (sem) is shown. Overall survival (OS) analysis was calculated using the Kaplan-Meier method and compared with the treatment group using the log-rank test (95% confidence interval (CI)). The statistical significance level was set at p<0.05, and results were reported as *p<0.05, **p<0.01, and ***p<0.001. The statistical tests used and sample sizes (n) are described in the captions for each figure.

[0305] research approval Tumor tissue used to generate glioblastoma stem cells (GSCs) was excised from patients who signed written informed consent and collected in accordance with the University of Texas MD Anderson Cancer Center (Houston) IRB protocols LAB04-0001, LAB03-0687, and 2012-0441. All tissue samples were anonymized. All studies were conducted in accordance with the Declaration of Helsinki. All animal experiments were conducted in accordance with the recommendations of the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and were approved by MD Anderson Cancer Center's IACUC protocol number 00001263-RN01.

[0306] Example 1 NK cells manipulated with cytokines showed potent anti-GSC activity. Cytokine manipulation has been used as a tool to improve the proliferation, persistence, and cytotoxic activity of T cells and NK cells against cancer. 22GSCs (glioblastoma stem cells) possess unique transcriptional, epigenetic, and metabolic characteristics and have tumor regeneration capabilities. 23 To clarify the effect of cytokine administration on the antitumor response of NK cells, particularly their response to GSCs, umbilical cord blood (CB)-derived NK cells were manipulated to express and autonomously release secreted IL-15 (SEQ ID NO: 29) or secreted IL-21 (SEQ ID NO: 31) (e.g., the constructs shown in Figure 1A (SEQ ID NOs: 44 and 43)). Transduction efficiency was analyzed by flow cytometry, with CD56 expression used for NK cell identification and co-transduction marker IgG1 Fc expression used as a surrogate indicator of IL-15 or IL-21 expression. The transduction efficiency of representative donor cells was approximately 84.7% for IL-15 NK cells and approximately 89.1% for IL-21 NK cells (Figure 1B). The concentrations of IL-21 or IL-15 secreted into the supernatant of non-transduced (NT) NK cells (NTNK), IL-21 NK cells (IL-21NK), and IL-15 NK cells (IL-15NK) were analyzed by ELISA 5 days after transduction (measured in pg / mL). IL-15 NK cells strongly expressed IL-15 (~1500 pg / mL; n=3 donors), and IL-21 NK cells strongly expressed IL-21 (~2,200 pg / mL; n=5 donors) (Figure 1C). Subsequently, the response of cytokine-contributing NK cells to GSCs was evaluated. NTNK cells were used as a control. After ex vivo culture with K562-based feeder cells and IL-2, no significant difference in short-term NK cell proliferation was observed among the three groups (Figure 7A). However, IL-15NK and IL-21NK cells showed significantly higher cytotoxicity in vitro compared to NTNK cells against GSC20 (n=6 donors) and GSC8-11 (n=3 donors) cells (Figure 1D and Figure 7B).

[0307] The inventors previously showed that GSCs suppress NK cell function through multiple mechanisms. 24To determine whether "arming" NK cells with cytokines such as IL-15 or IL-21 (cytokine conferral) could prevent GSC-induced suppression, NTNK cells and cytokine-transduced NK cells (IL-15NK or IL-21NK) were cultured for 48 hours in co-culture with GSC20 (Figure 1E) or without co-culture (Figure 1F). The NK cells were then purified, and their cytotoxicity against NK-sensitive K562 cells (phosphoblast cells isolated from the bone marrow of male patients with chronic myeloid leukemia (CML)) was evaluated. Co-culture with GSC20 significantly reduced the cytotoxicity of NTNK cells against K562 cells, while IL-15NK and IL-21NK cells retained their cytotoxic function and were able to kill K562 cells (Figure 1E-1F). Furthermore, in a three-dimensional (3D) spheroid system, two GSC models (GSC20 and GSC272) were used to form spheroids by culturing them for 72 hours before NK cell introduction, and the efficacy of cytokine-contributing NK cells was evaluated (Figure 1G-1I and Figure 23A-23S). As a result, cytokine-transduced NK cells were found to be more resistant to GSC-induced suppression than NTNK cells. These experiments demonstrated that NK cells expressing IL-15 or IL-21 more potently killed GSC targets and were more resistant to tumor-induced dysfunction than control NTNK cells.

[0308] Example 2 IL-21NK cells retained cytotoxicity and demonstrated higher metabolic adaptability against multiple GSC rechallenges. The short-term cytotoxicity test shown in Example 1 confirmed that IL-15NK and IL-21NK cells exhibited higher cytotoxicity against GSCs compared to NTNK cells. Therefore, the long-term cytotoxicity of NTNK, IL-15NK, and IL-21NK cells was evaluated in an in vitro tumor restimulation test using multiple types of GSCs. Here, NK cells were co-cultured with mCherry transduced GSCs (e.g., GSC20, GSC272, GSC267, or GSC8-11) (red) at a 1:1 E:T ratio, and then restimulated at least five times with 200,000 additional mCherry transduced GSCs every 2-3 days. Both IL-15NK and IL-21NK cells effectively eliminated GSCs at a 1:1 E:T ratio with a single GSC restimulation during the initial 3-day co-culture (Figures 2D-2G, 7C, and 8A-8B). However, unlike IL-21 NK cells, IL-15 NK cells, despite maintaining high viability, lost the ability to respond to additional GSC restimulation (Figures 2D-2G and 7C-7E). Furthermore, short-term priming of NT NK cells with exogenous human IL-21 (3 ng / ml) for 48 hours followed by co-culture with GSCs did not effectively suppress tumor growth (Figure 7F). This supports the strategy of genetically modifying NK cells to sustainably secrete IL-21 at stable levels, as shown in Figure 7G. Stable autonomous IL-21 expression allowed IL-21 transduced NK cells to continue producing inflammatory cytokines (e.g., IFNγ and TNFα) and cytotoxic molecules such as Granzyme B and Perforin over repeated restimulation (Figures 2H and 7H-7K). In contrast, NT NK and IL-15 NK cells lost the ability to secrete these important effector cytokines over time. Furthermore, NK cells expressing IL-21 were shown to produce significantly lower levels of neurotoxicity-related cytokines and chemokines (e.g., IL-6, IL-1β, MCP-1) compared to IL-15 transduced cells (Figure 2H).

[0309] Multifunctionality, cellular diversity, and metabolic adaptability are key determinants of an effective antitumor NK cell response. 25Therefore, the inventors used the single-cell IsoPlexis® platform to measure cytokine secretion to GSC20 by IL-15NK, IL-21NK, and NTNK cells. As a result, IL-21NK cells showed significantly higher pluripotency compared to IL-15NK or NTNK cells (e.g., significantly more NK cells showing 2, 3, 4, and / or 5 or more protein secretion profiles), which was confirmed in three donors (Figures 2I-2J and 7L). Furthermore, IL-21NK cells showed an increased pluripotency index (%), indicating increased expression profiles of effectors (e.g., GrB, IFNγ, MIP-1α, Perforin, TNFα, TNFβ) and chemotaxis (e.g., CCL-11, IP-10, MIP-1β, RANTES) compared to NTNK cells (Figure 2H).

[0310] Using mass cytometry, single-cell proteome profiles of IL-15NK, IL-21NK, and NTNK cells (n=3 CB donors) cultured for 48 hours with or without GSC20 cells were analyzed (Figure 2A-2C). Several clusters that appeared to be specific to IL-21NK cells were observed (e.g., clusters 4, 10, 11, 14). Some of these clusters (e.g., clusters 4, 10) were further expanded in co-culture with GSC cells (Figure 2A-2C). These clusters (Clusters 4 and 10) were characterized by high expression of functional / cytotoxic markers such as Granzyme A (GrA), Granzyme B (GrB), Perforin, and Zap70, low expression of inhibitory markers such as LAG3, CD95, and KLRG1, and upregulation of activation and proliferation-related markers such as NKp30, CD25, DNAM, Ki67, CD3ζ, EOMES, T-bet, and / or FCεRG (Figure 2C). Cluster 14 was found only in IL-21NK cells after co-culture with GSCs and showed high expression of cytotoxic markers (e.g., GrA, GrB, Perforin, TRAIL, CD95) and activation markers / receptors (e.g., CD25, CD69, DNAM, NKG2D, NKp44, NKp46).

[0311] Arming NK cells with cytokines can also affect intrinsic metabolic adaptability. Therefore, the mitochondrial metabolism and glycolytic ability of NTNK, IL-15NK, and IL-21NK cells co-cultured with GSC20 cells for 48 hours were analyzed (Figures 2K-2P). IL-21NK cells showed significantly higher basal and maximal oxygen consumption rates (OCR) compared to IL-15NK cells (Figures 2K-2M). Furthermore, IL-21NK cells showed a significantly opposite decrease in the extracellular acidification rate (ECAR), an indicator of glycolysis, compared to IL-15NK cells (Figures 2N-2P). These results indicate that IL-21NK cells mainly produce ATP through oxidative phosphorylation, which is a characteristic related to long-term persistence and anti-tumor activity. 26 。

[0312] Example 3 IL-21NK cells showed cytotoxicity against GSCs in vivo and a memory response to in vivo rechallenge. [[ID=##]] To examine and compare the in vivo anti-tumor functions of IL-21NK, IL-15NK, and NTNK cells, three orthotopic patient-derived GSC PDX mouse models were used (GSCs are described in Materials and Methods and in a previous report 5 ). Briefly, on day 0, 0.5×10 6 individual GSC20 luciferase-expressing cells were intracranially (I.C.) transplanted into 5-week-old NSG mice. Seven days after tumor transplantation, mice with established tumors were either singly locally intratumorally administered (IT) 0.1×10 [[ID=##]] 6 individual IL-15NK, IL-21NK, or NTNK cells or mock-treated. Similar to the in vitro results described above, short-term tumor control was observed in both the IL-15NK and IL-21NK groups, although weaker than that of NTNK cells (Figures 3A-3B). The best tumor control and survival rates were observed in the IL-21NK cell-administered group. Long-term tumor eradication was achieved with a single IT administration in the GSC20 model, and the survival period was significantly extended without observing toxicity or weight loss in the GSC8-11 and GSC267 models either (Figures 3A-3D, Figures 11A-11H).

[0313] The persistence of IL-21 NK cells was analyzed in an in vivo GSC tumor restimulation model. For IL-21 NK-treated mice (n = 4) that survived the first GSC20 tumor challenge, 0.25 × 10 6 GSC20 tumor cells were re-injected 400 days after the first treatment (Figure 3E). This group was compared with a group of naive control mice without tumors (n = 5) that were similarly injected intracranially with 0.25 × 10 6 GSC20 cells. All animals were euthanized 2 weeks after tumor challenge and necropsied. Tumors were confirmed in the brain tissues of the control group (Figure 12A - 12B). In contrast, clear infiltration of human CD45 + cells was observed in the brains of the IL-21 NK cell restimulation group, all of which were CD56 + CD3 - CD16 + NK cells, and very few tumors were detected (Figure 3F - G; Figure 12A - 12D). Furthermore, flow cytometry and IHC analysis revealed no infiltration of NK cells into secondary organs such as the lung, spleen, liver, and bone marrow (Figure 13A - 13B), suggesting the possibility of brain-endogenous long-lived IL-21 NK cells.

[0314] In contrast to the IL-21 NK or NTNK groups, IT administration of 0.5 × 10 6 IL-15 NK cells caused significant toxicity, insufficient GSC20 tumor control, and early death (Figure 3A - 3D). This was accompanied by a high infiltration of NK cells into the brain and marked gliosis (Table 4; Figure 9A - 9E), specifically significant weight loss, strong infiltration of activated NK cells shown by Granzyme B staining, increased Iba1 staining, and strong activation of microglia shown by morphological changes according to previously reported 27 findings, and marked astrocytosis shown by GFAP staining (Figure 9A - 9E). Similar toxicity was observed at a lower dose (0.1 × 10 6This was observed in IL-15 NK cells (Figures 3A-3D, 9F-9I) and under conditions of low IL-15 transduction efficiency (21.4% vs. approximately 80% under normal conditions) (Figures 25A-25J). Notably, in normal brain tissue surrounding the tumor, NK cells were located in close proximity to Ki67-positive microglia, but this was not the case within the tumor. This suggests the possibility of NK-microglia crosstalk in microglial proliferation and subsequent neuroinflammation (Figures 25H-25I). The underlying mechanism is described in the literature. 28 As shown, the expression of the IL-15 receptor (IL-15R) on microglia and the ability of human IL-15 to bind to mouse IL-15R may be involved, supporting the idea that IL-15 released from NK cells triggers microglial activation and proliferation. The tumor control ability of the IL-15NK cell group was inferior to that of the IL-21NK cell group (Figure 25J). Neurotoxicity and early death induced by IL-15NK cells observed after IT administration were also confirmed in a second model using GSC262 cells (Figure 10A). To investigate whether the administration route affects the degree of toxicity, IT administration and intravenous (IV) administration were compared (Figures 10A-10D). When IL-15NK cells were administered IV (Figures 10A-10B), no toxicity was observed, but NK cell infiltration into the brain was minimal and tumor control was not achieved (Figures 10C-10D). This suggests that the absence of toxicity after IV administration is due to the limited migration of IL-15NK cells to the brain. Serum analysis of mice that died after ITIL-15NK cell therapy showed no elevation in cytokine release syndrome markers (e.g., IL-6, IL-1β, MIP-1α, IFNγ, TNFα, IL-10; Table 1), supporting localized IL-15 toxicity in the brain rather than systemic inflammation. Similarly, IV administration of IL-21NK cells did not show toxicity, but it also failed to suppress tumor growth, which was thought to be due to the limited migration of NK cells to the brain (Figures 10E-10F). In summary, the antitumor effects of IL-21NK cells and the toxicity of IL-15NK cells are suggested to be limited to localized effects exerted in the brain when administered intratumorally. [Table 3] [Table 4]

[0315] Example 4 IL-21 NK cells exhibited transcriptional and epigenetic signatures. To elucidate the underlying mechanisms of the persistence of IL-21NK cells and their memory response (immunological memory, recall) to tumor restorative stimulation, the inventors investigated the epigenetic and transcriptional changes of cytokine-transduced NK cells (IL-21NK or IL-15NK) in the short term (e.g., after one GSC stimulation [day 3]) and long term (e.g., after three GSC restorative stimulations [day 9]). IL-15NK and IL-21NK cells not exposed to GSCs were also included as controls. Single-cell ATAC sequencing (scATAC-seq) identified epigenetically distinct clusters between IL-15NK and IL-21NK cells, as well as across different time points. At baseline (before GSC addition), IL-21NK and IL-15NK cells showed similar cluster distributions (Figure 4A-4B). After one GSC stimulation / short-term co-culture (day 3), some cluster reductions (e.g., clusters 2, 4, and 7) and expansions of other clusters (e.g., clusters 1 and 8) were observed in both groups. However, after three restimulations (day 9), significant epigenetic differences emerged between IL-15 and IL-21 NK cells, with cluster 3 being dominant in the IL-15 NK cell group, while cluster 6 was dominant in the IL-21 NK cell group (Figure 4A-4B).

[0316] To further characterize the epigenetic landscape of IL-21 NK cells, 7,117 differentially accessible peaks (DAPs) were identified in a comparison of cluster 6 with all other clusters. Analysis of cluster 6-specific peaks identified IRF1, TBX21, EOMES, IRF9, and STAT1, which are transcription factors (TFs) important for NK cell maturation and immune effector function. 29-31Furthermore, enrichment of AP-1 complex TFs such as JUN, JUNB, JUND, FOS, and FOSL1 was revealed. Interestingly, motifs of the CCAAT enhancer-binding protein (C / EBP; CEBP) family were also enriched. While the CEBP family is known to play important roles in cell proliferation and differentiation in various tissues, data on its role in NK cells has been limited until now. 33 (Figure 4C and Tables 5, 6). This enrichment was also observed in DAP analysis comparing IL-21NK and IL-15NK cells as a whole after short-term co-culture with GSCs (day 3), with enrichment confirmed in particular of C / EBP family motifs such as CEBPD (CEBPδ), CEBPA (CEBPα), CEBPG (CEBPγ), and CEBPE (CEBPε), AP-1 family complexes, and RUNX1 TFs, which have been shown to promote the adaptive behavior of NK cells against viruses (Figure 4D left panel). Notably, after multiple tumor restimulations (day 9), several C / EBP family TFs were significantly enriched in IL-21NK cells compared to IL-15NK cells, with CEBPD being the most prominently enriched (Figure 4D right panel and Figure 4E). Specifically, CEBPD was enriched 4.82 times, CEBPG 4.61 times, CEBPE 4.31 times, CEBPB (CEBPβ) 4.15 times, and CEBPA 3.33 times. The consensus binding motifs of these CEBP family proteins are shown in Figure 4E. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 5-14 Table 5-15 Table 5-16 Table 6

[0317] To further characterize the transcriptional landscape / heterogeneity in IL-21NK cells, unbiased single-cell RNA (scRNA) profiling was performed. Clustering of scRNA profiles revealed that differences between IL-21NK and IL-15NK cells increased over time, regardless of the presence or absence of GSCs, and, similar to epigenetic profiles, the most significant differences / divergences were observed on day 9 after multiple GSC retries (Figures 4F and 14). Differential gene expression analysis comparing dominant clusters in IL-15 (cluster 3) and IL-21NK cells (cluster 4) (Figures 4F-4G and 14) showed that 176 genes were upregulated in cluster 3 and 163 genes were upregulated in cluster 4 (e.g., CEBPD, CLIC3, EOMES, BIRC3, and NFKBIA). A major characteristic of cluster 4 in IL-21NK cell products was the upregulation of TFs (e.g., CEBPD, ETS1, IRF1, EOMES) previously identified in the scATAC-seq dataset (Figure 4H-4J). Additionally, genes important for T cell memory and NK cell survival (e.g., KLRD1, ITGA1, GZMK) were also present. 35-37 ) was also confirmed. Furthermore, genes such as NFKBIA, REST, and E2F3 were upregulated. In contrast, in cluster 3 (IL-15NK cells), genes related to antitumor immunosuppression and cell exhaustion (e.g., DUSP2) were upregulated. 38 , CISH 39 BAX 40 ) was upregulated (Figure 4H).

[0318] Example 5 C / EBP regulates IL-21NK cells at the transcriptional and epigenetic levels. To validate the aforementioned findings regarding scATAC-seq TFs, we estimated gene regulatory networks from scRNA-seq data using the pySCENIC pipeline. As a result, 146 differentially activated regulons were identified between IL-21 and IL-15NK samples (Figure 5A). Notably, many of the TF motifs enriched in differentially accessible peaks (DAPs) between IL-21NK and IL-15NK cells in the scATAC-seq data were also confirmed in this analysis (Figure 5B). Both CEBPD and CEBPB were included among the top 50 differentially activated regulons, supporting the possibility that these TFs play a crucial role in regulating IL-21NK cell activity at both transcriptional and epigenetic levels.

[0319] The gene-level chromatin accessibility profiles for CEBPD (106 target genes including the target gene CEBPB) and CEBPB (85 target genes) were significantly higher in IL-21NK cells compared to IL-15NK cells on day 3 and / or day 9 after GSC challenge or rechallenge (Figure 5C-5D). Pathways enriched in CEBPD and CEBPB regulons included TNF-α signaling (via NF-κB), mTORC1 signaling, and hypoxia response, all of which have long been known to be involved in memory formation / long-term persistence in T cells. 41、42(Figures 5E-5F). Genes involved in the representative pathways of CEBPB (mTORC1 signaling, protein folding response, hypoxia response, p53 pathway, NF-κB-mediated TNFα signaling, apoptosis, estrogen response) showed significantly higher accessibility (Figure 5E). Genes involved in the representative pathways of CEBPD (NF-κB-mediated TNFα signaling, hypoxia response, apoptosis, mTORC1 signaling, p53 pathway, UV response enhancement, homeostasis, IL-2 / STAT5 signaling, KRAS signaling enhancement, adipogenesis, bile acid metabolism, myogenesis, complement system, epithelial-mesenchymal transition) also showed significantly higher accessibility (Figure 5F). Interestingly, cluster 4 (Figure 5G), which defined IL-21NK cells on day 9 of co-culture with GSCs, was enriched with CEBPD at both the transcriptional level (Figures 5H and 15A) and in the target genes of the CEBPD regulon (Figures 5I and 15B). Taken together, these data support the idea that CEBPD is important as a TF that regulates multiple downstream genes such as CEBPB, JUN, FOSL2, KLF2, ETS1, NFIL3, and BNIP3L, and suggest its potential involvement in the memory / cellular persistence and mitochondrial adaptability phenotypes observed in IL-21NK cells (Figure 5I).

[0320] Furthermore, an integrated analysis of bulk RNA-seq and ATAC-seq from four other umbilical cord blood donors using NK cells designed to express IL-15 or IL-21 was performed. 47This revealed 1,146 differential gene expression (DEG) sequences, identifying NK cell signaling as the most activated representative pathway in IL-21NK cells (Figures 17A-17B). Furthermore, analysis of TF motif activity revealed different regulatory network activities in IL-21 and IL-15NK cells (Figure 17C). Specifically, the STAT1 gene network was upregulated in IL-21NK cells and downregulated in IL-15NK cells. On the other hand, the MYC gene network was upregulated in IL-15NK cells compared to IL-21NK cells. This data also independently supported the identification of JUN as the top-level upstream regulator and CEBPD as the primary target regulator activated in IL-21NK cells.

[0321] Finally, the inventors confirmed CEBPD upregulation at both the transcriptional and proteomic levels in IL-21NK cells after multiple GSC rechallenges, but not in NTNK cells or IL-15NK cells (Figure 16A-D). This upregulation was also confirmed at the proteomic level in NK cells extracted from the brains of GSC-transplanted mice treated with IL-21NK cells, but not in those treated with NTNK cells or IL-15NK cells, further highlighting the possibility that CEBPD plays a crucial role in mediating the potent antitumor activity of IL-21NK cells in vivo (Figure 16E-16F).

[0322] These results further support the crucial role that CEBP family proteins, such as CEBPD, play in mediating a potent antitumor NK cell response in vivo. Taken together, these data support the role of CEBPD as a key transformer mediating the effector function of IL-21 NK cells against GBM.

[0323] Example 6 CEBPD knockout reversed the advantages granted by IL-21. C / EBP is a family of six structurally homologous TF genes that can promote the expression of genes involved in various cellular processes such as cell proliferation, differentiation, and death. 48 In cancer, C / EBPs have been shown to play both pro-carcinogenic and anti-cancer roles. 49 The specific role of NK cells in NK cell function at the molecular level is unclear or previously unknown. 50 Since the results reported herein identified CEBPD as a regulator of CEBPB and as the most differentially enriched TF in IL-21NK cells, the inventors first investigated the effects of CEBPD deletion (knockout, CEBPD-KO) or introduction / overexpression (knock-in, CEBPD-KI, CEBPD-OE) in IL-21NK cells and IL-15NK cells.

[0324] Gene deletion of CEBPD reduced the in vitro proliferative capacity of IL-21 NK cells without impairing cell viability compared to IL-21 Cas9 NK cell controls (Figure 18A-18D). Notably, CEBPD-KO IL-21 NK cells rapidly lost cytotoxicity and effector responses to multiple in vitro GSC rechallenges and showed significant impairment in metabolic adaptability compared to control IL-21 Cas9 NK cells (Figure 6A-D and Figure 19A-19C). Transcriptome analysis after CEBPD knockout confirmed downregulation of IFN signaling, the JAK / STAT pathway, and the hypoxia response, accompanied by reverse upregulation of MYC target genes (Figure 19C). In contrast, CEBPD overexpression (CEBPD OE) in NTNK cells using a vector encoding full-length CEBPD (Figures 20A-20B) significantly increased the cytotoxic response to GSC rechallenge compared to NTNK cells and was associated with improved mitochondrial adaptability and reduced mitochondrial reactive oxygen species (ROS) (Figures 6E-H and 21A-21B). RNA-seq analysis confirmed upregulation of the same pathways that were downregulated after CEBPD knockout (Figure 19C).

[0325] Bulk RNA-seq and ATAC-seq analyses from IL-15 NK cells or IL-21 NK cells revealed 1,146 differentially expressed genes (DEGs), and the representative pathway most activated by IL-21 was identified as NK cell signaling (Figure 17A). In silico activity prediction of this pathway revealed that both IL-15 (1,122 DEGs) and IL-21 (248 DEGs) activate basic cytotoxic NK functions, while the antitumor functions of NK cells (e.g., tumor cell apoptosis, apoptosis, cytotoxicity, i.e., mediated by ERK1 / 2, NFκB, IFNG, TNFSF10, FASLG, Nfat activity, etc.) are primarily activated by IL-21 stimulation (Figure 17B). Furthermore, CEBPD-KI (SEQ ID NO: 20, translated sequence is SEQ ID NO: 19) (1,832DEG) enhanced the antitumor function of NK cells in IL-15 NK cells, while CEBPD-KO (1,032DEG) abolished the antitumor function of NK cells in IL-21 NK cells (Figure 17B). The transcriptional differences observed in network analysis were associated with changes in chromatin accessibility patterns in the antitumor response of NK cells, and analysis of TF motif activity by bulk ATAC-seq showed that STAT1 functions as a master regulator. In addition, this data independently supported JUN as a higher upstream regulator that was activated in IL-21 but not in IL-15, and CEBPD as a major target regulator.

[0326] The inventors investigated whether gene deletion of CEBPD was sufficient to eliminate the memory response of IL-21 NK cells to GSC rechallenge in vitro. CRISPR / Cas9-mediated CEBPD-KO did not significantly adversely affect the short-term in vitro proliferation of IL-21 NK cells (Figure 18A-18D) or the short-term cytotoxicity against GSCs (Figure 6A). However, the long-term in vitro proliferation and killing ability of IL-21 CEBPD-KO NK cells to tumor rechallenge was significantly impaired compared to control IL-21 Cas9 NK cells (Figure 6A, Figures 18A-18D, Figure 19A). Furthermore, IL-21 CEBPD-KO NK cells showed significantly reduced release of effector molecules (e.g., IFNγ, TNFα, MIP-1α, MIP-1β, Granzyme A, Granzyme B, Perforin, sCD137) compared to control IL-21 Cas9 NK cells (Figure 19B). Similarly, mitochondrial adaptability of IL-21 CEBPD-KO NK cells was significantly reduced compared to control IL-21 Cas9 NK cells (e.g., reduced maximal respiration) (Figures 6B-6D). Next, to investigate whether overexpression of CEBPD in NK cells was sufficient to confer long-term GSC-killing ability, the inventors generated CEBPD-KI ("CEBPD-OE") NK cells using a retroviral vector encoding full-length codon-optimized CEBPD (SEQ ID NO: 20, translated sequence is SEQ ID NO: 19) (Figures 20A-20B). Transduction efficiency of CEBPD-KI was measured by flow cytometry and averaged approximately 80% in five independent donor samples on day 5 post-transduction. Compared to NTNK cells, CEBPD-KI NT NK cells showed significantly improved GSC-killing ability for multiple tumor rechallenges (Figure 6E). Furthermore, compared to NTNK cells, CEBPD-KI NT NK cells showed significantly improved mitochondrial adaptability (e.g., significantly increased basal and maximal respiration) (Figures 6F-6H).After 20 days of co-culture and multiple GSC retries, CEBPD-KI NK cells had significantly lower mitochondrial reactive oxygen species (ROS) levels compared to control NK cells, while IL-21 CEBPD-KO NK cells had significantly higher ROS levels compared to control IL-21 Cas9 NK cells (Figures 21A-21B).

[0327] Next, these in vitro results were validated in a highly invasive orthotopic GBM model (using GSC272 cells). NSG mice were surgically prepared for the experiment (e.g., intracranial bolt implantation). After recovery, NSG mice showed 0.5 × 10⁶ chromosomes on day 0. 6 Tumors were intracranially inoculated with GSC272 luciferase cells. Mice with tumors were either left untreated or 2 × 10⁶ with or without CEBPD-KI. 6 NTNK cells (NTNK or CEBPD-KI NTNK) or IL-21NK cells (IL-21NK or IL-21 CEBPD-KO NK) with or without CEBPD-KO were administered intratumorally (Figure 6I). As mentioned above, IL-15NK cells were not included as a control in this in vivo experiment because they had been shown to cause severe toxicity and death in several orthotopic mouse models. Potent and significant antitumor activity was observed in mice treated with IL-21NK cells (Figures 6I-6K), and was significant compared to tumors alone (GSC272 alone) and NTNK cells. In contrast, mice treated with IL-21 CEBPD-KO NK cells did not show a significant improvement in survival compared to the tumor-only control group. Furthermore, CEBPD-KI NT-NK cells improved in vivo antitumor activity (Figure 6J) and animal survival (Figure 6K) compared to NTNK cells.

[0328] Taken together, these data demonstrate that arming NK cells with IL-21 induces stable epigenetic changes in NK cells, and that CEBPD plays a crucial and essential role in establishing an enhanced and sustained antitumor memory response, particularly against GBM. Furthermore, it was suggested that overexpression of CEBP family proteins such as CEBPD is sufficient to replicate the IL-21-induced phenotype in NK cells.

[0329] Example 7 TI-NK cells were rich in CEBPB, CEBPD, and AP-1 complex-related transcription factors. In a scRNA dataset derived from GBM patients (obtained from Shaim, H., et al., Targeting the alphav integrin / TGF-beta axis improves natural killer cell function against glioblastoma stem cells. J. Clin Invest 131 (2021)), the expression of CEBPD, CEBPB, and AP-1 complex genes in tumor-infiltrating NK cells (TI-NK) was analyzed, revealing that their expression was significantly higher compared to NK cells derived from peripheral blood mononuclear cells from healthy donors (HC-NK) (Figure 22A-22B). These results support the clinical relevance that CEBPD is an important factor in the transcriptional and epigenetic coordination of NK cell memory against cancer, and provide a basis for advancing IL-21 engineered NK cells for evaluation in future clinical trials in GBM immunotherapy.

[0330] Example 8 STAT3 signaling induced the expression of CEBPD target genes in IL-21 NK cells. IL-21 is known to contribute to the activation of the JAK3 / STAT3 pathway, which is important for transmitting signals ...

Claims

1. Engineered natural killer (NK) cells modified to overexpress CCAAT / enhancer-binding protein (CEBP) transcription factor family proteins.

2. The engineered NK cells according to claim 1, wherein the expression of the CEBP transcription factor is compared to that of unengineered NK cells and / or NK cells engineered to express IL-15.

3. The engineered NK cells according to claim 1, wherein the CEBP protein is CEBPD (CEBP-delta, CEBPδ), CEBPA (CEBP-alpha, CEBPα), CEBPB (CEBP-beta, CEBPβ), CEBPG (CEBP-gamma, CEBPγ), CEBPE (CEBP-epsilon, CEBPε), and / or CEBP homolog protein (CHOP).

4. The engineered NK cell according to claim 1, wherein the CEBP protein is CEBPD and / or CEBPB.

5. The engineered NK cells according to claim 1, wherein the NK cells overexpress the transgenic CEBPD and / or CEBPB proteins.

6. The engineered NK cells according to claim 1, wherein the NK cells transgenically express activators of transcription and / or translation of CEBP protein and / or are treated with them.

7. The engineered NK cells according to claim 1, wherein the NK cells transgenically express and / or undergo inhibition of the transcription and / or translation of the CEBP protein.

8. The engineered NK cell according to claim 1, wherein the CEBP protein contains at least 80%, 85%, 90%, 95%, 99%, or 100% identical sequences to SEQ ID NO:

19.

9. The engineered NK cell according to claim 1, wherein the CEBP protein is encoded by a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 20 or 21.

10. The engineered NK cell according to claim 1, wherein the CEBP protein comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any of SEQ ID NOs: 23, 25, or 27.

11. The manipulated NK cell according to claim 1, wherein the CEBP protein is encoded by a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any of SEQ ID NOs: 24, 26, or 28.

12. The engineered NK cells according to claim 1, wherein the engineered NK cells have enhanced mitochondrial aptitude and / or memory-like characteristics compared to unengineered NK cells and / or NK cells engineered to express IL-15.

13. The engineered NK cell according to claim 12, wherein the enhanced mitochondrial fitness includes an increase in basal and / or maximal oxygen consumption rate (OCR) and / or a decrease in glycolysis (for example, if glycolysis is measured by extracellular oxidation rate (ECAR)) compared to an unengineered NK cell and / or an NK cell engineered to express IL-15.

14. The engineered NK cells according to claim 1, wherein the engineered NK cells have enhanced antitumor cytotoxic activity compared to unengineered NK cells and / or NK cells engineered to express IL-15.

15. The engineered NK cells according to claim 1, wherein the engineered NK cells have enhanced antitumor memory compared to unengineered NK cells and / or NK cells engineered to express IL-15.

16. The engineered NK cells according to claim 1, wherein the engineered NK cells have higher expression of functional markers, lower expression of repressive markers, higher expression of survival genes, lower expression of exhaustion genes, and / or upregulation of activating receptors and markers, compared to unengineered NK cells and / or NK cells engineered to express IL-15.

17. The engineered NK cells according to claim 16, wherein the functional marker comprises granzyme A (GrA), granzyme B (GrB), perforin, and / or Zap70.

18. The engineered NK cells according to claim 16, wherein the inhibitory markers include LAG3 and / or KLRG1.

19. The engineered NK cell according to claim 16, wherein the activation receptor and markers include NKp30, CD25, DNAM, Ki67, CD3ζ, T-bet, and / or FCεRG.

20. The engineered NK cell according to claim 16, wherein the survival genes include KLRD1, ITGA1 and / or GZMK.

21. The engineered NK cell according to claim 16, wherein the exhaustion gene comprises DUSP2, CISH and / or BAX.

22. The engineered NK cells according to claim 1, wherein the engineered NK cells exhibit higher expression of the cytotoxic markers GrA, GrB, perforin, TRAIL and / or CD95, and / or higher expression of the activating markers / receptors CD25, CD69, DNAM, NKG2D, NKp44 and / or NKp46, compared to unengineered NK cells and / or NK cells engineered to express IL-15.

23. The engineered NK cells according to claim 1, wherein the engineered NK cells exhibit higher levels of NK cell maturation, NK cell immune function, NK cell cytotoxicity, IFN-γ response, memory formation, and / or higher levels of transcription factors important to AP-1 complex members, compared to unengineered NK cells and / or NK cells engineered to express IL-15.

24. The engineered NK cell according to claim 23, wherein the transcription factor comprises CEBPD, CEBPB, ETS1, IRF1, TBX21, EOMES, IRF9, STAT1, ETS1, JUN, JUNB, JUND, FOS, and / or FOSL1.

25. The engineered NK cells according to claim 1, wherein the cells are engineered to provide one or more interleukins (ILs).

26. The engineered NK cell according to claim 25, wherein the IL is an artificially linked IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, and / or the p35 and p40 subunits of IL-12.

27. The engineered NK cell according to claim 26, wherein IL is IL-21 and / or IL-15.

28. The engineered NK cells according to claim 26, wherein the NK cells acquire tumor cell apoptosis, apoptosis, and / or enhanced cytotoxicity.

29. The manipulated NK cells according to claim 26, wherein the NK cells have increased levels and / or activity of ERK1 / 2, NFκB, IFNG, TNFSF10, FASLG, and / or Nfat.

30. The manipulated NK cell according to claim 25, wherein the IL is secreted, bound, or membrane-bound within the cell.

31. The engineered NK cells according to claim 1, wherein the NK cells are derived from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, NK cell lines, or a combination thereof.

32. The engineered NK cells according to claim 1, wherein the NK cells are primary NK cells and are not derived from stem cells and / or induced pluripotent stem cells (iPSCs).

33. The engineered NK cells according to claim 1, wherein the NK cells form complexes with one or more monospecific, bispecific, and / or multispecific antibodies.

34. The manipulated NK cells according to claim 33, wherein the NK cells express one or more antibodies.

35. The engineered NK cells according to claim 1, wherein the NK cells are further modified to express one or more additional heterologous proteins selected from antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof.

36. The manipulated NK cell according to claim 35, wherein the manipulated receptor is a manipulated antigen receptor.

37. The manipulated NK cell according to claim 33, wherein the target antigen is a cancer antigen.

38. The manipulated NK cells according to claim 1, wherein the K cells contain a suicide gene.

39. The engineered NK cells according to claim 1, wherein the NK cells further include engineered mutations in one or more endogenous genes.

40. The engineered NK cell according to claim 39, wherein the endogenous genes are TGFBR2, CISH, GR, and / or CD38.

41. The manipulated NK cells according to claim 1, wherein the NK cells are pre-activated with one or more cytokines.

42. The engineered NK cell according to claim 41, wherein one or more cytokines include IL-2.

43. A composition comprising the manipulated NK cells described in claim 1.

44. The composition according to claim 43, further comprising a pharmaceutically acceptable excipient.

45. The composition according to claim 43, wherein the composition is included in a delivery device.

46. A method for treating a disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of the manipulated NK cells or composition described in any one of the preceding claims.

47. The method according to claim 46, wherein the disease is an autoimmune disease, an infection, and / or cancer.

48. The method according to claim 46, wherein the disease is cancer.

49. The method according to claim 48, wherein the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, cervix, or hematological.

50. The method according to claim 48, wherein the cancer is glioblastoma.

51. The method according to claim 50, wherein the glioblastoma is a TCGA mesenchymal, neural, classical, or preneurial subtype.

52. The method according to claim 51, wherein the glioblastoma is of the mesenchymal or anterior nerve subtype.

53. The method according to claim 50, wherein the glioblastoma is MGMT unmethylated, methylated, or in an undefined state.

54. The method according to claim 50, wherein the glioblastoma is primary or recurrent.

55. The method according to claim 48, wherein engineered NK cells exhibit enhanced antitumor function in vivo compared to unengineered NK cells and / or NK cells engineered to express IL-15.

56. The method according to claim 55, wherein the enhanced in vivo antitumor function includes long-term tumor eradication, a significant extension of the subject's survival time, and / or a reduction in toxicity or weight loss.

57. The method according to claim 50, wherein administration is by intracranial injection.

58. The method according to claim 50, wherein the administration is by intratumor injection.

59. The method according to claim 50, wherein the method provides immunological memory against glioblastoma.

60. The method according to claim 50, wherein the method provides immunological memory against glioblastoma stem cells.

61. A method for providing an immunological memory against cancer to a subject, comprising administering to an individual a therapeutically effective amount of manipulated NK cells according to any one of claims 1 to 42 or a composition according to any one of claims 43 to 45.

62. Isolated nucleic acids encoding CEBP proteins fused with heterologous transcriptional regulators.

63. The isolated nucleic acid according to claim 62, wherein the CEBP protein comprises a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

19.

64. The isolated nucleic acid according to claim 62, wherein the CEBP protein is encoded by a sequence that includes at least 80%, 85%, 90%, 95%, 99%, or 100% identical to sequence number 20 or 21.

65. The isolated nucleic acid according to claim 62, wherein a heterogeneous transcriptional regulatory element is the promoter.

66. A method for treating glioblastoma, comprising administering to an individual a therapeutically effective dose of NK cells engineered to autonomously and / or constitutively express secreted IL-21.

67. The method according to claim 66, wherein administration is by intracranial injection.

68. The method according to claim 66, wherein the administration is by intratumor injection.

69. The method according to claim 66, wherein NK cells engineered to constitutively and / or autonomously express secreted IL-21 include a transgenic polynucleotide sequence encoding and / or containing at least 80%, 85%, 90%, 95%, or 100% identical sequences of SEQ ID NOs. 31-32.

70. The method according to claim 66, wherein the method provides immunological memory against glioblastoma.

71. The method according to claim 66, wherein the method provides immunological memory against glioblastoma stem cells.

72. The method according to claim 66, wherein NK cells are manipulated to stably secrete IL-21 at a rate suitable for reaching an extracellular concentration of 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 pg / mL or higher.

73. The method according to claim 66, wherein NK cells are manipulated to stably secrete IL-21 at a rate suitable for reaching an extracellular concentration of approximately 200–800 pg / mL, 250–750 pg / mL, 300–700 pg / mL, or 350–650 pg / mL or higher.

74. The method according to claim 66, wherein the glioblastoma is a TCGA mesenchymal, neural, classical, or preneurial subtype.

75. The method according to claim 66, wherein the glioblastoma is a mesenchymal or preneurial subtype.

76. The method according to claim 66, wherein the glioblastoma is MGMT unmethylated, methylated, or in an undefined state.

77. The method according to claim 66, wherein the glioblastoma is primary or recurrent.

78. The method according to claim 66, wherein engineered NK cells exhibit enhanced antitumor function in vivo compared to unengineered NK cells and / or NK cells engineered to express IL-15.

79. The method according to claim 78, wherein the enhanced in vivo antitumor function includes long-term tumor eradication, a significant extension of subject survival, and / or reduction of toxicity or weight loss.

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