Expansion of natural killer and chimeric antigen receptor modified cells

Abstract

The present invention discloses methods and compositions for generating immunotherapy cells (e.g., NK and T cells) with enhanced cytotoxicity and expansion capacity. The methods and compositions disclosed herein can be further used for the enhanced expansion of CAR-modified NK and T cells with increased cytotoxicity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 808,031, filed February 20, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to methods of producing modified feeder cells, compositions comprising modified feeder cells, and methods of using the same.

[0004] Thank you for the government's support

[0005] This invention was made with government support under Grants 1R01AI130197-01A1, HL125018, AI124769-01, and AI129594 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0006] NK cells are an important subset of lymphocytes that provide the body's first line of defense. NK cells were initially described as being able to spontaneously kill tumor cells (Rosenberg et al. J Natl Cancer Inst 52:345-52 (1974); Kiessling et al. Eur J Immunol 5:117-21 (1975); Kiessling et al. Eur J Immuol 5:112-7 (1975); Herberman et al. Int J Cancer 16:230-9 (1975); Herberman et al. Int J Cancer 16:216-29 (1975)) and are different from T cells, which require prior sensitization. NK cells kill tumor cells or virus-infected cells through several pathways (Liu et al. Immunity 31:99-109 (2012); Liu et al. Immunity 36:600-11 (2012); Long et al. Annu Rev Immunol 31:227-58 (2013)), including direct cytotoxicity (natural cytotoxicity and ADCC) and indirect effects (e.g., cytokine production and interaction with adaptive immunity). Among these functions, an important application of NK cells is the use of primary ex vivo expanded NK cells or genetically modified NK cells to treat various cancers. A large number of clinical trials have shown that NK cell infusion has a milder graft-versus-host disease (GvHD) than T cell infusion.

[0007] NK cells have two main clinical applications. The first is to use NK cells with genetically modified primary ex vivo expansion to treat cancer. Specifically, clinically, NK cells are used to treat ALL and AML (Miller et al. Blood 105: 3051-7 (2005); Rubnitz et al. J Clin Oncol 28: 955-9 (2010)). The second is that genetically modified NK cells such as chimeric antigen receptor (CAR) modified NK cells have become emerging tools for cancer immunotherapy (Liu et al. Leukemia 32: 520-31 (2018); Liu et al. Protein Cell 9: 902 (2018)). Clinical studies on immunotherapy based on CAR-modified NK cells have been widely used in several types of cancer (Rezvani K and Rouce RH, Front Immunol 6: 578 (2015)). Similar to CAR-T cell-based immunotherapy, different research groups have studied the use of various CAR molecules to redirect genetically modified NK cells with different antigen specificities (Rezvani K and Rouce RH, Front Immunol 6:578 (2015); Hermanson DL and Kaufman DS, Front Immunol 6:195 (2015); Glienke et al. Front Pharmacol 6:21 (2015)).

[0008] CAR-modified T cell therapy has become a potential immunotherapy strategy for the treatment of blood cancer (Porter et al. N Engl J Med 365:725-33 (2011); Kim et al. Arch Pharm Res 39:437-52 (2016); Maude S and Barrett DM, Br J Haematol 172:11-22 (2016)) and has received great attention from academic and industry researchers (Glienke et al. Front Pharmacol 6:21 (2015)). The adoptive transfer of CAR-modified immune cells (including CAR-T, CAR-NK and CAR-NKT cells) into patients has shown significant success in the treatment of various blood cancers. Clinical trials using CAR products to treat multiple myeloma (Garfall et al. N Engl J Med 373:1040-7 (2015); Atanackovic et al. Br J Haematol 172:685-98 (2016)), leukemia (Porter et al. N Engl J Med 365:725-33 (2011); Maude et al. N Engl J Med 371:1507-17 (2014); Lee et al. Lancet 385:517-28 (2015)), sarcoma (Ahmed et al. J Clin Oncol 33:1688-96 (2015)), and neuroblastoma (Pule et al. Nat Med 14:1264-70 (2008); Louis et al. Blood 118:6050-6 (2011)) have shown promising results. Scientists and pharmaceutical companies around the world have invested considerable effort and money in the development and optimization of CARs (Casucci et al. Cancer Immunol Immunother 64:123-30 (2015); Gottschalk et al. Ernst Schering Found Symp Proc 69-82 (2006); Ramos et al. Cancer J 20:112-8 (2014); Savoldo B and Dotti G, Cancer J 20:112-8 (2014)).

[0009] Adoptive CAR T-cell therapy combines tumor antigen specificity with immune cell activation in a single recipient. The treatment involves isolating the patient's own T cells, engineering them to express a chimeric antigen receptor (CAR) that recognizes tumor proteins, and re-infusing them into the patient. A potential problem with adoptive CAR T-cell therapy is the use of autologous T cells isolated from the patient. Autologous T cells isolated from patients face two major problems. 1) T cells isolated directly from immunocompromised cancer patients generally have poor cytotoxicity and functionality, so they are not used. 2) Autologous T cells cannot be used in other patients due to potential GVHD. Summary of the invention

[0010] There is still a need for improved cytotoxic cell-mediated immunotherapy, for example, to alleviate the shortcomings of CAR-modified cell immunotherapy, such as poor cytotoxicity. Disclosed herein are methods and compositions for expanding cells such as NK and T cells for immunotherapy, with improved cytotoxicity and cell expansion capacity.

[0011] Disclosed herein are modified 721.221 cells. In some examples, the modified 721.221 cells express at least one of membrane-bound IL-21 (mIL-21), IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, membrane-bound IL-15 (mIL-15), TLR ligands, UL16 binding protein (ULBP)-1, ULPB-2, and / or major histocompatibility complex (MHC) class I chain-associated protein A (MIC-A). In a specific non-limiting example, the modified 721.221 cells express mIL-21, such as comprising an amino acid sequence having 90% or 95% sequence identity to SEQ ID NO: 2 (and / or such as encoded by a nucleic acid sequence having 90% or 95% sequence identity to SEQ ID NO: 1), for example using a viral (such as a retroviral) vector (e.g., a lentiviral such as a Moloney murine leukemia virus (MoMLV) vector, such as a SFG retroviral vector). The modified 721.221 cells (e.g., IL-15 receptor alpha (IL-15Rα)) may include additional heterologous cytokines, including activating receptor ligands, TRL ligands, or receptors thereof. In some examples, the modified 721.221 cells include a heterologous nucleic acid encoding at least one of mIL-21, IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, TLR ligands, ULBP-1, ULPB-2, and / or MIC-A. In specific examples, the modified 721.221 cells express mIL-21 or mIL-21 and IL-15Rα.

[0012] Also disclosed herein is a method for producing modified 721.221 cells, for example, comprising transducing or transfecting a 721.221 cell population with a nucleic acid encoding mIL-21, IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, membrane-bound IL-15 (mIL-15), TLR ligands, ULBP-1, ULPB-2, and / or MIC-A; isolating cells expressing mIL-21, IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, membrane-bound IL-15 (mIL-15), TLR ligands, ULBP-1, ULPB-2, and / or MIC-A; and irradiating the isolated cells to produce modified 721.221 cells. In some examples, the cells are modified by transduction (e.g., using a viral vector such as a retrovirus or a lentivirus). In a specific non-limiting example, the modified 721.221 cells express mIL-21, such as comprising an amino acid sequence having 90% or 95% sequence identity to SEQ ID NO: 2 (and / or such as encoded by a nucleic acid sequence having 90% or 95% sequence identity to SEQ ID NO: 1), for example using a retroviral vector (e.g., a Moloney murine leukemia virus (MoMLV) vector, such as an SFG retroviral vector). The method may also include modifying the 721.221 cells to express one or more additional heterologous cytokines, activating receptor ligands, TRL ligands or receptors thereof (e.g., IL-15Rα).

[0013] Also disclosed herein is a method for expanding a natural killer (NK) cell population or a T cell population, for example, by contacting a lymphocyte population with a modified 721.221 cell disclosed herein and at least one cytokine (e.g., interleukin, such as IL-5 or IL-2) for 1-40 days (e.g., 14-21 days) under conditions sufficient for cell expansion. The lymphocyte population can be from any sample type, such as peripheral blood, cord blood, ascites, menstrual blood, or bone marrow, and can, for example, include peripheral blood mononuclear cells (PBMC). The cell population contacted with the modified 721.221 cells may also include modified cells for immunotherapy, such as chimeric antigen receptor (CAR)-modified cells (e.g., CAR-NK or CAR-T cells, such as CD19 CAR modified NK cells). In some examples, the NK or T cell population (e.g., after contacting the modified 721.221 cells for at least 14-21 days under conditions sufficient for cell expansion) increases by at least 5000-90000 times.

[0014] Also disclosed herein is a method for treating cancer or infectious disease or immune disease, for example, by applying NK cells or T cells (such as NK cells or T cells modified by CAR, such as CD19 CAR modified NK cells) produced using the methods described herein to a subject suffering from cancer or infectious disease or immune disease, so as to treat cancer or immune disease. In some instances, cancer or immune disease or infectious disease include autoimmune disease, graft rejection, solid tumors (such as lymphoma, breast cancer, hepatocellular carcinoma (HCC) and pancreatic cancer), sarcoma, neuroblastoma, blood cancer (such as multiple myeloma; Lymphoma, such as non-Hodgkin lymphoma; Or leukemia; Such as acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML)), HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), tuberculosis (TB) or malaria.

[0015] The foregoing and other features of the present invention will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A-1F . Characterization of K562 and 721.221 cells expressing membrane IL-21. Figure 1A ), representative histograms showing IL-21 and 4-1BBL expression of K562 (green) and K562 transduced with IL-21 (K562-mIL21, red) detected by flow cytometry. The mean fluorescence intensity (MFI) is recorded in each histogram. ( Figure 1B ), representative histograms showing the expression of IL-21 and 4-1BBL on 721.221 (green) and 721.221 transduced with IL-21 (721.221-mIL21, red) detected by flow cytometry. MFI is recorded in each histogram. ( Figure 1C ), Confocal images of IL-21 expression on K562 cells transduced with IL-21 (K562-mIL21). ( Figure 1D ), Confocal images of IL-21 expression on 221 cells transduced with IL-21 (221-mIL21). ( Figure 1E ), Representative histograms showing the expression of ICAM-1, PD-L1, HLA-E, and MICB on K562 (green) and K562-mIL21 (red) cells detected by flow cytometry. MFI is recorded in each histogram. ( Figure 1F ), Representative histograms showing the expression of ICAM-1, PD-L1, HLA-E, and MICB on 721.221 (green) and 721.221-mIL21 (red) cells detected by flow cytometry. MFIs are recorded in the corresponding histograms.

[0017] Figure 2A-2E .Expansion of primary human NK cells using four different feeder cell types. Figure 2A ), Representative dot plots showing the purity of NK cells expanded with different types of feeder cells at the indicated day after expansion as determined by flow cytometry. PBMCs were stimulated with irradiated K562, K562-mIL21, 721.221, and 721.221-mIL21 on day 0. NK cell purity was measured on day 7 and every 3 to 5 days thereafter. ( Figure 2B and 2C ), quantitative data show the fold expansion of NK cells from 11 donors expanded for 21 days with irradiated K562, K562-mIL21, 721.221 and 721.221-mIL21 ( Figure 2B ) and purity ( Figure 2C ). Figure 2D and 2E ), quantitative data showing the fold expansion of NK cells obtained from 11 donors on day 21 with the indicated feeder cells ( Figure 2D ) and purity ( Figure 2E ). Figure 2B )and( Figure 2C ) shows the mean value (solid line) with 95% CI (grey band). *p<0.05. **p<0.01, ***p<0.001.

[0018] Figures 3A-3E .Phenotypes of NK cells expanded from different feeder cells. Figure 3A ), Representative histograms showing the expression of CD16, NKG2D, NKp46, 2B4, and DNAM-1 on NK cells expanded with K562, K562-mIL21, 721.221, and 721.221-mIL21. ( Figure 3B ), Representative histograms showing the expression of CD69, CD94, CD8a, and NKG2C on NK cells expanded with K562, K562-mIL21, 721.221, and 721.221-mIL21. ( Figure 3C ), Representative histograms showing the expression of NKG2A, CTLA-4, KLRG1, and PD-1 on NK cells expanded with K562, K562-mIL21, 721.221, and 721.221-mIL21. ( Figure 3D ), Representative histograms showing the expression of LIR1, TIM-3, TIGIT, and LAG-3 on NK cells expanded with K562, K562-mIL21, 721.221, and 721.221-mIL21. ( Figure 3E), representative histograms showing the expression of KIR, KIR2DL1, KIR2DL2 / L3, KIR3DL1 and KIR3DL2 on NK cells expanded with K562, K562-mIL21, 721.221 and 721.221-mIL21. MFI is shown in each histogram.

[0019] Figures 4A-4H .Comparison of the functions of NK cells and susceptible target cells. ( Figure 4A ), using CFSE / 7-AAD cytotoxicity assay, quantitative data showed the cytotoxic activity of expanded NK cells and K562 cells. K562 cells were labeled with CFSE and then incubated with expanded NK cells at an E:T ratio of 1:4 to 4:1 for 4 hours. Next, lysis of K562 cells was measured using 7-ADD. ( Figure 4B ), quantitative data indicate the percentage of expanded NK cells expressing CD107a after no stimulation, stimulation with K562, and stimulation with PMA / ionomycin for 2 hours. ( Figure 4C ), using CFSE / 7-AAD cytotoxicity assay, quantitative data demonstrated the cytotoxic activity of expanded NK cells and 721.221 cells. 721.221 cells were labeled with CFSE and then incubated with expanded NK cells for 4 hours at an E:T ratio of 1:4 to 4:1. Next, lysis of 721.221 cells was measured using 7-ADD. ( Figure 4D ), Percentage of expanded NK cells expressing CD107a after no stimulation, stimulation with 721.221, and stimulation with PMA / ionomycin for 2 hours. ( Figure 4A )and( Figure 4C Mean ± SD is shown in ), and ( Figure 4B )and( Figure 4D ) shows the mean ± SD. ( Figure 4E ) Gating strategy for NK cell-mediated cytotoxicity using the CFSE / 7-AAD method. After 4 h of incubation of NK cells with CFSE-labeled target cells, dead cells were gated on the 7-AAD positive subpopulation. Figure 4F ) Representative flow cytometry dot plots of the percentage of 7-AAD-positive cells in CFSE-labeled K562 cells after incubation with expanded NK cells at different effector:target (E:T) cell ratios. ( Figure 4G ) Gating strategy for cell surface CD107a determination. Figure 4H ) Representative dot plots of the percentage of expanded NK cells expressing CD107a after no stimulation (NK cells only, negative control group), stimulation with K562, and stimulation with PMA / ionomycin (positive control group). ***p<0.001, ns p>0.05.

[0020] Figures 5A-5F . Figure 5A An exemplary method for amplifying CD19-CAR NK cells with 721.221-mIL21 is schematically shown. Briefly, 221.mIL21 cells were irradiated with a dose of 100 Gray (10,000 rads). PBMCs were then co-cultured with irradiated feeder cells in the presence of IL-2 and IL-15. In parallel, CD19-CAR retrovirus was produced by transfecting 293T cells. The amplified NK cells were transduced with CD19-CAR retrovirus on day 7. The cells were cultured for 21 days. ( Figure 5B ), Representative dot plots show the percentage of NK cells expanded in CD19-CAR positive cells on the indicated day after expansion. PBMCs were stimulated with irradiated 721.221-mIL21 on day 0 and transduced with CD19-CAR retrovirus on day 7. The purity of NK cells in CD19-CAR positive cells was tested every 3 to 4 days. ( Figure 5C ) Dynamic time-lapse expansion data of fold expansion of CD19-CAR NK cells from 3 donors. CD19-CAR modified NK cells were expanded for 21 days using irradiated K562, K562-mIL21, 221 and 221-mIL21 feeder cells. ( Figure 5D ) Quantitative data of fold expansion of CD19-CAR NK cells from 3 donors at day 21 of expansion. ( Figure 5E ) Dynamic time-lapse expansion data of the purity of NK cells 544 in CD19-CAR positive cells from 3 donors. NK cells were expanded using irradiated 545K562, K562-mIL21, 221 and 221-mIL21 feeder cells, respectively. ( Fig. 5F ) Quantitative data of the percentage of NK cells among CD19-CAR positive cells from 3 donors on day 21 after expansion. ( Figure 5C )and( Figure 5D ) shows the mean value (solid line) with 95% CI (grey band).

[0021] Figures 6A-6D .Umbilical cord blood (CB)-derived NK and CAR-NK cells were expanded using 721.221-mIL21. Fig. 6A ) Flow cytometry dot plots of the percentage of CD19-CAR positive cells in NK cells on the indicated days. CBMCs were stimulated with irradiated feeder cells on day 0 and transduced with CD19-CAR retrovirus on day 7. ( Figure 6B ) Quantitative data of the percentage of CD19-CAR positive cells in NK cells expanded from CBMCs (n=3). Figure 6C) Quantitative data of the cytotoxic activity of expanded CD19-CAR CB-NK cells against Raji cells using CFSE / 7-AAD cytotoxicity assay. Target cells were labeled with CFSE and then incubated with expanded CD19-CAR CB-NK cells for 4 hours at E:T ratios ranging from 5:1 to 0.3125:1. Next, lysis of target cells was detected using 7-ADD. ( Fig.6D ) Quantitative data of the cytotoxic activity of expanded CD19-CAR CB-NK cells against Daudi cells using CFSE / 7-AAD cytotoxicity assay.

[0022] Figures 7A-7I .Excellent antitumor activity of 221-mIL21-expanded CD19-CAR NK cells in a lymphoma xenograft model. Fig. 7A ) Experimental design of the Daudi lymphoma xenograft model. Male and female NSG mice (n=5) were injected intravenously via the tail vein on day -4 with 2×10 6 Starting on day 0, mice were injected (iv) with 1×10 Daudi-FFLuc cells in 100 μL PBS on days 0, 3, 7, and 10. 7 221-mIL21-expanded or K562-mIL21-expanded CD19-CAR NK cells were injected (ip) with 150 μL PBS in IL-2 (50,000 units / mouse) and IL-15 (10 ng / mouse). Animals were imaged twice a week using the IVIS system for tumor cell tracking. Figure 7B ) Representative images of tumor burden at the indicated time points. Fluorescence intensity range is 1×10 5 Up to 2×10 6 Unit: photon / sec / cm 2 / sr. ( Figure 7C ) Quantitative data of tumor burden at the indicated time points. Mice were imaged on the indicated days to assess tumor burden expressed as quantitative bioluminescence (mean light intensity), indicative of tumor growth. ( Fig.7D ) Quantitative data of mouse body weight on the indicated days. ( Fig. 7E ) Experimental design of the Raji lymphoma xenograft model. Male and female NSG mice (n=10) were injected intravenously via the tail vein on day 0 with 2×10 6 On days 2 and 4, mice were injected (intravenously) with 1×10 7K562-mIL21-expanded CD19-CAR NK cells, 221-mIL21-expanded CD19-CAR NK cells, and 221-mIL21-expanded CD19-CAR-IL15 NK cells were injected (intraperitoneally) with IL-2 (50,000 units / mouse) and IL-15 (10 ng / mouse) in 150 μL PBS. Animals were imaged once a week using the IVIS system for tumor cell tracking. Figure 7F ) Representative images of tumor burden at the indicated time points. The fluorescence intensity range on day 7 was 5 × 10 5 Up to 1×10 7 Unit: photon / sec / cm 2 / sr, the fluorescence intensity range on the 14th and 21st days was 2×10 7 Up to 5×10 8 Unit: photon / sec / cm 2 / sr. ( Figure 7G ) Kaplan–Meier survival curves of tumor-bearing mice after treatment with PBS, K562-mIL21-expanded CD19-CAR NK cells, 221-mIL21-expanded CD19-CAR NK cells, and 221-mIL21-expanded CD19-CAR-IL15 NK cells. p values ​​were analyzed by log-rank (Mantel-Cox) test. ( Figure 7H ) Quantitative data of tumor burden at indicated time points. Mice were imaged on indicated days to assess tumor burden expressed as quantitation, representing tumor growth. ( Fig.7I ) Quantitative data of mouse body weight on the indicated days.

[0023] Figures 8A-8B Schematic representation of an exemplary recombinant retroviral vector encoding human IL-21 and an exemplary method for expanding NK cells using 721.221.mIL-21 feeder cells. Fig. 8A ), the IL-21 construct contains the human IgG1 Fab' domain, the CD28 transmembrane domain, the intracellular domain of 4-1BB and the intracellular domain of CD3ζ. ( Figure 8B ) feeder cells were irradiated with a dose of 100 Gy (10,000 rads), and then PBMCs were co-cultured with irradiated feeder cells with IL-2 and IL-15 for NK cell expansion.

[0024] Fig. 9 Human primary NK cells express cell surface IL-21 receptor. Representative histograms show the expression of IL-21R on primary NK cells from PBMCs. MFI is recorded in each histogram.

[0025] Figures 10A-10C . Primary human NK cell expansion using 721.221 cells expressing membrane IL-15 receptor α (221-Mil-15Rα). Fig. 10A ), Representative dot plots show the purity of NK cells expanded with two different types of feeder cells at the indicated day after expansion as detected by flow cytometry. PBMCs were stimulated with irradiated wild-type 721.221 (top) and 721.221-mIL-15Rα on day 0, respectively. The purity of NK cells was checked on days 7, 14, and 21. ( Fig. 10B and 10C ), quantitative data show the fold expansion of NK cells from 7 donors expanded for 21 days with irradiated wild-type 721.221 and 721.221-mIL-15Rα, respectively ( Fig. 10B ) and purity ( Fig. 10C ).

[0026] Figures 11A-11C . Primary human T cell expansion using 721.221 cells expressing membrane IL-21. Fig.11A ) Representative dot plots show the purity of T cells expanded with two different types of feeder cells on the indicated day after expansion detected by flow cytometry. PBMCs were stimulated with irradiated K562-mIL21 (upper figure) and 721.221-mIL21 (lower figure) on day 0, respectively. The purity of NK cells was checked on days 7, 14, and 21. Quantitative data show the fold expansion (left figure) and purity (right figure) of T cells from 11 donors expanded for 21 days with irradiated K562-mIL21 and 721.221-mIL21, respectively. ( Fig. 11B ) Representative dot plots show the purity of T cells expanded with two different types of feeder cells at the indicated day after expansion as detected by flow cytometry. Cord blood mononuclear cells were stimulated with irradiated K562-mIL21 (upper panel) and 721.221-mIL21 (lower panel) at day 0, respectively. The purity of NK cells was checked at day 7, day 14, and day 21. Quantitative data show the fold expansion (left panel) and purity (right panel) of T cells from 11 donors expanded for 21 days with irradiated K562-mIL21 and 721.221-mIL21, respectively. ( Fig. 11C ) Representative dot plots show the purity of T cells expanded with two different types of feeder cells at the indicated day after expansion as detected by flow cytometry. PBMCs from a patient with anaplastic large cell lymphoma were stimulated with irradiated 721.221-mIL21 feeder cells. The purity of T cells was checked at day 7, day 20, and day 28, respectively.

[0027] Fig.12Primary human NK cell expansion using four different feeder cell types. PBMCs were stimulated with irradiated K562, K562-mIL21, 721.221, and 721.221-mIL21, and quantitative data demonstrate fold expansion of NK cells.

[0028] Figures 13A-13N .221-mIL21-expanded NK cells showed enriched metabolic pathways and immature phenotypes. ( Fig.13A ) PBMCs were stimulated with irradiated K562-mIL21 and 221-mIL21 feeder cells. NK cells were purified from the expanded cells using flow cytometry for RNA sequencing (RNA-Seq) at days 7 and 14. Principal component analysis (PCA) of inter-sample distances of NK cells expanded with K562-mIL21 or 221-mIL21 feeder cells at days 7 and 14. ( Fig. 13B ) Mean (MA) plot of differentially expressed genes (DEGs) in NK cells expanded with 221-mIL21 feeder cells compared to NK cells expanded with K562-mIL21 feeder cells at day 7; p-values ​​were calculated using DESeq2. The top 15 significant DEGs were marked on the MA plot. Upregulated, upregulated DEGs with adjusted p<0.05 and log2-fold change ≥1; Downregulated, downregulated DEGs with adjusted p<0.05 and log2-fold change ≤-1; NS, not significant. ( Fig. 13C ) MA plot of DEGs in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 14. The top 15 significant DEGs are marked on the MA plot. Upregulated, upregulated DEGs with adjusted p < 0.05 and log2-fold change ≥ 1; Downregulated, downregulated DEGs with adjusted p < 0.05 and log2-fold change ≤ -1; NS, not significant. ( Fig.13D ) Gene set enrichment analysis (GSEA) of cellular amino acid metabolic processes in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 7 using the Gene Ontology (GO) biological process (BP) dataset from the Molecular Signature Database (MSigDB). NES, normalized enrichment score; p.adjusted, false discovery rate (FDR)-adjusted p-value. Fig.13E ) GSEA of glycolysis in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 7 using the Hallmark dataset in MSigDB. NES, normalized enrichment score; p.adjusted, FDR-adjusted p-value. ( Fig.13F) Dynamic levels of glucose in the culture medium during NK cell expansion using K562-mIL21 or 221-mIL21 as feeder cells. Arrows indicate the time points of culture medium changes. ( Figure 13G ) Comparison of quantitative glucose uptake by NK cells expanded with K562-mIL21 feeder cells or 221-mIL21 feeder cells at day 7 and day 14. ( Fig.13H ) GSEA of lymphocyte activation in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 7 using the GO_BP dataset in MSigDB. NES, normalized enrichment score; p.adjusted, FDR-adjusted p-value. ( Fig.13I ) GSEA of lymphocytic differentiation in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 7 using the GO_BP dataset in MSigDB. NES, normalized enrichment score; p.adjusted, FDR-adjusted p-value. ( Fig.13J ) GSEA of cell-cell adhesion in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 7 using the GO_BP dataset in MSigDB. NES, normalized enrichment score; p.adjusted, FDR-adjusted p-value. ( Figure 13K ) Heat map of NK cell inhibitory receptors. Figure 13L ) Heat map of NK cell activation receptors. Figure 13M ) Heat map of genes associated with NK cell cytotoxicity. Fig.13N ) Heatmap of genes associated with NK cell development and maturation. The heatmap was generated from z-scores obtained from transformed RNA-seq counts using regularized-logarithm transformation (rlog). Each column represents a biological replicate.

[0029] Figures 14A-14F . Kinetics of expansion of different cell populations in different types of feeder cell expansion systems. Fig.14A ) Dynamic time-lapse expansion data of the percentage of T cells (CD3+CD56-) from PBMCs (n=11) expanded for 21 days with irradiated K562, K562-mIL21, 221, and 221-mIL21 feeder cells. ( Fig. 14B ) Quantitative data of the percentage of T cells (CD3+CD56-) from PBMCs (n=11) expanded with the indicated feeder cells at day 21. ( Fig. 14C) Dynamic time-lapse expansion data of the percentage of CD3+CD56+ from PBMCs (n=11) expanded for 21 days with irradiated K562, K562-mIL21, 221 and 221-mIL21 feeder cells. ( Fig.14D ) Quantitative data of the percentage of CD3+CD56+ from PBMCs (n=11) at day 21 of expansion with the indicated feeder cells. ( Fig.14E ) Dynamic time-lapse expansion data of the percentage of CD3-CD56- from PBMCs (n=11) expanded for 21 days with irradiated K562, K562-mIL21, 221 and 221-mIL21 feeder cells. ( Fig.14F ) Quantitative data of the percentage of CD3-CD56- from PBMCs (n=11) at day 21 of expansion with the indicated feeder cells. ( Fig.14A , 14C Means (solid lines) with 95% CI (grey bands) are shown in 14A and 14E). *p<0.05, **p<0.01, ***p<0.001, ns p>0.05.

[0030] Figures 15A-15K Figure S7. Improved expansion of umbilical cord blood-derived NK cells using 221-mIL21 cells. Fig.15A ) Representative flow cytometry dot plots of the purity of NK cells expanded with different feeder cells on the indicated day after expansion. Umbilical cord blood mononuclear cells (CBMCs) were stimulated with irradiated K562-mIL21 or 221-mIL21 on day 0, and the purity of NK cells was checked on day 7 and then every 3 to 4 days. ( Fig. 15B ) Dynamic time-lapse expansion data of fold expansion of NK cells from CBMCs from 9 donors expanded for 21 days with irradiated K562-mIL21 or 221-mIL21 feeder cells. ( Fig. 15C ) Quantitative data of fold expansion of NK cells from CBMCs of 9 donors on day 21. ( Fig.15D ) Dynamic time-lapse expansion data of the purity of NK cells from CBMCs from 9 donors expanded for 21 days using irradiated K562-mIL21 or 221-mIL21 feeder cells. ( Fig.15E ) Quantitative data of the purity of NK cells from CBMCs of 9 donors on day 21. ( Fig.15F ) Dynamic time-lapse expansion data of the percentage of T cells (CD3+CD56-) in CBMCs (n=9) expanded for 21 days with irradiated K562, K562-mIL21, 221 and 221-mIL21 feeder cells. ( Figure 15G ) Quantitative data of the percentage of T cells (CD3+CD56-) in CBMCs (n=9) expanded with the indicated feeder cells at day 21. ( Fig.15H ) Dynamic time-lapse expansion data of the percentage of CD3+CD56+ in CBMCs (n=9) expanded for 21 days with irradiated K562, K562-mIL21, 221 or 221-mIL21 feeder cells. ( Fig.15I ) Quantitative data of the percentage of CD3+CD56+ of CBMCs (n=9) expanded with the indicated feeder cells at day 21. ( Fig.15J ) Dynamic time-lapse expansion data of the percentage of CD3-CD56- in CBMCs (n=9) expanded for 21 days with irradiated K562, K562-mIL21, 221 and 221-mIL21 feeder cells. ( Figure 15K ) Quantitative data of the percentage of CD3-CD56- in CBMCs (n=9) expanded with the indicated feeder cells at day 21. ( Fig. 15B , 15D Means (solid lines) with 95% CI (grey bands) are shown in 15F, 13H and 15J). **p<0.01, ns p>0.05.

[0031] Figures 16A-16D Phenotype and function of NK cells expanded from umbilical cord blood mononuclear cells using different feeder cell systems. Fig.16A ) Representative histograms of the expression of NKG2D, NKp46, 2B4, and CD226 on NK cells expanded from cord blood mononuclear cells using 221-mIL21 (red) and K562-mIL21 (green) feeder cells. Also shown are NK cells (blue) from freshly isolated cord blood mononuclear cells from the same donor. ( Fig. 16B ) Representative histograms of the expression of CD69, CD94, CD8a, and CD16 on NK cells expanded from cord blood mononuclear cells using 221-mIL21 (red) and K562-mIL21 (green) feeder cells. Also shown are NKs from freshly isolated cord blood mononuclear cells from the same donor (blue). ( Fig. 16C ) Representative histograms of the expression of NKG2A, NKG2C, KIR and KIR3DL1 on NK cells expanded from cord blood mononuclear cells using 221-mIL21 (red) and K562-mIL21 (green) feeder cells. Also shown are NKs from freshly isolated cord blood mononuclear cells from the same donor (blue). ( Fig.16D ) Quantitative data of the cytotoxic activity of expanded CB-NK cells against K562 cells using CFSE / 7-AAD cytotoxicity assay. K562 cells were labeled with CFSE and then incubated with expanded CB-NK cells for 4 hours at E:T ratios ranging from 5:1 to 0.3125:1. Next, lysis of K562 cells was detected using 7-ADD.

[0032] Figures 17A-17H . Expanding PBMC-derived CD19-CAR NK cells using different feeder cell systems. Fig.17A ) Representative flow cytometry dot plots of the percentage of CD19-CAR positive cells in NK cells at the indicated time points. PBMCs were stimulated with irradiated feeder cells on day 0 and transduced with CD19-CAR retrovirus on day 7. ( Fig. 17B ) Quantitative data of the percentage of CD19-CAR positive cells in NK cells expanded from PBMC (n=3). Fig. 17C ) Dynamic time-lapse expansion data of the percentage of T cells (CD3+CD56-) in CD19-CAR positive cells (n=3). Fig.17D ) Quantitative data of the percentage of T cells (CD3+CD56-) in CD19-CAR positive cells (n=3) on day 21. ( Fig.17E ) Dynamic time-lapse expansion data of the percentage of CD3+CD56+ in CD19-CAR positive cells (n=3) on day 21. ( Fig.17F ) Quantitative data of the percentage of CD3+CD56+ in CD19-CAR positive cells (n=3) on day 21. ( Figure 17G ) Dynamic time-lapse amplification data of the percentage of CD3-CD56- in CD19-CAR positive cells (n=3). Fig.17H ) Quantitative data of the percentage of CD3-CD56- in CD19-CAR positive cells (n=3) on day 21. ( Fig. 17C , 17E Means (solid lines) with 95% CI (grey bands) are shown in 17G and 17B ).

[0033] Figures 18A-18F .221 Enriched metabolic pathways and immune phenotypes of expanded NK cells. ( Fig.18A ) Dot plots of GSEA of genes between NK cells expanded with 221-mIL21 and K562-mIL21 feeder cells at day 7 (left) and day 14 (right) using the Gene Ontology (GO) biological process (BP) dataset in the Molecular Signature Database (MSigDB). ( Fig.18B ) Gene set enrichment analysis (GSEA) of cellular amino acid metabolism processes in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 14. NES, normalized enrichment score; p.adjusted, FDR-adjusted p value. Fig.18C) GSEA of glycolysis in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 14 using the Hallmark dataset from the Molecular Signature Database (MSigDB). NES, normalized enrichment score; p.adjusted, FDR-adjusted p-value. ( Fig.18D ) GSEA of lymphocyte activation in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 14. NES, normalized enrichment score; p.adjusted, FDR-adjusted p value. ( Fig.18E ) GSEA of lymphocytic differentiation in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 14. NES, normalized enrichment score; p.adjusted, FDR-adjusted p-value. ( Fig.18F ) GSEA of cell-cell adhesion in NK cells expanded with 221-mIL21 feeder cells compared with NK cells expanded with K562-mIL21 feeder cells at day 14. NES, normalized enrichment score; p.adjusted, FDR-adjusted p value.

[0034] Figures 19A-19I .Heatmap of enriched metabolic pathways and immune phenotypes of expanded NK cells. Figures 19A-19B ) Heat map of GSEA-identified genes in cellular amino acid metabolism. Fig.19C ) Heat map of GSEA-identified genes for glycolysis. Figures 19D-19E ) Heat map of GSEA-identified genes for lymphocyte activation. Figures 19E-19F ) Heat map of GSEA-identified genes for lymphocyte differentiation. Figures 19G-19I ) Heatmap of GSEA-identified genes for cell-cell adhesion. Heatmap was generated from z-scores obtained from transformed RNA-seq counts using regularized logarithmic transformation (rlog). Each column represents a biological replicate.

[0035] sequence

[0036] The nucleic acid and amino acid sequences provided herein are shown using standard letter abbreviations for nucleotide bases and amino acids as defined in 37 CFR § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in any reference to the displayed strand.

[0037] SEQ ID NO: 1 is an exemplary nucleic acid sequence from the extracellular domain of interleukin (IL)-21.

[0038] SEQ ID NO: 2 is an exemplary amino acid sequence from the extracellular domain of IL-21.

[0039] SEQ ID NO: 3 is an exemplary nucleic acid sequence of a construct for transducing cells with membrane-bound (m) IL-21.

[0040] SEQ ID NO: 4 is an exemplary nucleic acid sequence of IL-15Rα.

[0041] SEQ ID NO: 5 is an exemplary amino acid sequence of IL-15Rα.

[0042] SEQ ID NO: 6 is an exemplary nucleic acid sequence of IL-15.

[0043] SEQ ID NO: 7 is an exemplary amino acid sequence of IL-15.

[0044] SEQ ID NO: 8 is an exemplary nucleic acid sequence of IL-2.

[0045] SEQ ID NO: 9 is an exemplary amino acid sequence of IL-2.

[0046] SEQ ID NO: 10 is an exemplary nucleic acid sequence of IL-27.

[0047] SEQ ID NO: 11 is an exemplary amino acid sequence of IL-27.

[0048] SEQ ID NO: 12 is an exemplary nucleic acid sequence of IL-12B.

[0049] SEQ ID NO: 13 is an exemplary amino acid sequence of IL-12B.

[0050] SEQ ID NO: 14 is an exemplary nucleic acid sequence of IL-12p35.

[0051] SEQ ID NO: 15 is an exemplary amino acid sequence of IL-12p35.

[0052] SEQ ID NO: 16 is an exemplary nucleic acid sequence of IL-12p40.

[0053] SEQ ID NO: 17 is an exemplary amino acid sequence of IL-12p40.

[0054] SEQ ID NO: 18 is an exemplary nucleic acid sequence of IL-18.

[0055] SEQ ID NO: 19 is an exemplary amino acid sequence of IL-18.

[0056] SEQ ID NO: 20 is an exemplary nucleic acid sequence of IL-18.

[0057] SEQ ID NO:21 is an exemplary amino acid sequence of IL-18.

[0058] SEQ ID NO: 22 is an exemplary nucleic acid sequence of IL-33.

[0059] SEQ ID NO: 23 is an exemplary amino acid sequence of IL-33.

[0060] SEQ ID NO: 24 is an exemplary nucleic acid sequence of IL-7.

[0061] SEQ ID NO: 25 is an exemplary amino acid sequence of IL-7.

[0062] SEQ ID NO:26 is an exemplary nucleic acid sequence of MICA.

[0063] SEQ ID NO: 27 is an exemplary amino acid sequence of MICA. DETAILED DESCRIPTION

[0064] Disclosed herein are modified 721.221 cells expressing one or more cytokines or cytokine receptors (e.g., IL-15 receptor alpha (IL-15Rα) and / or membrane-bound IL-21) and methods of using modified 721.221 cells to expand immune cells. As shown herein, modified 721.221 cells can be used to effectively expand NK cells or T cells (including CAR-modified NK cells or T cells).

[0065] In combination with recombinant IL-15 and IL-2 and modified 721.221 cells, primary NK cells expanded approximately 39,663-fold after three weeks of expansion. Moreover, transduction with retroviruses encoding CAR molecules specific for the CD19 protein allowed the expansion of primary NK cells from peripheral blood and umbilical cord blood. Thus, a platform for the expansion of human primary NK cells and genetically modified CAR-NK cells is described.

[0066] Compared with previous NK expansion systems (Denman et al. PLoS One 7:e30264 (2012); Fujisaki et al. Cancer Res 69:4010-7 (2009)), the 721.221-mIL-21 cells for NK expansion described herein include three unique advantages. Using the technology described herein and in combination with two soluble cytokines in membrane form of IL-21 and cell culture, the number of amplified NK cells is significantly higher (approximately 39663 times in 721.221-mIL-21 cells, 3588 times using K562-mIL-21 cells), wherein NK cells are efficiently propagated in vitro. Compared with K562-mIL-21-expanded NK cells, 721.221-mIL-21-expanded NK cells also have the characteristics of higher purity and enhanced cytotoxicity. In addition, herein, 721.221-mIL-21NK was utilized to expand CAR-NK cells derived from umbilical cord blood (CB), which is easily obtained from a CB bank, and 2) CB-derived CAR-NK cells were used as ready-made CAR products.

[0067] the term

[0068] Unless otherwise indicated, technical terms are used conventionally. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); George P. Rédei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics, Second Edition, 2003 (ISBN: 0-471-26821-6).

[0069] Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" refer to one or more than one. For example, the term "comprising an interleukin" includes a singular or plural interleukin and is considered equivalent to the phrase "comprising at least one interleukin". Unless the context clearly indicates otherwise, the term "or" refers to a singular element or a combination of two or more elements of the stated optional elements. As used herein, "comprising" means "including". Therefore, "comprising A and B" means "comprising A, B, or A and B", without excluding additional elements.

[0070] It is further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​for nucleic acids or polypeptides are approximate and are provided for illustration only. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The same is true for accession numbers (for sequences appearing on February 20, 2019). In the event of a conflict, the present specification controls, including explanations of terms. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0071] In order to facilitate evaluation of various embodiments of the present disclosure, specific terms are explained below.

[0072] 721.221 cells: also called LCL 721.221 or CRL-1855 TM Cells, 721.221 cells are B lymphocytes derived from a cell line transformed by human Epstein-Barr virus. 721.221 cells do not express class I histocompatibility antigens (also known as major histocompatibility complex (MHC) class I molecules). Methods for producing 721.221 cells are known in the art (see, e.g., Shimiz et al., Proc Natl Acad Sci US A., 85(1):227-31, 1988, which is incorporated herein by reference in its entirety).

[0073] Activating receptor ligand: A ligand that binds to a receptor of a natural killer (NK) or T cell, thereby activating the NK or T cell. Examples of activating receptor ligands include UL16 binding protein (ULBP)-1, ULPB-2, and / or major histocompatibility complex (MHC) class I chain-associated protein A (MIC-A).

[0074] Autoimmune disorder: A disorder in which the immune system mounts an immune response (e.g., a B-cell or T-cell response) to a foreign antigen, resulting in tissue damage. The damage may be localized to certain organs, as in thyroiditis, or may involve specific tissues at various locations, as in Goodpaster's disease, or may be systemic, as in lupus erythematosus.

[0075] In some examples, the autoimmune diseases include systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, type I diabetes, Wegener's granulomatosis, inflammatory bowel disease, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, Graves' disease, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, pre-Alzheimer's disease, demyelinating disease, multiple sclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, DeLay's disease, Sler syndrome, myasthenia gravis, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia), adult-onset diabetes mellitus (type 2 diabetes), autoimmune infertility in men and women, ankylosing spondylitis, ulcerative colitis, Crohn's disease, mixed connective tissue disease, polyarteritis, systemic necrotizing vasculitis, juvenile rheumatoid arthritis Rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpaster syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird lover's lung, allergic diseases, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis Mannosis, trypanosomiasis, Takayasu arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter syndrome, eczema, lymphomatoid granuloma, Behcet's disease, Kaplan syndrome, Kawasaki disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema protuberans, psoriasis, erythroblastosis fetalis, eosinophilic facial inflammation, Schulman syndrome, Felty syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis cyclitis), Fuch's cyclitis, IgA nephropathy, Henoch-Schönlein purpura, glomerulonephritis, graft-versus-host disease, transplant rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, postvaccinia syndrome, congenital rubella infection, Hodgkin's lymphoma and non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulinemia, Epstein-Barr virus infection, mumps virus, and Evan's syndrome.

[0076] Cancer: Also known as "malignant tumor" or "malignant neoplasm", cancer refers to any of a variety of diseases characterized by uncontrolled, abnormal cell proliferation. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system (i.e., metastasis) and have many characteristic structural and / or molecular features. A "cancer cell" is a cell with special structural properties, lack of differentiation, and the ability to invade and metastasize. Both indolent and advanced forms are included. In some examples, the cancer is a solid cancer (such as a sarcoma (e.g., rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, and alveolar soft part sarcoma); a carcinoma (e.g., colorectal cancer and hepatocellular carcinoma (HCC)); and a lymphoma, such as Hodgkin's lymphoma or a non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma); neuroblastoma; gynecological cancer (e.g., ovarian cancer); breast cancer; liver cancer (e.g., hepatocellular carcinoma (HCC)); lung cancer; prostate cancer; skin cancer; bone cancer; pancreatic cancer; brain cancer (neuroblastoma); head and neck cancer; kidney cancer (such as Wilms' tumor); retinoblastoma; adrenal cortical tumors; desmoid tumors; desmoplastic small round cell tumors; endocrine tumors; and / or blood cancers (such as a myeloma, such as multiple myeloma; a lymphoma, such as Hodgkin lymphoma or a non-Hodgkin lymphoma, for example, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or a central nervous system lymphoma; or a leukemia, such as acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML)).

[0077] Chimeric antigen receptor (CAR): a chimeric fusion protein with an extracellular domain, which is fused to an intracellular signal transduction domain capable of activating T cells through a transmembrane domain. CAR molecules may include an extracellular domain (extracellular domain) and two (or more) targeting domains that are functionally different from each other (multi-specific CAR) and bind to two different sites on the target (multi-targeting). For example, a targeting domain of a multi-specific CAR can be a cell surface receptor, such as CD19 (e.g., a multi-specific CD19-based CAR). In another example, a targeting domain of a multi-specific CAR can be a cell surface receptor, such as CD19, and a second targeting domain can be an antibody or a fragment thereof, such as scFv (i.e., multi-specific CD19-scFv CAR). In some embodiments, CD19-scFv CAR binds to two different target sites (i.e., multi-targeted CD19-scFv). Single-function CAR contains only a single functional element in the targeted extracellular domain. In certain specific embodiments, a portion of the extracellular binding domain of CAR is derived from a mouse or humanized monoclonal antibody.

[0078] The intracellular signal transduction domain of the CAR molecule includes two different cytoplasmic signal transduction domains. For example, a signal transduction domain can be a cytoplasmic effector function signal transduction domain, and a second signal transduction domain can be a cytoplasmic co-stimulatory signal transduction domain. The joint can connect the domains to each other (such as two targeting domains) or they can connect one domain to another domain (such as connecting the ligand binding domain to the transmembrane domain). CAR is also called chimeric immune receptor, zetakines and universal T cell receptor.

[0079] Methods for making CARs are available (see, e.g., Park et al. Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Publications WO 2012 / 079000, WO 2013 / 059593; and US Publication 2012 / 0213783, each of which is incorporated herein by reference in its entirety).

[0080] Contact: In direct physical association, including solid and liquid forms. In one example, contact includes association between a substance or cell in a liquid culture medium (such as a cytokine or feeder cell) and one or more other cells (such as NK cells or T cells in culture). Contact with isolated cells or tissues can occur in vitro or in vivo by administration to a subject.

[0081] Culture or cell culture: In vitro growth of a cell population under a set of specified conditions (such as culture medium, extracellular matrix, temperature and / or culture time). In some examples, cell cultures include substantially pure cultures (e.g., isolated 721.221 cells or isolated NK cells). In other examples, cell cultures include mixed cultures, such as co-cultures of two or more types of cells (e.g., cultures of NK cells and feeder cells). In other examples, cell cultures include cells grown in contact with an extracellular matrix.

[0082] Culture medium: A set of synthetic culture conditions containing the nutrients necessary to support the survival, function and / or growth of a specific cell population, such as 721.221 cells. The culture medium generally includes components such as a carbon source, a nitrogen source, and a buffer to maintain pH. Additional components in the culture medium may also include one or more of serum, cytokines, hormones, growth factors, protease inhibitors, protein hydrolysates, shear force protectants, proteins, vitamins, glutamine, trace elements, inorganic salts, minerals, lipids and / or attachment factors.

[0083] Cytokine: A protein produced by cells such as lymphocytes that affect the behavior of other cells. In one embodiment, the cytokine is an interleukin, a molecule that regulates cell growth, differentiation, and motility (e.g., to stimulate immune responses, such as inflammation). In other embodiments, the cytokine can be an activating receptor ligand, a TRL ligand, or its receptor. In some examples, cytokines include molecules known to stimulate or costimulate cell expansion (e.g., NK or T cell expansion). The term "cytokine" can be used as a generic name for different groups of soluble proteins and peptides that act as humoral regulators at sodium molar to picomolar concentrations and regulate the functional activity of various cells and tissues under normal or pathological conditions. These proteins also directly mediate interactions between cells and regulate processes that occur in the extracellular environment. Examples of cytokines include, but are not limited to, tumor necrosis factor alpha (TNF-α), interleukin (IL-2), IL-7, IL-15, IL-21 including membrane-bound IL-21 (mIL-21), interferon (IFN) γ, IFNα, IFNβ, IL-12, IL-33, IL-27, IL-18, IL-1 family molecules (e.g., IL-1α, IL-1β, IL-1Rα, IL-18, IL-36Rα, IL36α, IL36β, IL-36γ, IL-37, IL-38, IL-33, toll receptor (TLR) ligands, activating receptor ligands (e.g., UL16 binding protein (ULBP)-1, ULPB-2, major histocompatibility complex (MHC) class I chain-related protein A (MIC-A)), IL-1 family molecules, Fc receptors, intercellular adhesion molecule 1 (ICAM-1), CD8α, 2B4 (also known as cluster of differentiation 244 (CD244)), intercellular adhesion molecule 1 (ICAM-1), CD8α, CD40, CD28, 4-1BB ligand (4-1BBL), OX40L, TRX518, CD3 antibodies, and CD28 antibodies.

[0084] Effective amount: The amount of a specified agent sufficient to achieve the desired effect, for example, in a subject being treated with an agent. In some examples, the effective amount of the amplified NK cells or T cells (e.g., chimeric antigen receptor (CAR)-NK cells or CAR-T cells) disclosed herein is sufficient to treat or inhibit a disease or disorder (such as a tumor, viral infection, autoimmune disease, or transplant rejection) in a subject. In other examples, the effective amount is the amount of amplified NK cells or T cells (e.g., chimeric antigen receptor (CAR)-NK cells or CAR-T cells) sufficient to reduce or alleviate one or more symptoms of a disease or disorder in a subject. The effective amount (e.g., the amount of a disorder in a subject to be alleviated, inhibited, and / or treated) will depend on, for example, the specific disorder being treated, the subject being treated, the mode of administering the composition, and other factors.

[0085] Expression: The process of converting the coded information of a gene into the operable, inoperable, or structural parts of a cell, such as protein synthesis. Gene expression can be affected by external signals. For example, exposing cells to hormones can stimulate the expression of hormone-induced genes. Different types of cells may respond differently to the same signal. Gene expression can also be regulated anywhere in the DNA to RNA to protein pathway. Regulation can include control of transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or through the activation, inactivation, compartmentalization, or degradation of specific protein molecules after their production.

[0086] Feeder cells: cells that provide support to another cell type in an ex vivo or in vitro culture. Feeder cells can provide one or more factors required for the survival, growth and / or differentiation (or inhibition of differentiation) of cells cultured with feeder cells. Typically, feeder cells are irradiated or otherwise treated to prevent them from proliferating in culture. In certain examples described herein, NK cells are cultured with feeder cells such as irradiated modified 721.221 cells (e.g., 721.221 cells expressing mIL-21).

[0087] Heterologous nucleic acid: A nucleic acid that is introduced into a cell, for example, by transduction or transfection. A "heterologous" nucleic acid or protein refers to a nucleic acid or protein that originates from a different genetic source. For example, a nucleic acid or protein that is heterologous to a cell originates from an organism or individual other than the cell in which it is expressed, including synthetic nucleic acids (e.g., mRNA). In other examples, a heterologous nucleic acid or protein originates from a cell type other than the cell in which it is expressed (e.g., a nucleic acid or protein that is not normally present in a 721.221 cell is heterologous to the 721.221 cell). In further examples, a heterologous nucleic acid includes a recombinant nucleic acid, such as a protein-coding nucleic acid operably linked to a promoter from another gene and / or two or more operably linked nucleic acids from different sources.

[0088] Immune system disorder: A disease or disorder associated with a pathological immune response in a subject (see International Patent Publication No. WO 2013 / 192294 and U.S. Patent Publication No. 2011 / 00811323, both of which are incorporated herein by reference). Examples include immunodeficiencies (e.g., primary or inherited immunodeficiencies and immunodeficiencies associated with other conditions, such as immunosuppression associated with HIV, aging, and cancer), cytokine storms, allergies, asthma, various types of inflammation, and autoimmune disorders.

[0089] Infectious Diseases: Also known as infectious diseases or contagious diseases, infectious diseases are pathological conditions caused by infection. Infections are caused by infectious agents including viruses, viroids, prions, bacteria; nematodes, such as parasitic roundworms and pinworms; arthropods, such as ticks, mites, fleas, and lice; fungi, such as ringworms; and other large parasites such as tapeworms and other worms. The host fights infection through the immune system, such as the innate response (e.g., mammals), which includes inflammation followed by an adaptive response. Drugs used to treat infections include antibiotics, antivirals, antifungals, antiprotozoal agents, and anthelmintics. Specific examples of infectious diseases include human immunodeficiency syndrome (HIV), hepatitis B virus (HBV), tuberculosis (TB), and malaria.

[0090] Inhibit or treat a condition: "Inhibit" a condition refers to inhibiting the full development of a condition or disease, such as a tumor. Inhibition of a condition can range from partial inhibition to substantially complete inhibition (e.g., including but not limited to prevention) of a disease (e.g., a tumor, a viral infection, an autoimmune disease, or transplant rejection). In some examples, the term "inhibit" refers to reducing or delaying the occurrence or development of a condition. "Treatment" refers to therapeutic intervention to alleviate the signs or symptoms of a disease or condition after it has begun to develop. A subject to whom an effective amount of the disclosed NK cells or T cells (e.g., CAR-NK cells or CAR-T cells) is to be administered can be identified by standard diagnostic techniques for such disorders (e.g., whether a disease or disorder is present or a risk factor for developing a disease or disorder).

[0091] Isolated: An "isolated" or "purified" biological component (such as a cell, nucleic acid, peptide, protein, protein complex, or virus-like particle) has been substantially separated, isolated, or purified from other components (such as other biological components in the cell or organism in which the component naturally occurs). Thus, cells, nucleic acids, peptides, and proteins that have been "isolated" or "purified" include cells, nucleic acids, and proteins purified by standard purification methods.

[0092] The term "isolated" or "purified" does not require absolute purity; rather, it is considered a relative term. Thus, for example, an isolated biological component refers to a component in which the biological component is more enriched than in the natural environment of a cell, organism, sample, or production vessel (e.g., cell culture system). Preferably, the preparation is purified such that the biological component accounts for at least 50% of the total biological component content of the preparation, such as at least 70%, at least 80%, at least 90%, at least 95% or more.

[0093] Natural Killer (NK) Cell: A cell of the immune system that kills target cells in the absence of specific antigenic stimuli and without restriction according to MHC class. Target cells can be tumor cells or cells carrying viruses. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. NK cells usually account for about 10% to 15% of the mononuclear cell fraction in normal peripheral blood. Historically, NK cells were first identified by their ability to lyse certain tumor cells in the absence of previous immunity or activation. NK cells are thought to provide a "backup" protection mechanism against viruses and tumors that may evade cytotoxic T lymphocyte (CTL) responses by downregulating MHC class I presentation. In addition to participating in direct cytotoxic killing, NK cells also play a role in cytokine production, which may be important for controlling cancer and infection. Memory NK cells that reside in tissues are included.

[0094] In some examples, a "CAR-NK cell" is a NK cell transduced with a heterologous nucleic acid encoding or expressing a CAR.

[0095] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carrier (vehicle) that can be used in the present invention is a conventional carrier. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition (2005) describes compositions and formulations suitable for drug delivery of one or more therapeutic compositions such as one or more modified NK cells and / or additional pharmaceutical agents.

[0096] Generally, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally include injectable liquids, which include pharmaceutically and physiologically acceptable liquids such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as carriers. For solid compositions (e.g., powders, pills, tablets, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain a small amount of non-toxic adjuvant substances, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0097] Subject: A living multicellular vertebrate organism, a category that includes humans and non-human mammals (e.g., veterinary animals, including dogs and cats, as well as mice, rats, rabbits, sheep, horses, cattle, and non-human primates).

[0098] T cell: A type of white blood cell that is critical to the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. CD4+ T lymphocytes are immune cells that express CD4 on their surface. These cells, also known as helper T cells, help coordinate immune responses, including antibody responses as well as killer T cell responses. Th1 and Th2 cells are functional subsets of helper T cells. Th1 cells secrete a group of cytokines, including interferon-γ, whose main function is to stimulate phagocyte-mediated defense against infections, especially those associated with intracellular microorganisms. Th2 cells secrete a group of cytokines, including interleukin (IL)-4 and IL-5, whose main function is to stimulate IgE and eosinophil / mast cell-mediated immune responses and downregulate Th1 responses. In other examples, T cells may include regulatory T cells (Tregs), NKT cells, tumor-infiltrating lymphocytes (TILs), other unconventional T cells (e.g., MAITs, γδT cells, and CD8αα+IELs), innate lymphocytes (ILCs), tissue-resident memory T cells, or any vaccine-induced T cells. Similar to CD4+ T cells, Tregs also express CD4 but can be distinguished by the expression of TGFβ. Tregs can help treat immune disorders such as autoimmune diseases, chronic graft-versus-host disease (GVHD), diabetes, systemic lupus erythematosus, obesity and encephalitis as well as promote organ transplant acceptance. NKT cells co-express αβT cell receptors and a variety of molecular markers commonly associated with NK cells, such as CD161. NKT cells can recognize lipids and glycolipids presented by CD1d molecules, and therefore, NKT cells can be used to recognize glycolipids from organisms that cause tuberculosis, such as Mycobacterium.

[0099] In some examples, genetically modified T cells, such as "CAR-T cells", are T cells transduced with a heterologous nucleic acid encoding or expressing a CAR, or can be T cells expressing a chimeric cytokine receptor (CCR), which are T cells transduced with a heterologous nucleic acid encoding a CCR (see, e.g., PCT Patent Publication WO 2017 / 029512, which is incorporated herein by reference in its entirety).

[0100] Toll-like receptor (TLR) ligands: TLR ligands are evolutionarily conserved and include pathogen-associated molecules such as bacterial cell surface lipopolysaccharide (LPS), lipoproteins, lipopeptides, and lipoarabinomannan; proteins such as flagellin from bacterial flagella; double-stranded RNA from viruses; non-methylated CpG islands of bacterial and viral DNA; CpG islands in eukaryotic DNA promoters; and other RNA and DNA molecules.

[0101] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation includes all techniques by which nucleic acid molecules can be introduced into such cells, including viral vector transduction, plasmid vector transformation, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration (e.g., "transfection").

[0102] Vector: A nucleic acid molecule that allows an exogenous or heterologous nucleic acid to be inserted into a cell (without destroying the ability of the vector to replicate and / or integrate in the host cell). A vector may include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the required regulatory sequences to allow transcription and / or translation of the inserted gene or genes. In some non-limiting examples, the vector is a viral vector, such as a retroviral vector or a lentiviral vector.

[0103] II. Overview of Several Implementation Schemes

[0104] Described herein are modified (e.g., genetically engineered) 721.221 cells and modified 721.221 cell lines using irradiation (e.g., B cell lines obtained by mutagenesis that do not express MHC class I molecules or express low levels of MHC class I molecules (Shimizu et al., Proc Natl Acad Sci USA 85:227-31 (1988)) method for expanding immune cells (such as NK cells, T cells or genetically modified NK cells or T cells), wherein the irradiated modified 721.221 cell line expresses at least one of membrane-bound IL-21 (mIL-21), IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, IL-1 family cytokines, TLR ligands, ULBP-1, ULPB-2, Fc receptors, 2B4 (also known as CD244), intercellular adhesion molecule 1 (ICAM-1), CD8α and / or MIC-A. Also disclosed is a method for expanding immune cells (such as NK cells, T cells or genetically modified NK cells or T cells) by using a modified 721.221 cell line that encodes expression of membrane-bound IL-21 (mIL-21), IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, IL-1 family cytokines, TLR ligands, ULBP-1, ULPB-2, Fc receptors, 2B4 (also known as CD244), intercellular adhesion molecule 1 (ICAM-1), CD8α and / or MIC-A. 2. A method of producing modified 721.221 cells by nucleic acid transduction or transfection of IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, IL-1 family cytokines, TLR ligands, ULBP-1, ULPB-2, Fc receptors, 2B4 (also known as CD244), intercellular adhesion molecule 1 (ICAM-1), CD8α and / or MIC-A. Modified 721.221 cells are used in a method of amplifying primary NK cells or T cells or modified NK cells or T cells (such as CAR-NK or CAR-T cells). Finally, the amplified cells are used in a method of treating a disease or disorder such as cancer, an infectious disease or an immune disease.

[0105] Recent studies have shown that NK cells can be rapidly expanded in vitro using K562 cells expressing 4-1BB (also known as CD137) ligand (4-1BBL / CD137L) and IL-21 as feeder cells (Denman et al. PLoS One 7:e30264 (2012)). However, the characterization and application of these cells for treatment of patients is important to ensure that the cells are functional and healthy. In addition, specific NK cell expansion is required to advance in vivo NK cell immunotherapy. A potential concern regarding the use of irradiated feeder cells to expand NK cells in vitro in the presence of the cytokine IL-2 is naivety. Immune cells are exhausted or aged after rapid proliferation and differentiation (Keir et al. Annu Rev Immunol 26: 677-704 (2008)). Indeed, CAR-modified immune cells express exhaustion markers such as PD-1 (John et al. Oncoimmunology 2: e26286 (2013); Cherkassky et al., J Clin Invest 126: 3130-44 (2016); Chong et al., Blood (2016); Gargett et al., Mol Ther 24: 1135-49 (2016)). To solve the problem of immune cell exhaustion, one method is to block PD-1 signaling in CAR-modified T cells (Cherkassky et al., J Clin Invest 126: 3130-44 (2016)). Given the essential metabolic signaling in T cells (Buck et al., J Exp Med 212: 1345-60 (2015)), another possible strategy is to alter metabolic pathways in CAR-modified T cells (Ping et al., Protein Cell (2017)) or enhance lymphocyte metabolism (Lim WA and June CH, Cell 168: 724-40 (2017)).

[0106] In the prior art, the expansion of CAR-modified T cells and NK cells requires the use of antibodies and cytokines to stimulate genetically modified T cells and NK cells in vitro. This antibody and cytokine-driven activation and expansion can negatively change CAR-T / NK cell function. For example, at the end of a large number of expansion programs, CAR-modified immune cell exhaustion may be induced, which can be demonstrated by the upregulation of PD-1, TIM-3 and LAG-3 in CART cells (Long et al. Nat Med 21: 581-90 (2015)). Therefore, given that immune cell exhaustion is the main factor in the impaired immune response to tumors and viruses during chronic antigen stimulation, new modification and expansion strategies can be developed in vivo, but exhaustion is not induced (Wherry EJ, Nat Immunol 12: 492-9 (2011); Virgin et al. Cell 138: 30-50 (2009)). In addition, it takes at least 2-3 weeks to expand CAR-modified immune cells for clinical application, which is an obvious obstacle for some patients. The "Sleeping Beauty transposon" or piggBac system (Guerrero et al., Chin J Cancer 33:421-33 (2014); Singh et al., Immunol Rev 257:181-90 (2014); Maiti et al., J Immunother 36:112-23 (2013)), which is capable of delivering large (9.1-14.3 kb) transposable elements without significantly reducing T cell potency, combined with genetically engineered artificial cells expressing membrane-bound IL-15 and 4-1BB ligands, has been used for CAR-modified T cell immunotherapy.

[0107] III. Modified 721.221 Cells and Production Methods

[0108] Disclosed herein are modified (e.g., genetically engineered) 721.221 cells that express one or more of the following: a cytokine (e.g., membrane-bound interleukin-21 (mIL-21), IL-21, IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, a toll-like receptor (TLR) ligand, or an activating receptor ligand (e.g., UL16 binding protein (ULBP)-1, ULBP -2, major histocompatibility complex (MHC) class I chain-related protein A (MIC-A)), IL-1 family molecules, Fc receptors, intercellular adhesion molecule 1 (ICAM-1) and CD8α, 2B4 (also known as differentiation cluster 244 (CD244)), intercellular adhesion molecule 1 (ICAM-1) and CD8α), including CD40, CD28, 4-1BB ligand (4-1BBL), OX40L, TRX518, CD3 antibody and CD28 antibody. Herein, these cells are referred to as "modified 721.221 cells". In some embodiments, the modified 721.221 cells also express IL-15 receptor alpha (IL-15Rα). In one example, the modified 721.221 cells express mIL-21. In other examples, the modified 721.221 cells express mIL-21 and IL-15Rα.

[0109] 721.221 cells are B lymphocytes that are characterized by being transformed with human Epstein-Barr virus and expressing no class I histocompatibility antigens (also known as major histocompatibility complex (MHC) class I molecules) or expressing low levels of MHC I molecules. 721.221 cells are also known as LCL 721.221 (formerly also known as CRL-1855 TM Cells). 721.221 cells can be generated by any method used in the art. An exemplary method for generating 721.221 cells is described in Shimiz et al., Proc Natl Acad Sci US A., 85(1):227-31, 1988 (incorporated by reference in its entirety).

[0110] In some embodiments, the modified 721.221 cells include heterologous nucleic acids encoding one or more of mIL-21, IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, TLR ligands, ULBP-1, ULPB-2, MIC-A, IL-1 family molecules, Fc receptors, 2B4 (also known as CD244), intercellular adhesion molecule 1 (ICAM-1), and / or CD8α. In some embodiments, the nucleic acid encodes a protein that promotes the expansion of immune cells such as natural killer (NK) cells or T cells. In some examples, the nucleic acid encodes a cytokine or cytokine receptor (e.g., an interleukin or interleukin receptor), such as mIL-21, mIL-15, IL-7, IL-2, IL-12, IL-33, IL-27, IL-18, IFNα, IFNβ, IFNγ, an IL-1 family molecule or its receptor (e.g., IL-15Rα; see, e.g., Wu et al. Front Immunol, 8:930, 2017, incorporated herein by reference in its entirety), a toll-like receptor (TLR) ligand, an activating receptor ligand (e.g., ULBP-1, ULPB-2, MIC-A, Fc receptor, 2B4 (also known as CD244), intercellular adhesion molecule 1 (ICAM-1) and / or CD8α), CD40, CD28, 4-1BB ligand (4-1BBL), OX40L, TRX518, a CD3 antibody, and a CD28 antibody. In other examples, the cytokine or cytokine receptor is membrane-bound (e.g., membrane-bound IL-21 or membrane-bound IL-15). In specific non-limiting examples, the modified 721.221 cells include a nucleic acid encoding mIL-21. In other non-limiting examples, the modified 721.221 cells include heterologous nucleic acids encoding mIL-21 and IL-15Rα. In additional non-limiting examples, the modified 721.221 cells include heterologous nucleic acids encoding membrane-bound ICAM-1, Fc receptors, CD8α, ULBP-1, ULPB-2, or MIC-A.

[0111] In some examples, the nucleic acid encoding mIL-21 comprises or consists of a nucleic acid that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:1 and / or encodes a protein that comprises or consists of an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:2. In some examples, the nucleic acid encoding IL-15α comprises or consists of a nucleic acid that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:3 and / or encodes a protein that comprises or consists of an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:5.

[0112] Also disclosed herein is a method for producing modified 721.221 cells as described herein. Modified or recombinant 721.221 cells are produced by using at least one heterologous nucleic acid (such as encoding mIL-21, IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, TLR ligands, ULBP-1, ULPB-2, MIC-A, IL-1 family molecules, Fc receptors, 2B4, ICAM-1, CD8α, CD40, CD28, 4-1BB ligands (4-1BBL), OX40L, TRX518, one or more nucleic acids in CD3 antibodies or CD28 antibodies) and in some examples, IL-15Rα is also used to transduce or transfect 721.221 cells. In a specific non-limiting example, the modified 721.221 cells include heterologous nucleic acids encoding mIL-21. In other non-limiting examples, the modified 721.221 cells include heterologous nucleic acids encoding mIL-21 and IL-15Rα. In additional non-limiting examples, the modified 721.221 cells include heterologous nucleic acids encoding membrane-bound ICAM-1, Fc receptor, CD8α, ULBP-1, ULPB-2, or MIC-A.

[0113] In some examples, 721.221 cells are transduced or transformed with a vector (e.g., a lentiviral or retroviral vector) comprising at least one heterologous nucleic acid. For example, 721.221 cells are transduced or transfected with at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more heterologous nucleic acids, or about 1-2, 1-3, 1-5, 1-7, or 1-10 heterologous nucleic acids, or about 1, 2, or 3 heterologous nucleic acids.

[0114] Any transduction or transfection method can be used, such as viral transduction (e.g. using retroviruses such as MoMLV or lentiviruses) or non-viral transduction, mRNA transfection, or nanoscale nucleic acid delivery (e.g. chemical dendrimers, DNA dendrimers, nanospheres, nanolayers, nanorods and nanotubes).

[0115] In some embodiments, the disclosed methods utilize a viral vector to deliver at least one heterologous nucleic acid into 721.221 cells. Examples of suitable viral vectors include retroviral (e.g., MoMLV or lentiviral) vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and fowlpox vectors. In a specific example, a retroviral system is used to introduce one or more heterologous nucleic acids into 721.221 cells. In some examples, a MoMLV vector, such as a SFG retroviral vector, can be used. The SFG vector is derived from the murine leukemia virus (MLV) backbone. This type of murine leukemia virus (MLV)-based retroviral vector is a commonly used gene delivery vehicle and has been widely used in clinical trials. The current SFG vector is fully optimized for gene expression for lymphocyte genetic modification, protein expression, and viral titer.

[0116] In some examples, the SFG vector is a pseudotyped gamma retrovirus with an RD114 envelope. RD114 pseudotyped transient retrovirus supe can be generated by triple transfecting cells (e.g., 293T cells using GeneJuice (Novagen)) with Peq-Pam plasmid (Moloney GagPol; e.g., about 4.69 μg), RDF plasmid (RD114 envelope; e.g., about 3.125 μg), and SFG-VRC01 plasmid (e.g., about 4.69 μg). The supernatant can be collected (e.g., after about 48 and 72 hours). High titer production cell lines are generated by multiple transductions of monkey and human lymphocytes.

[0117] The introduced heterologous nucleic acid can be a nucleic acid encoding any cytokine, an activating receptor ligand or its receptor or fragment such as IL-21 (e.g., to produce mIL-21), IL-15Rα, IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, TLR ligand, ULBP-1, ULBP-2, MIC-A, IL-1 family molecules, Fc receptors, 2B4, ICAM-1, CD8α, CD40, CD28, 4-1BB ligand (4-1BBL), OX40L, TRX518, CD3 antibody and / or CD28 antibody. In a specific example, nucleic acid encoding mIL-21, IL-15Rα or a combination thereof. In other non-limiting examples, nucleic acid encoding membrane-bound ICAM-1, Fc receptor, CD8α, ULBP-1, ULBP-2 or MIC-A.

[0118] In embodiments where the at least one heterologous nucleic acid comprises a nucleic acid encoding a membrane-bound cytokine, the at least one heterologous nucleic acid can comprise the cytokine of interest and additional heterologous nucleic acid sequences (e.g., in the same or different vectors), e.g., to form a membrane-bound cytokine. For example, the at least one heterologous nucleic acid can comprise at least one extracellular sequence, at least one transmembrane sequence, and / or at least one intracellular sequence (e.g., in the same vector).

[0119] In some examples, at least one heterologous nucleic acid comprises at least two extracellular sequences, at least three extracellular sequences, at least four extracellular sequences or at least five extracellular sequences or about 1-2, 1-3 or 1-5 extracellular sequences. The at least one extracellular sequence may include a cytokine of interest for the membrane, such as an interleukin. In a specific example, the interleukin is IL-21. In some examples, at least one extracellular sequence may include an extracellular fragment from an IgG sequence. In some examples, at least one extracellular sequence may include an extracellular fragment from CD8α. In some examples, at least one heterologous nucleic acid comprises at least two extracellular sequences. In a specific example, the at least two extracellular sequences include a cytokine of interest such as IL-21 and an extracellular fragment from an IgG sequence.

[0120] In some examples, at least one heterologous nucleic acid comprises at least two transmembrane sequences or at least three transmembrane sequences or about 1-2 or 1-transmembrane sequences. In some examples, at least one transmembrane sequence may include a transmembrane fragment from a CD28 sequence. Other transmembrane sequences, such as transmembrane sequences from CD40L or 2B4, may also be used. In some examples, at least one heterologous nucleic acid comprises at least two intracellular sequences, at least three intracellular sequences, at least four intracellular sequences, at least five intracellular sequences, or at least six intracellular sequences, or about 1-2, 1-3 or 1-6 intracellular sequences. In some examples, at least one intracellular sequence may include an intracellular fragment from a CD28 sequence, an intracellular fragment from a 4-1BB sequence, and / or an intracellular fragment from a CD3ξ sequence. In some examples, the nucleic acid construct comprises or consists of a nucleic acid that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to SEQ ID NO:3.

[0121] This article describes techniques for isolating and enriching 721.221 cells in vitro. In some examples, a large number of 721.221 cells or a subpopulation of 721.221 cells can be isolated by an enrichment process, such as by using immunomagnetic beads or flow sorting. The isolated 721.221 cells can be grown in a cell culture medium. In one example, the culture medium is a medium containing 10% (v / v) fetal bovine serum (FBS) and 100U / mL penicillin-streptomycin. RPMI-1640 The isolated 721.221 cells can be analyzed by flow cytometry for expression of at least one transgene, such as mIL-21 and / or IL-15Rα. In some examples, the method includes preventing proliferation of the 721.221 cells, such as by contacting with a preventing agent or condition. In some examples, proliferation of 721.221 cells is prevented by irradiation (e.g., gamma irradiation, such as at a dose of at least 1,000, at least 2,000, at least 3,000, at least 5,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 11,000, at least 12,000, or at least about 15,000, or about 1,000-15,000, 2,000-12,000, 1,000-5,000, 5,000-10,000, or 8,000-12,000, or about 10,000 rads) or by contact with mitomycin-C (MC).

[0122] Various techniques known to those skilled in the art can be used to identify modified 721.221 cells. In some examples, flow cytometry or immunomagnetic methods are used to identify modified 721.221 cells. For example, a detectable antibody can be used to bind to a modified 721.221 that expresses, for example, a surface-expressed cytokine of interest, a TRL ligand, or an activating receptor ligand. In some examples, flow cytometry or magnetic beads can also be used to identify modified 721.221 cells.

[0123] IV. Methods for expanding immune cells using modified 721.221 cells

[0124] Disclosed herein is a method for expanding NK or T cells using the modified 721.221 cells disclosed herein. In a specific example, CAR-modified NK or T cells are expanded using the methods disclosed herein.

[0125] Techniques for isolating and enriching NK or T cells in vitro or ex vivo are described herein. Exemplary methods are described in U.S. Patent Application Publication 2014 / 0086890, International Patent Application Publication WO 2017 / 127729, and U.S. Patent Publication 2013 / 0315884, which are incorporated herein by reference in their entirety. One skilled in the art may identify additional methods for expanding NK or T cells, such as described in Childs et al. Hematol. The Education Program 2013:234-246, 2013, which are incorporated herein by reference in their entirety; U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication 2006 / 0121005.

[0126] Mononuclear cells are collected from a subject (e.g., a healthy subject, a donor subject, or a subject with cancer, immune disorder, or infectious disease) or from a donor that is HLA matched to the subject to be treated. In some examples, mononuclear cells are collected by apheresis. For example, mononuclear cells are enriched for NK or T cells by negative depletion using an immunomagnetic bead strategy. In other examples, mononuclear cells include PBMCs, which are prepared, for example, using a method based on Separation method ( Healthcare) and other polysaccharide technologies.

[0127] In some examples, a mononuclear cell sample is optionally depleted of T cells, B cells, monocytes, dendritic cells, platelets, macrophages, and erythrocytes using a mixture of biotinylated monoclonal antibodies to enrich for NK cells. In some examples, magnetic beads coupled to streptavidin are used to remove non-NK cells from the sample, thereby obtaining an enriched preparation of NK cells. An exemplary commercially available kit for this method is Untouched TM Human NK Cell Kit (ThermoFisher Scientific, Waltham, MA).

[0128] In some examples, a mixture of biotinylated monoclonal antibodies is used to deplete NK cells, B cells, monocytes, dendritic cells, platelets, macrophages, and erythrocytes of a mononuclear cell sample and enrich for T cells. In some examples, magnetic beads coupled to streptavidin are used to remove non-NK cells from the sample, thereby obtaining an enriched preparation of NK cells. An exemplary commercially available kit for this method is EASYSEP TM Human T cell isolation kit (STEMCELL TM In some examples, magnetic beads coupled to streptavidin are used to remove non-NK cells from the sample, thereby obtaining an enriched preparation of NK cells.

[0129] In some examples, NK or T cells are enriched by positive selection. In some examples, the method includes, for example, selecting CD56 cells by using magnetic beads conjugated to anti-CD56 antibodies (e.g., CD56 MicroBeads, Miltenyi Biotec, Inc., Auburn, CA). + NK cells are positively selected to enrich for NK cells. In other examples, negative depletion (such as T cell depletion) and subsequent CD56 + A two-step method of positive selection of NK cells is used to enrich NK cells. In other examples, the method includes, for example, using magnetic beads conjugated to anti-CD4 or anti-CD8 antibodies (such as CD4 or CD8 MicroBeads, Miltenyi Biotec, Inc., Auburn, CA) to enrich CD4 + T cells or CD8 + T cells are positively selected to enrich for T cells. In other examples, negative depletion (such as NK cell depletion) and subsequent CD4 + T cells or CD8 +A two-step method of positive selection of T cells is used to enrich T cells. One skilled in the art can identify other methods that can be used to prepare enriched NK or T cell populations.

[0130] The expression of the marker of the NK or T cell separated by flow cytometry analysis. In some examples, the purity of the separated cells can be determined using a marker. In some examples, CD56 can be used as a marker, for example, to analyze NK cells. In some examples, CD8 or CD4 can be used as a marker, for example, to analyze T cells.

[0131] In some embodiments, NK cells or T cells are expanded in vitro. In some examples, the NK cells or T cells of enrichment can be used for expansion. In other examples, NK cells or T cells are expanded using a heterologous cell pool (such as a cell group derived from a sample such as a tissue, body fluid or blood sample). In some examples, a cell group includes peripheral blood mononuclear cells (PMBC). Cell groups (such as PMBC) can be generated from any tissue, body fluid or blood sample, for example, peripheral blood, cord blood, ascites, menstrual blood or bone marrow. In a specific example, a cell group includes PMBC from a healthy donor, cord blood mononuclear cells from a healthy donor or PBMC from a patient with non-Hodgkin's lymphoma (NHL).

[0132] In some examples, to enhance expansion, NK cells or T cells are expanded using modified 721.221 cells disclosed herein (e.g., 721.221 cells expressing mIL-21). Modified 721.221 cells disclosed herein are used as feeder cells for NK or T cells. Any amount of cells and feeder cells for expansion can be used. In some examples, the amount of cells (e.g., PMBCs) used for expansion can include at least about 10 1 , at least about 10 2 , at least about 10 3 , at least about 10 4 , at least about 10 5 , at least about 10 6 , at least about 10 7 , at least about 10 8 , at least about 10 9 or at least about 10 10 , about 10 1 -10 10 , 10 4 -10 8 or about 10 6 For example, 5x10 6 In some examples, the cells used for expansion (e.g., a cell population including NK cells or T cells, such as PMBCs) can be combined with at least about 10 1 , at least about 102 , at least about 10 3 , at least about 10 4 , at least about 10 5 , at least about 10 6 , at least about 10 7 , at least about 10 8 , at least about 10 9 or at least about 10 10 , about 10 1 -10 10 , 10 5 -10 9 or about 10 6 Such as 1x10 7 In some examples, the ratio of cells (e.g., PMBCs) used for amplification to feeder cells can be at least about 1:1 to about 1:50, such as at least about 1:1, at least about 1:2, at least about 1:5, at least about 1:6, at least about 1:7, at least about 1:8, at least about 1:9, at least about 1:10, at least about 1:15, at least about 1:20, at least about 1:25, at least about 1:30, at least about 1:35, at least about 1:40, at least about 1:45 or at least about 1:50 or about 1:2, about 1:7, about 3:20 or about 1:20. In some examples, other reagents are used to enhance amplification, such as additional cytokines, such as IL-2, IL-5, IL-7, IL-8 and / or IL-12.

[0133] The cells for expansion (e.g., a cell population comprising NK cells or T cells, such as PMBCs) are contacted with feeder cells and / or other expansion enhancing agents (e.g., IL-2, IL-5, IL-7, IL-8 and / or IL-12) for at least about 1-40 days, such as at least about 1 day, at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 35 days, about 10-30 days, 10-20 days, 20-30 days or 15-25 days, or about 14 days (e.g., for T cell expansion) or about 21 days (e.g., for NK cell expansion, such as CAR-NK cells).

[0134] The amplified NK cells or T cells (e.g., enriched or in a heterologous cell population such as PMBC) produced using the techniques disclosed herein (e.g., by contacting NK cells or T cells with, for example, feeder cells expressing mIL-21, such as modified 721.221 cells) may be superior to control amplification techniques that do not use feeder cells such as modified 721.221 cells (e.g., expressing mIL-21). In some examples, the amplification performed using the techniques disclosed herein can enhance the amplification by at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, 1-20 times, 5-15 times, 1-5 times, 5-10 times, 10-15 times, or about 10 times.

[0135] In some examples, the cytotoxicity of the expanded NK cells or T cells can be evaluated. Cytotoxicity can be evaluated at any time, such as after the expanded NK cells or T cells are expanded, or optionally, after the expanded NK cells or T cells are transduced (e.g., to express a chimeric antigen receptor (CAR)). In some examples, to evaluate cytotoxicity to tumor cells, an animal model can be used, such as an animal model that expresses a detectable tumor marker (e.g., a bioluminescent tumor marker, such as fluorescein, such as ffluc.Daudi tumor cells). In a specific example, the NK or T cells, for example, exhibit superior cytotoxicity to tumor cells compared to control NK or T cells not produced using the methods disclosed herein. Example For example, the NK or T cells produced using the disclosed method, for example, show greater cytotoxicity to tumor cells, which is at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times or at least about 10 times, about 0.5-10 times, 1-5 times or 5-10 times or about 3 times more toxicity. In some examples, chromium release assays can be used to assess the cytotoxicity of NK cells to cell targets. Those skilled in the art can identify other methods to assess the NK cell populations (e.g., purity, survival and / or activity) of separation.

[0136] In some embodiments, NK or T cells can be further transduced to express a protein of interest. In a specific example, NK or T cells can be transduced to express CAR. Then, modified NK or T cells are amplified using modified 721.221 cells and methods disclosed herein. NK or T cells can be transduced at any time throughout the method described herein, such as before or during amplification. In a specific example, NK or T cells are transduced with CAR during amplification (such as at least about 1 / 4, 1 / 3, 1 / 2 or 3 / 4 of the amplification process duration). In a specific non-limiting example, NK or T cells are transduced with CAR at about 1 / 3 of the amplification duration, for example, the amplification process is more than 21 days, and NK or T cells can be transduced with CAR on about the 7th day. In other examples, the modified 721.221 cells disclosed herein are subsequently modified to modify the amplified NK or T cells to express CAR.

[0137] In a specific example, NK or T cells are transduced with a viral vector (for delivery therein) containing a CAR of interest. Examples of suitable viral vectors include retroviruses (e.g., MoMLV or lentivirus), adenovirus, adeno-associated virus, vaccinia virus, and fowlpox vectors. In a specific example, CAR is introduced into NK or T cells using a retroviral system. In some examples, MoMLV vectors such as SFG retroviral vectors can be used. In some examples, CAR may include a protein or fragment thereof from at least one heterologous nucleic acid, may include at least one extracellular sequence, at least one transmembrane sequence, and / or at least one intracellular sequence (e.g., in the same or different vectors).

[0138] In some examples, at least one heterologous nucleic acid comprises at least two extracellular sequences, at least three extracellular sequences, at least four extracellular sequences, or at least five extracellular sequences or about 1-2, 1-3, or 1-5 extracellular sequences. The at least one extracellular sequence may include any CAR of interest, such as CD19 or κ light chain sequence. In some examples, at least one extracellular sequence may include an extracellular fragment from an IgG sequence. Other extracellular sequences may be used, including extracellular sequences from CD8α or CD28. In some examples, at least one heterologous nucleic acid comprises at least two extracellular sequences. In a specific example, the at least two extracellular sequences include CAR of interest such as CD19 or κ and an extracellular fragment from an IgG sequence.

[0139] In some examples, at least one heterologous nucleic acid comprises at least two transmembrane sequences or at least three transmembrane sequences or about 1-2 or 1- transmembrane sequences. In some sequences, at least one transmembrane sequence may include a transmembrane fragment from a CD28 sequence. Other transmembrane sequences, such as 4-1BB sequences, can be used. In some examples, at least one heterologous nucleic acid comprises at least two intracellular sequences, at least three extracellular sequences, at least four intracellular cells, at least five intracellular sequences or at least six intracellular sequences or about 1-2, 1-3 or 1-6 intracellular sequences. In some examples, at least one intracellular sequence may include an intracellular fragment from a CD28 sequence, an intracellular fragment from a 4-1BB sequence and / or an intracellular fragment from a CD3 ζ sequence.

[0140] Additional CARs may be used, such as LL1 (anti-CD74), GD2 antigen, CD5 antigen, CD57 antigen, LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituximab (anti-CD20), atezolizumab (GA101, anti-CD20), lambrolizumab (anti-PD1), nivolumab (anti-PD1), MK-3475 (anti-PD1), AMP-224 (anti-PD1), pidilizumab (anti-PD1), MDX-1105 (anti-PD-L1), MEDI4736 (anti-PD-L1), MPDL3280A (anti-PD-L1), BMS-936559 (anti-PD-L1), ipilimumab (anti-CTLA4), trevilizumab (anti-CTL4A), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known as TROP-2)), PAM4, or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e or CEACAM-5), MN-15 or MN-3 (anti-CEACAM-6), Mu-9 (anti-colon specific antigen-p), Immu-31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g. CC49), Tn, 1591 or HuJ591 (anti-PSMA (prostate specific Membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);PAM4 (also known as crizotinib, anti-mucin), BWA-3 (anti-histone H2A / H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1), PRI-1 (anti-histone H2B), LG11-2 (anti-histone H2B), LG2-2 (anti-histone H2B), and trastuzumab (anti-ErbB2), carbonic anhydrase IX, B7, CCL19, CCL21, CSAp, HER-2 / neu, BrE3, CDI, CD1a, CD2, CD3, CD4, CDS, CDS, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD72, GPC-3, CD74, CD79a, CDS0, CD83, CD95, CD126, CD133, CD137, D138, CD147, CD154, CD127 (also known as B7-H3), CEACAM-5, CEACAM-6, CTLA4, alpha-fetoprotein (AFP), VEGF (e.g., fibronectin splice variants), ED-B fibronectin (e.g., L19), EGP-1 (TROP-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1) (e.g., ), ErbB2, ErbB3, factor H, FHL-1, Flt-3, folate receptor, Ga 733, GRO family proteins, HMGB-1, hypoxia-inducible factor (HIF), HMl.24, HER-2 / neu, insulin-like growth factor (ILGF), IFN family proteins, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HMl.24, gangliosides, Fas-L, HCG, L243-bound HLA -DR antigen, CD66 antigen (i.e., CD66a-d or a combination thereof), MAGE, mCRP, MCP-1, MIP-1A, MIP-18, macrophage migration inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac, placental growth factor (PlGF), PSA (prostate specific antigen), PSMA, PAM4 antigen, PD1 receptor, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le (y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas Friedenreich antigen, tumor necrosis antigen, tumor angiogenesis antigen, TNF-a, TRAIL receptor (R1 and R2), TROP-2, VEGFR, RANTES and TI01. It is also possible to use other CARs, such as multispecific CARs (e.g., bispecific or trispecific CARs, such as including one or more CARs disclosed herein).

[0141] IV. Methods and Compositions for Treating or Inhibiting Conditions

[0142] Disclosed herein is a method for treating a subject with a disease or disorder by administering NK or T cells produced by the methods described herein to a subject (e.g., CAR-modified NK or T cells). In a specific non-limiting example, NK cells are administered. Unmodified NK or T cells or modified (e.g., CAR-modified) NK or T cells described herein can be administered to an animal or human subject. In a specific example, NK or T cells (or CAR-NK or CAR T cells) are from non-HLA-matched donors, including unrelated individuals. In other examples, NK or T cells (or CAR-NK or CAR T cells) are from a subject (e.g., autologous) being treated. In some embodiments, the disease or disorder is cancer (e.g., solid cancer (e.g., sarcoma (e.g., rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, and alveolar soft part sarcoma); carcinoma (e.g., colorectal cancer); lymphoma, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma); neuroblastoma; gynecological cancer (e.g., ovarian cancer); breast cancer; liver cancer; lung cancer; prostate cancer; skin cancer; bone cancer; pancreatic cancer; brain cancer (neuroblastoma); head and neck cancer; kidney cancer (e.g., Wilms' tumor); retinoblastoma adrenocortical tumor; desmoid tumor; desmoplastic small round cell tumor; endocrine tumor; and / or blood cancer (such as a myeloma, such as multiple myeloma; a lymphoma, such as Hodgkin lymphoma or non-Hodgkin lymphoma, such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma; or a leukemia, such as acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML)), an immune disorder (such as an autoimmune disease or transplant rejection) or an infectious disease (such as cytomegalovirus, adenovirus, respiratory syncytial virus, Epstein-Barr virus, or HIV infection).

[0143] The expanded NK or T cells described herein can be incorporated into a pharmaceutical composition. Such a composition generally includes a population of NK or T cells (such as modified NK or T cells) and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are compatible with drug administration (see, for example, Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition, 2005). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, balanced salt solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as fixed oils, may also be used. Supplementary active compounds may also be incorporated into the composition. Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in, for example, Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st In a non-limiting example, the transduced NK cells are suspended in PLASMA-LYTE TM in multiple electrolyte solutions.

[0144] In some examples, the composition comprises about 10 4 Up to 10 12 NK or T cells (e.g., about 10 4 Up to 10 8 cells, about 10 6 Up to 10 8 cells or about 10 6 Up to 10 12 For example, the composition can be prepared so that about 10 4 Up to 10 10 NK or T cells / kg (e.g. approximately 10 4 , 10 5 , 10 6 , 10 7 or 108 In a specific example, the composition comprises at least 10 4 , 10 5 , 10 6 or 10 7 NK cells. NK or T cell populations are usually administered parenterally, such as intravenously; however, injection or infusion into the tumor or close to the tumor (local administration) or administration to the peritoneal cavity can also be performed. A person skilled in the art can determine a suitable route of administration.

[0145] Multiple doses of NK or T cell populations can be administered to a subject. For example, NK or T cell populations can be administered daily, every other day, twice a week, weekly, every other week, every three weeks, monthly or less frequently. A skilled clinician can select an administration plan based on the subject, the condition being treated, previous treatment history, and other factors.

[0146] In another example, at least one, at least one, at least two, at least three or at least four cytokines (such as IL-2, IL-15, IL-21 and / or IL-12) are also administered to the subject to support the survival and / or growth of NK or T cells. In a specific non-limiting example, at least one cytokine includes IL-2 and IL-15 (for example, to support the survival and / or growth of NK cells). The cytokine is administered before, after or substantially simultaneously with NK or T cells. In a specific example, at least one cytokine (such as IL-2 and / or IL-2) is administered simultaneously with, for example, NK cells.

[0147] In some examples, the method includes treating or inhibiting a hyperproliferative disorder, such as a hematological malignancy or a solid tumor. Examples of hematological malignancies include leukemias, including acute leukemias (such as 11q23 positive acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), T-cell large granular lymphocytic leukemia, true erythroleukemia, Cell hyperplasia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (indolent and high-grade forms; including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma or central nervous system lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. Unmodified or modified (e.g., CAR-modified) NK or T cells can be administered. In a specific example, unmodified NK or T cells expanded using the methods herein can be administered to treat or inhibit lymphomas, such as B-cell lymphomas; gynecological cancers, such as ovarian cancer; breast cancer; liver cancer; lung cancer; or blood cancers, such as myeloma or leukemia, such as multiple myeloma, ALL or AML).

[0148] Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoma (including indolent and high-grade types; Hodgkin lymphoma; and non-Hodgkin lymphomas such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma), pancreatic cancer, breast cancer (including basal breast cancer, ductal carcinoma, and lobular breast cancer) ), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, and CNS tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0149] In specific examples, hematological malignancies that can be inhibited or treated by the methods disclosed herein include, but are not limited to, multiple myeloma, chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, prolymphocytic / myeloid leukemia, plasma cell leukemia, NK cell leukemia, Waldenstrom's macroglobulinemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma (indolent and advanced forms; including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma or central nervous system lymphoma. In other specific examples, solid tumors that can be treated or inhibited by the methods disclosed herein include lung cancer, prostate cancer, pancreatic cancer (such as insulinoma), breast cancer, colorectal adenocarcinoma or squamous cell carcinoma, neuroblastoma, testicular cancer (such as seminoma) and ovarian cancer. In specific, non-limiting examples, the subject has chronic myeloid leukemia, acute monocytic leukemia, or non-Hodgkin's lymphoma (indolent and high-grade forms; including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma). One skilled in the art can select NK cells or T cells expressing an appropriate transgene to treat a subject with a specific tumor or other disorder.

[0150] In some examples, one or more chemotherapeutic agents and / or radiation therapy may also be administered to a subject (e.g., a subject with a tumor or hyperproliferative disorder). These agents include alkylating agents such as nitrogen mustards (e.g., nitrogen mustard, cyclophosphamide, melphalan, uracil nitrogen mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, cemustine, streptozocin, or dacarbazine); antimetabolites such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine), and purine analogs such as mercaptopurine or thioguanine; or natural products such as vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase). Additional agents include platinum coordination complexes (such as cis-diamine dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocortical suppressants (such as mitotane and aminoglutethimide); hormones and antagonists, such as adrenocortical steroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include adriamycin, melphalan, and phenoxyprogesterone. Ara-C (cytarabine), carmustine, busulfan, lomustine, carboplatin, cisplatin, cyclophosphamide Daunorubicin, dacarbazine, 5-fluorouracil, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes such as docetaxel), vinblastine, vincristine, VP-16, and newer drugs including gemcitabine Trastuzumab Irinotecan (CPT-11), leustatin, navelbine, rituximab Imatinib (STI-571), topotecan Capecitabine, ibritumomab ) and calcitriol.

[0151] In some examples, the method includes treating or inhibiting blood cancer (including inert and advanced forms; including myeloma, such as multiple myeloma; lymphoma, such as Hodgkin lymphoma or non-Hodgkin lymphoma, such as diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma or central nervous system lymphoma; or leukemia, such as acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML)). For example, the method may include selecting a subject with blood cancer. The method may also include administering lymphocytes modified with any CAR disclosed by the method disclosed herein, thereby treating blood cancer. For example, NK cells modified with CD19-CAR produced using 721.221 cells. In some examples, the administered CD19-CAR modified NK cells can be generated using modified 721.221 cells expressing mIL-21 and / or IL-15Rα.

[0152] In a specific non-limiting example, the method includes treating or inhibiting leukemia (such as acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML)). For example, the method may include selecting a subject with leukemia. The method may also include administering lymphocytes modified by any CAR disclosed using the methods disclosed herein to treat leukemia, for example, using 721.221 cells to produce CD19-CAR modified NK cells. In some examples, modified 721.221 cells expressing mIL-21 and / or IL-15Rα can be used to produce administered CD19-CAR modified NK cells.

[0153] In some examples, the method includes treating or inhibiting solid tumors (indolent and high-grade forms; including sarcomas, carcinomas, and lymphomas (such as Hodgkin's or non-Hodgkin's lymphomas)). For example, the method may include selecting a subject with a solid tumor. The method also includes administering any CAR-modified lymphocytes disclosed using the methods disclosed herein to treat solid tumors. For example, 721.221 cells are used to produce CD19-CAR-modified NK cells. In some examples, modified 721.221 cells expressing mIL-21 and / or IL-15Rα can be used to produce the administered CD19-CAR-modified NK cells.

[0154] In a specific non-limiting example, the method includes treating or inhibiting lymphoma (including indolent and high-grade forms; including Hodgkin or non-Hodgkin lymphoma). For example, the method may include selecting a subject with lymphoma. The method also includes administering any CAR-modified lymphocytes disclosed using the methods disclosed herein to treat lymphoma. For example, NK cells modified with CD19-CAR produced using 721.221 cells. In some examples, modified 721.221 cells expressing mIL-21 and / or IL-15Rα can be used to produce the administered CD19-CAR-modified NK cells.

[0155] In a specific non-limiting example, the method includes treating or inhibiting non-Hodgkin's lymphoma (including indolent and high-grade forms; including diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma, or central nervous system lymphoma). For example, the method may include selecting a subject with non-Hodgkin's lymphoma. The method also includes administering any CAR-modified lymphocytes disclosed using the methods disclosed herein to treat non-Hodgkin's lymphoma. For example, CD19-CAR modified NK cells produced using 721.221 cells. In some examples, modified 721.221 cells expressing mIL-21 and / or IL-15Rα can be used to produce the administered CD19-CAR modified NK cells.

[0156] In some examples, the method includes treating or suppressing an immune system disorder. An immune system disorder can be any type of immune system disorder, such as a cytokine storm, an immune system disorder (e.g., inflammatory or autoimmune disorder) or can be an immune system disorder related to another disorder and / or disease (e.g., human immunodeficiency virus infection or microgravity exposure). In some non-limiting examples, an immune system disorder is an inflammatory disorder. In a specific embodiment, an inflammatory disorder can be rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel disease, or systemic lupus erythematosus. In other examples, an immune system disorder is an autoimmune disorder. In some embodiments, an autoimmune disorder is type I diabetes, multiple sclerosis, lupus erythematosus, myasthenia gravis, ankylosing spondylitis, celiac disease, Crohn's disease, Graves' disease, Hashimoto's thyroiditis, transplant rejection, or autoimmune uveitis. Modified or unmodified NK or T cells amplified by the method disclosed herein can be used. In specific examples, modified (e.g., CAR-modified) NK or T cells can be used, for example, to treat or inhibit rheumatoid arthritis, Crohn's disease, or transplant rejection.

[0157] In some examples, one or more immunomodulatory therapies (e.g., immunomodulatory biologics such as muromonab, ipilimumab, abatacept, belatacept, tremelimumab, BMS-936558, CT-011, MK-3475, AMP224, BMS-936559, MPDL323280A, MEDI4736, MGA271, IMP321, BMS-663513, PF-05082566, CDX-111127, anti-OX40, huMAb, OX40L, and TRXX518, such as Yao et al. Nat Rev Drug Discov, 12(2):130–146, 2013, and Kamphorst et al. Vaccine, 33(0 2):B21–B28, 2015, both of which are incorporated herein by reference in their entirety; regulatory cytokines, such as IL-7; mTOR modulators, such as rapamycin; antimicrobial therapies, such as vaccination, antifungals, and / or antibiotics), anti-inflammatory agents (NSAIDS, anti-leukotrienes, immunoselective anti-inflammatory derivatives, ImSAIDs, bioactive compounds with anti-inflammatory activity, such as plumbagin and frangipani; and / or steroids), disease-modifying antirheumatic drugs (DMARDs, such as methotrexate, sulfasalazine, leflunomide, hydroxychloroquine, tofacitinib, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, anakinra, abatacept, and / or rituximab), antimalarial drugs (such as chloroquine and hydroxychloroquine), medical procedures (including surgery and stem cell transplantation); immunosuppressants (e.g., for preventing rejection of transplanted organs or tissues, treating autoimmune diseases, immune and / or inflammatory diseases; for example, glucocorticoids, such as prednisone, dexamethasone, and hydrocortisone; cytostatics, such as alkylating agents and antimetabolites; antibodies, such as Atgam, thymoglobulin, and antibodies against T cell receptor- and IL-2 receptors; immunophilin targeting agents, such as cyclosporine, tacrolimus, sirolimus, and everolimus; interferons (IFNs), such as IFNλ and IFNβ; opioids; TNF binding proteins, such as infliximab, etanercept, and Adalimumab; mycophenolate mofetil; and small biologics, such as fingolimod and myriocin), immune tolerance therapy (e.g., for treatment of subjects at risk for tissue or organ transplant rejection, allergic subjects, and / or subjects with autoimmune diseases; for example, T- or B-cell-targeted or T- or B-cell-inhibitory drugs, such as CAMPATH-1H, calcineurin inhibitors, rituximab, epratuzumab, belimumab, and atacicept; anti-cluster of differentiation (CD) 3 antibodies; abatacept;Induction of hematopoietic chimerism, such as mixed hematopoietic chimerism, in which the bone marrow of an organ or tissue recipient is replaced with the bone marrow of a donor or a mixture of donor and recipient bone marrow to reduce organ or tissue transplant rejection; Antigen desensitization; see Nepom et al. Immuneno Rev; 241(1):49–62, 2011, incorporated herein by reference), antihistamines, helminthic therapy (e.g., intentionally infecting a subject with helminths or helminth eggs to treat an immune disorder). ;

[0158] In some examples, the method includes treating or inhibiting an infectious disease by administering to a subject a therapeutically effective amount of a composition disclosed herein. In some aspects, the infectious disease is selected from arbovirus infection, botulism, brucellosis, candidiasis, campylobacteriosis, chickenpox, chlamydia, cholera, coronavirus infection, staphylococcal infection, coxsackievirus infection, Creutzfeldt-Jakob disease, cryptosporidiosis, cyclospora infection, cytomegalovirus infection, Epstein-Barr virus infection, dengue fever, diphtheria, ear infection, encephalitis, influenza virus infection, parainfluenza virus infection, giardiasis, gonorrhea, Haemophilus influenzae infection, Hantavirus infection, viral hepatitis, herpes simplex virus infection, HIV / AIDS, Helicobacter infection, human papillomavirus (HPV) infection, infectious mononucleosis, Legionnaires' disease, leprosy, leptospirosis, listeriosis, Lyme disease, lymphocytic choriomeningitis, malaria, measles , Marburg hemorrhagic fever, meningitis, monkeypox, mumps, mycobacterial infection, mycoplasma infection, Norwalk virus infection, pertussis, pinworm infection, pneumococcal disease, Streptococcus pneumoniae infection, Mycoplasma pneumoniae infection, Moraxella catarrhalis infection, Pseudomonas aeruginosa infection, rotavirus infection, psittacosis, rabies, respiratory syncytial virus infection (RSV), ringworm, Rocky Mountain spotted fever, rubella, salmonellosis, SARS, scabies, sexually transmitted diseases, shigellosis, herpes zoster, sporotrichosis, streptococcal infection, syphilis, tetanus, trichinosis, tuberculosis, tularemia, typhoid fever, viral meningitis, bacterial meningitis, West Nile virus infection, yellow fever, adenovirus-mediated infections and diseases, retrovirus-mediated infectious diseases and yersiniosis zoonotic diseases. For example, the infectious disease can be influenza, parainfluenza, respiratory syncytial virus.

[0159] Unmodified or modified (e.g., CAR-modified) NK or T cells amplified by the methods disclosed herein can be used to treat or inhibit infectious diseases. In a specific example, CAR-modified NK or T cells amplified by the disclosed methods can be used to treat or inhibit HIV, such as using CARs based on HIV antibodies VRC01, 2G12, 2F5, 4E10, 3BNC117, 10–1074, VRC01LS, VRC07-532LS, 3BC176, PG16, NIH45-46G54W, PG9, PG16, PGT145, PGDM1400, PGT121, PGT124, PGT128, PGT135, 8ANC195, 10E8 and / or PD-1. In a specific example, NK or T cells modified by the disclosed methods can be used to treat or inhibit HBV, such as using CARs targeting HBsAg (e.g. nos. KP972453.1 or KP972454.1) and / or CAR of HB1.

[0160] In some examples, one or more anti-infective agents (e.g., antibodies, antifungals, antivirals, and / or antiparasitics) are also administered to a subject (e.g., a subject with an infectious disease such as HIV). In a specific example, the infectious disease is HIV, and an antiretroviral agent, such as a nucleoside and nucleotide reverse transcriptase inhibitor (nRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor, an entry inhibitor (or fusion inhibitor), a maturation inhibitor, or a broad-spectrum inhibitor, such as a natural antiviral, is also administered to the subject. Exemplary agents include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine, and efavirenz.

[0161] Example

[0162] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the invention to the specific features or embodiments described.

[0163] The clinical success of chimeric antigen receptor (CAR)-modified T cells requires engineering of autologous T cells obtained from patients, which limits the wider application of CAR cell therapy. The development of allogeneic universal cell products will significantly broaden its application and reduce costs. The unique biological characteristics of NK cells make it a safe and effective alternative immunotherapy strategy for CAR-modified T cells in the clinic.

[0164] This article describes a method for expanding NK and CAR-modified NK cells from peripheral blood and umbilical cord blood. Here, 721.221-based artificial antigen presenting cells (APCs) with membrane-bound interleukin (IL)-21 (mIL-21) are developed to breed clinical-grade NK and CAR-modified NK cells. In contrast to K562-based APCs with mIL-21, the ability to propagate NK cells with 721.221 feeder cells expressing mIL-21 (expansion 5335-94170 times; for example, Fig.12 ) is superior to K562 feeder cells with mIL-21 (amplification 662-7743 times). The NK cells amplified by 721.221-mIL-21 are phenotypically similar to the NK cells amplified by K562-mIL-21. However, it was observed that the NK cells amplified by 721.221-mIL-21 have excellent cytotoxicity. Therefore, the development of existing NK cell products with excellent functionality, persistence and value-added derived from cord blood or peripheral blood will support clinical applications for adoptive immunotherapy. This method provides a powerful tool for the field of immunotherapy to expand primary NK and CAR-modified NK cells for clinical applications.

[0165] Example 1: Methods and Materials

[0166] Antibodies and reagents. PE and APC anti-human CD3 antibodies (clone OKT3, ), FITC, BV605, PE / Cy7 and BV 510 anti-human CD56 antibodies (clone HCD56, ), PE anti-human CD69 antibody (clone FN50, ), PE / Cy7 anti-human CD8a antibody (clone HIT8a, ), AF647 anti-human IL-21 antibody (clone 3A3-N2, ), APC / Fire750 anti-human CD226 antibody (DNAM-1) (clone 11A8, ), APC / Fire 750 anti-human KLRG1 (MAFA) antibody (clone SA231A2, ), BV421 anti-human CD335 (NKp46) antibody (clone 9E2, ), PE / Cy7 anti-human CD158b (KIR2DL2 / L3, ) antibody (clone DX27, ), PE / Cy7 anti-human CD244 (2B4) antibody (clone C1.7, ), PE anti-human CD152 (CTLA-4) antibody (clone BNI3), APC anti-human CD366 (Tim-3) antibody (clone F38-2E2), PerCP / Cy5.5 anti-human TIGIT (VSTM3) antibody (clone A15153G), FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, ) and PerCP / Cy5.5 anti-human CD94 (clone DX22, )Purchased from (San Diego, CA, USA). APC anti-human CD16 antibody (clone B73.1, BD Biosciences TM Biosciences), FITC anti-human CD3 antibody (clone UCHT1, BD Biosciences), TM Biosciences), BV480 anti-human CD85j antibody (LIR-1) antibody (clone GHI / 75, BD Biosciences), TM Biosciences), BV711 anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences), TM Biosciences) and FITC anti-human CD107a antibody (clone H4A3, BD Biosciences) TM Biosciences) was purchased from BD TM Biosciences (San Jose, CA, USA). FITC anti-human KIR / CD158 antibody (clone 180704, R&D ), PE anti-human KIR2DL1 / KIR2DS5 antibody (clone 143211, R&D ), APC anti-human KIR3DL1 antibody (clone DX9, R&D ), AF405 anti-human KIR3DL2 / CD158k antibody (clone 539304, R&D ), APC anti-human NKG2A / CD159a antibody (clone 131411, R&D ) and PE anti-human NKG2C / CD159c antibody (clone 134591, R&D )Purchased from R&D AF647 goat anti-human IgG F(ab')2 fragment antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA).

[0167] Cell lines. 721.221 cell lines were donated by a gift. 293T, K562 and Daudi cell lines were purchased from AMERICAN TYPECULTURE To establish K562-mIL21 and 721.221-mIL21 cells, K562 and 721.221 cells were retrovirally transduced with IL-21, respectively, and then stained with AF647 mouse IgG1 anti-human IL-21 (clone 3A3-N2) using FACS ARIA TM II cell sorter (BD TM Biosciences) to sort membrane IL-21-positive cells. To establish Daudi-FFluc cells, CD19-positive Daudi cells were transduced with a lentiviral vector encoding FFIuc as previously described (Xiong et al. Mol Ther 26:963-75 (2018)). In 5% (v / v) CO 2 Incubate at 37°C in a humidified medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin. RPMI-1640 K562, 721.221, K562-mIL21, 721.221-mIL21, Daudi, and Daudi-FFluc cells were cultured in PBS. For NK cell expansion, K562, 721.221, K562-mIL21, and 721.221-mIL21 cells were irradiated at a dose of 10,000 rads, washed with PBS, and then used as feeder cells. The cells were cultured in 5% (v / v) CO 2 Incubate at 37°C in a humidified medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin. DMEM 293T was cultured in .

[0168] Primary NK cell expansion. Use lymphocyte separation medium PBMCs were isolated from buffy coats (Gulf Coast Regional Blood Center). In a 6-well cell culture plate (WilsonWolf), 200 U / ml IL-2 was added to 35 ml of and 5ng / ml IL-15 Complete RPMI-1640 medium was used with 1 × 10 7 5×10 feeder cells were cultured with 10,000 rads of irradiation 6 PBMC. The culture medium was changed every 3-4 days, leaving 2×10 7 The total number of cells was counted using trypan blue. To determine the percentage of NK cells, the cells were stained for CD3 and CD56 and then subjected to flow cytometry analysis.

[0169] Transduction of expanded NK cells with CD19-CAR. To generate CD19-CAR retrovirus, 293T cells were transfected with a combination of plasmids containing CD19-specific scFv, RDF, and PegPam3 as previously described (Xiong et al., Mol Ther 26:963-75 (2018)). NK cells were collected on day 7 of expansion and cultured on a plate coated with Two days later, the cells were transferred to 6-well cell culture plates and maintained in 35 ml complete RPMI-1640 medium containing 200 U / ml IL-2 (PeproTech) and 5 ng / ml IL-15 (PeproTech). The medium was changed every 3-4 days, leaving 2×10 cells in each well. 7 The total number of cells was counted using trypan blue. To determine the percentage of NK cells and expand CAR, the cells were stained with CD3, CD56 and anti-human IgG (H+L) F(ab')2 fragments and then analyzed by flow cytometry.

[0170] Flow cytometry analysis. PBMC and expanded NK cells were stained with fluorescently conjugated antibodies in FACS staining buffer (PBS containing 1% FBS) for 30 min on ice, washed with PBS, and plated on a FACS LSRII or Flow cytometer (BD TM ) were analyzed. PMT voltages were adjusted and compensation values ​​were calculated before data collection. TM Software (BD TM ) to obtain data and use Software (Tree Star) analysis.

[0171] NK cell cytotoxicity assay based on flow cytometry. NK cell cytotoxicity was determined using K562 and 721.221 cells as target cells, respectively. Target cells were collected and treated with 5 μM CELLTRACE TM CFSE The staining was continued for 20 minutes. The staining was terminated by adding complete RPMI-1640 medium and then washed twice with PBS. The expanded NK cells were collected and plated in complete RPMI-1640 medium on V-bottom 96-well plates at 5 different E:T ratios (effector:target; 4:1, 2:1, 1:1, 0.5:1, and 0.25:1, respectively). 5 CFSE-labeled target cells were co-cultured at 37°C and 5% CO 2 After incubation for 4 hours, cells were treated with 7-AAD (EBIOSCIENCE TM) staining and then analyzed by flow cytometry. + ), then use 7-ADD + The percentage of NK cell cytotoxicity was calculated by (experimental - spontaneous death) / (100 - spontaneous death) × 100%.

[0172] NK degranulation assay (CD107a). 1.5 × 10 5 NK cells (5×10 5 ) for 2 hours. Afterwards, the cells were collected, washed, and stained with GOLGISTOP TM CD3, CD56 and CD107a were stained for 30 minutes; and analyzed by flow cytometry. (See, e.g., Zheng et al. J Allergy Clin Immunol 135, 1293-1302, (2015), incorporated herein by reference).

[0173] Animal studies. All animal experiments were approved by the Houston Methodist Research Institute Institutional Animal Care and Use Committee (IACUC). NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice from the Jackson Laboratory were used for all in vivo experiments. To establish a human lymphoma xenograft model, male and female NSG mice (8 weeks old) were injected intravenously via the tail vein with 2×10 6 Starting from day 0, mice were injected (intravenously) with 1×10 FFLuc-Daudi cells in 100 μL PBS. 7 721.221-mIL21-expanded or K562-mIL21-expanded CD19-CAR NK cells were then injected (ip) with IL-2 (50,000 units / mouse) and IL-15 (10 ng / mouse) in 150 μL PBS on days 0, 3, 7, and 10. 150 mg / kg D-luciferin (GOLD St.Louis,MO,USA) 10 minutes later, use system( -200, Waltham, MA, USA) were used to image isoflurane-anesthetized animals. Software 64( Life Sciences, Hopkinton, MA, USA) quantifies the photons emitted from tumor cells expressing luciferase. A false color image (blue is the least intense, red is the most intense) representing the light intensity is generated and superimposed on a grayscale reference image. A constant region of interest (ROI) is drawn on the entire animal except the tail, and the intensity of the signal is measured in total photons per second. After effector CD19-CAR NK cell injection, animals are imaged twice a week at the Preclinical Imaging Center of the Houston Methodist Research Institute for tumor cell tracking.

[0174] RNA-seq sample preparation, sequencing and data analysis. NK cells were expanded in PBMCs with irradiated 221-mIL21 and K562-mIL21 cells as described above. On the 7th and 14th days of expansion, cells were collected and stained on ice for 30 minutes with PE-anti-CD3 and PE / Cy7-anti-CD56 antibodies. After washing twice with FACS staining buffer (PBS containing 2% FBS), the cells were respun in FACS staining buffer, and then CD3-CD56+ cells were sorted to a purity of> 98% for each replicate using a FACS Aria II cell sorter (BD Biosciences). Purified NK cells were directly lysed in Trizol reagent (Thermo Fisher Scientific) to extract RNA using the manufacturer's protocol. RNA side sequence (RNA-seq) was performed on the BGISEQ-500 platform by BGI Group (Shenzhen, Guangdong, China). After filtering low-quality reads (reads with more than 50% base quality less than 15), reads with adapters and reads with more than 10% unknown bases (N), clean reads in FASTQ format were obtained. FASTQ files were aligned to the hg38 human reference genome using STAR2.6.1d. The aligned files were processed using the GenomicAlignments package (v.1.20.0) to obtain a count matrix. As an initial step, genes with less than 10 read medians were pre-filtered in all comparisons. Differentially expressed genes were identified using the DESeq2 package (v.1.24.0) and defined as having an adjusted p-value <0.05 and a log2 fold change ≥1 or ≤-1. The log2 fold change was reduced using the lfcShrinkfunction, and then used to make an MA-map using the ggpubr package (v.0.2.1). GSEA was performed using MSigDB (Broad Institute) and clusterProfiler packages (v.3.12.0). Heatmaps were generated using z-scores derived from log-transformed counts. All data analyses were performed using R (v.3.6.0).

[0175] Statistical analysis. Data are expressed as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student's t-test, two-tailed paired Student's t-test, or two-way ANOVA, as indicated. P < 0.05 was considered statistically significant.

[0176] Example 2 Production of membrane-bound IL-21 on an artificial antigen presenting cell line

[0177] Previous studies have shown that IL-21 plays a key role in NK cell proliferation (e.g. Denman et al. PLoS One, 7(1): e30264, 2012). The artificial antigen presenting cell line was developed using 721.221 cells expressing membrane-bound IL-21, with no significant phenotypic changes ( Fig. 8A ). The expression of IL-21 receptor on human primary cells was also examined ( Fig. 9 ).

[0178] To expand human primary NK cells, PBMCs were isolated from peripheral blood or umbilical cord blood. Freshly isolated PBMCs were co-cultured with 721.221 cells expressing membrane IL-21 (221-mIL-21) in the presence of 200 U / mL IL-2 and 5 ng / mL IL-15 ( Figure 8B ).like Figures 8A-8B As shown, IL-21 was cloned into the SFG vector containing human IgG1, CD28 transmembrane (TM) domain, CD28 intracellular domain, 4-1BB ligand and CD3ζ ( Figures 8A-8B As controls, the assay included wild-type (WT) K562, K562-mIL-21, and WT 721.221. K562 and 721.221 cells were retrovirally transduced with IL-21 and sorted using FACS by staining with anti-human IL-21 antibodies. After 2 weeks of culture, FACS was used to examine the expression of IL-21 on K562-mIL21 and 721.221-mIL21. Figure 1A ) and 721.221-mIL21 cells ( Figure 1B ) expressed high levels of IL-21. Both K562-mIL21 and 721.221-mIL21 cells were stained with anti-IL-21 antibodies, and mIL-21 cells were also stained with anti-IL-21 antibodies and assessed for proper plasma membrane localization of IL-21 protein by confocal microscopy ( Figure 1C-1D ). The membrane-bound IL-21 molecule is expressed on the surface of K562-mIL21 and 721.221-mIL21 cells ( Figure 1D ). Verification of human primary NK cell expression of IL-21 receptor (IL-21R) Fig. 9 To determine whether IL-21 transduction of K562 and 721.221 cells alters the expression of activating and inhibitory NK cell ligands, flow cytometry was used to examine ICAM-1 (ligand for LFA-1), PD-L1 (ligand for PD-1), HLA-E (ligand for CD94 / NKG2A / C), and MICB (ligand for NKG2D). The expression levels before and after transduction were comparable ( Figure 1E and 1F). Thus, membrane expression of IL-21 in K562 and 721.221 cells with similar surface ligands was established.

[0179] Example 3: Excellent proliferation of NK cells by 721.221-mIL21 cells among different types of feeder cells

[0180] After establishing K562-, K562-mIL21, 721.221- and 721.221-mIL21 cell lines as feeder cells, the best feeder cell lines for amplifying human NK cells were investigated. To amplify primary human NK cells, PBMCs were isolated from the buffy coat of healthy donors and cultured with feeder cells plus 200U / ml IL-2 and 5ng / ml IL-15, as described above. To compare the ability of amplified NK cells, K562-mIL21 and 721.221-mIL21 were directly compared. WT-K562 and WT-221 cell lines were used as control groups. The initial number of NK cells and the ratio of NK cells were 5 million and 10-20%, respectively. Representative NK amplification spectra ( gated with CD3 and CD56) using flow cytometry data at different time points (day 0, day 7, day 12, day 17 and day 21) are shown. Figure 2A ). The dynamic number of NK cells was increased after 3 weeks of co-culture of PBMC with different feeder cells ( Figure 2B ) and ratio ( Figure 2C ) significantly increased. However, the fold increase of NK cells expanded with 721.221-mIL21 feeder cells ( Figure 2D ) and the purity of expanded NK cells ( Figure 2E ) was significantly higher than that of K562-mIL21 feeder cells. In addition, in the presence of feeder cells expressing mIL-21, non-NK cells (including CD3+CD56-, CD3+CD56+, and CD3-CD56- cells) were reduced ( Fig.14A -F). Therefore, 721.221-mIL-21 cells are superior to K562-mIL21 cells as feeder cells for expanding human primary NK cells.

[0181] Example 4 Characteristics of expanded NK cells from peripheral blood

[0182] To determine the immunophenotyping of the expanded NK cells, 562-, K562-mIL21-, 721.221-, and 721.221-mIL21-expanded NK cells were examined by flow cytometry using antibodies against activating and inhibitory receptors. Activating receptors include CD16, NKG2D, NKP46, 2B4, DNAM-1, CD69, CD94, CD8a, and NKG2C ( Figure 3A and 3BInhibitory receptors include NKG2A, CTLA-4, KIRG1, PD-1, LIR1, TIM-3, TIGIT, LAG-3, total KIR, KIR2DL1, KIR2DL2 / L3, KIR3DL1, and KIR3DL2 ( Figure 3C , 3D and 3E). The expression of these activating and inhibitory receptors on expanded NK cells was comparable. CD69, an activation marker for NK cells, was decreased in 221-mIL21-expanded NK cells.

[0183] To examine the functional similarity of the ex vivo expanded NK cells, we compared them with the NK susceptible target cells 721.221 ( Figure 4A and 4B ) and K562( Figure 4C and 4D ) were co-cultured to examine the cytotoxicity of expanded NK cells. Interestingly, NK cells expanded with 721.221-mIL-21 cells showed better cytotoxicity than NK cells expanded with K562-mIL-21 cells ( Figure 4A To further confirm this observation, the surface levels of CD107a molecules were examined using a CD107a assay after NK degranulation. Comparable degranulation was observed between K562-mIL-21- and 721.221-mIL-21-expanded NK cells ( Figure 4B When these expanded NK cells were co-cultured with WT K562 cells, no significant differences in cytotoxicity or degranulation were observed ( Figure 4C and 4D ).

[0184] To further examine whether NK cells isolated from umbilical cord blood (CB) can be expanded by this system, the number and purity of NK expansion of K562-mIL21 and 221-mIL21 were compared. Similar results were obtained ( Figures 15A-15K ). The immunophenotype of NK cells expanded by K562-mIL21 and 221-mIL21 was also examined by flow cytometry. In 221-mIL21-expanded NK cells with comparable cytotoxicity, CD69 expression was significantly reduced ( Figures 16A-16D ). In conclusion, the expanded NK cells displayed a phenotype similar to that of freshly isolated human primary NK cells and possessed superior cytotoxicity.

[0185] Example 5 Improving CAR-NK Expansion from Peripheral Blood Using 721.221-mIL-21 Cells

[0186] It was also examined whether 721.221-mIL-21 feeder cells could expand CAR-NK cells in a similar manner as non-genetically modified primary NK cells. To expand CAR-NK cells ex vivo, unfractionated PBMCs were stimulated with 721.221-mIL-21 feeder cells in the presence of soluble IL-2 and IL-15 for 7 days, inducing rapid proliferation of NK cells and, in some cases, nonspecific expansion of T cells ( Figure 5A ). On day 7, the expanded NK cells were transduced with CD19-CAR retroviral vector. The kinetics of NK cell number and purity were examined on days 0, 7, 11, 14, 18, and 21. Typical characteristics of ex vivo expanded NK cells from one donor showed superior NK number and purity ( Figure 5B ). Number of NK cells from 5 donors ( Figure 5C ) and purity ( Figure 5D ) showed that 721.221-mIL-21 feeder cells provided excellent CD19-CAR-NK cell expansion. The number of NK cells from 3 donors ( Figure 5C-5D ) and purity ( Figure 5E-5F ) showed that 221-mIL21 feeder cells provided excellent CD19-CAR-NK cell expansion.

[0187] However, the percentage of CD19-CAR-positive NK cells stimulated by K562-mIL21 was comparable to that of NK cells stimulated by 221-mIL21 ( Figures 17A-17H In contrast, non-NK cells (including CD3+CD56-, CD3+CD56+, and CD3-CD56- populations) were reduced in the presence of feeder cells expressing mIL-21 ( Figures 17A-17H ). In conclusion, 721.221-mIL-21 feeder cells showed superior CAR-NK cell expansion ability than CAR-NK cells expanded with K562-mIL-21 feeder cells.

[0188] Example 6 721.221-mIL-21 feeder cells demonstrate excellent ability to expand primary NK cells and CAR-NK cells derived from umbilical cord blood

[0189] In view of the multiple advantages of NK and CAR-NK cells derived from umbilical cord blood (Liu et al. Leukemia 32:520-31 (2018); Nahm et al. J Immunother 41:64-72 (2018); Balassa K and Rocha V, Expert Opin Bio Ther 18:121-34 (2018)), it was investigated whether 721.221-mIL-21 feeder cells can expand NK cells and CAR-NK cells derived from umbilical cord blood. To expand CB-NK cells in vitro, unfractionated CB-lymphocytes were stimulated with 721.221-mIL-21 feeder cells in the presence of soluble IL-2 and IL-15 for 7 days, inducing rapid proliferation of NK cells ( Figures 6A-6D ). As a control, in a separate group, unfractionated CB-lymphocytes were stimulated with K562-mIL21 feeder cells in the presence of soluble IL-2 and IL-15. On day 7, the expanded NK cells were retrovirally transduced with CD19-CAR. The kinetics and purity of NK cell numbers were examined on days 0, 7, 11, 14, 18, and 21. Typical characteristics of ex vivo expanded NK cells from one donor showed superior NK numbers and purity ( Fig. 6A ) NK cell quantity and purity from 3 donors ( Figure 6B ) showed that 721.221-mIL-21 feeder cells provided excellent CD19-NK cell expansion.

[0190] To examine whether these ex vivo expanded CD19-CAR.CB-NK cells have similar functions, the cytotoxicity of the expanded NK cells was examined by co-culturing them with NK-susceptible Raji and Daudi cells. CD19-CAR.CB-NK cells expanded with 221-mIL21 cells showed superior cytotoxicity than CD19-CAR.CB-NK cells expanded with K562-mIL21 cells ( Figure 6C-6D ). Therefore, the NK expansion method using 721.221-mIL-21 cells to improve umbilical cord blood-derived NK and CAR-NK expansion was successful.

[0191] Example 7 Effectiveness and side effects of in vivo expanded CAR-NK cells

[0192] To further evaluate whether the expanded CD19-CAR-NK cells can kill tumor cells in vivo, the anti-tumor activity of 721.221-mIL-21-expanded CD19-CAR-NK cells and K562-mIL-21-expanded CD19-CAR-NK cells was compared using the NSG mouse lymphoma xenograft model ( Figures 7A-7DLuciferase-labeled Daudi (FFluc-Daudi) was implanted using intravenous (iv) tail vein injection. Tumor burden was assessed at designated points by measuring tumor-derived bioluminescence followed by infusion of CD19-CAR-NK ( Fig. 7A After treatment, mice treated with 721.221-mIL-21-expanded CD19-CAR-NK cells showed superior anti-tumor activity than those treated with K562-mIL-21-expanded CD19-CAR-NK cells ( Figure 7B and 7C ). Similar tumor growth inhibition (TGI) results were obtained. To further evaluate the toxicity of CD19-CAR-NK cells expanded in vivo, the body weight of mice was quantified. No significant difference in body weight was observed ( Fig.7D ), indicating that the side effects of CD19-CAR-NK cells expanded in vitro were minimal. In summary, 721.221-mIL-21-expanded CD19-CAR-NK cells showed excellent in vivo anti-tumor activity with minimal side effects.

[0193] The NK cell expansion capacity of wild-type 221 cells and 221 cells expressing transmembrane presentation of IL-15 was compared. However, no significant difference in NK expansion fold and NK expansion purity was observed ( Figures 10A-10C ).

[0194] Example 8 Efficacy of T cells expanded by 221-mIL21 cells

[0195] Further testing was performed to expand T cells from different sources using 721.221-mIL21. CD3-positive T cell subsets from PBMC and cord blood were examined. Both K562-mIL21 cells and 721.221-mIL21 cells were able to expand T cells. However, using 721.221-mIL21 cells to expand T cells from PBMC and cord blood samples resulted in superior fold increases ( Figures 11A-11C The percentage of T cells from PBMCs expanded by 721.221-mIL21 cells was lower than that expanded using K562-mIL21 cells ( Fig.11A No difference in T cell purity from cord blood was observed between K562-mIL21 cells and 721.221-mIL21 cells ( Fig. 11B ). Further, 721.221-mIL21 feeder cells preferentially expand CD4+ T cells from PBMCs of patients with anaplastic large cell lymphoma (ALCL, a rare type of non-Hodgkin lymphoma). After two weeks of expansion by 721.221-mIL21 feeder cells, more than 90% of the cells were CD4+, CD3+, and CD56- subsets ( Fig. 11C).

[0196] Example 9 221-mIL21 feeder cell expansion system promotes the development of memory-like NK cells with a low degree of differentiation

[0197] To further investigate the superior NK cell expansion capacity and function induced by the 221-mIL21 feeder cell expansion system, RNA sequencing (RNA-Seq) experiments were performed using NK cells expanded with different feeder cell systems at different time points. Briefly, PBMCs were stimulated with irradiated K562-mIL21 or 221-mIL21 feeder cells. On days 7 and 14, NK cells expanded from these two different expansion systems were sorted for RNA-Seq using flow cytometry. The principal component analysis (PCA) plot of the inter-sample distances of NK cells expanded using K562-mIL21 or 221-mIL21 cells showed significant differences on day 7 compared to NK cells expanded on day 14 ( Fig.13A ).

[0198] Therefore, the following RNA-Seq data analysis focused on the data from day 7. Compared with NK cells expanded with K562-mIL21 on day 14, the number of differentially expressed genes (DEGs) in NK cells expanded with 221-mIL21 feeder cells on day 7 was significantly increased by the mean average (MA) plot ( Fig. 13B and 13C ). Gene set enrichment analysis (GSEA) using the Gene Ontology (GO) biological process (BP) dataset and the signature dataset in the Molecular Signature Database (MSigDB) showed that NK cells expanded using the 221-mIL21 feeder cell expansion system upregulated gene signatures related to cellular amino acid metabolic processes and glycolysis on day 7 compared with NK cells expanded using the K562-mIL21 feeder cell expansion system, which was further validated by glucose uptake analysis ( Figures 13D-13H ; Figures 18A-18C ; Figures 19A-19C ).

[0199] Unexpectedly, gene signatures for lymphocyte activation, lymphocyte differentiation, and cell-cell adhesion were significantly downregulated in NK cells expanded using the 221-mIL21 feeder cell expansion system at day 7 compared to NK cells expanded using the K562-mIL21 feeder cell expansion system ( Figures 13H-13J ; Fig.18A and 18D -18F), which can be further illustrated by heat maps of NK cell development and maturation, inhibitory receptors, activating receptors, and cytotoxic function ( Figures 13K-13N ; Figures 19D-19IIn conclusion, the 221-mIL21 feeder cell expansion system promotes the development of less differentiated, memory-like NK cells.

[0200] In view of the fact that the principles of the present invention can be applied to many possible embodiments, it should be recognized that the embodiments shown are only examples and should not be considered as limiting in scope. Instead, the scope is defined by the following claims. Therefore, we claim protection for all that is within the scope and spirit of these claims. Sequence Listing <110> Rutgers, The State University of New Jersey <120> Expansion of natural killer and chimeric antigen receptor modified cells <130> 7213-101957-02 <150> US 62 / 808,031 <151> 2019-02-20 <160> 27 <170> PatentIn Version 3.5 <210> 1 <211> 462 <212> DNA <213> Artificial sequence <220> <223> Exemplary nucleic acid sequences from the extracellular domain of interleukin (IL)-21 <400> 1 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgcgtc 60 gaccaaggtc aagatcgcca catgattaga atgcgtcaac ttatagatat tgttgatcag 120 ctgaaaaatt atgtgaatga cttggtccct gaatttctgc cagctccaga agatgtagag 180 acaaactgtg agtggtcagc tttttcctgc tttcagaagg cccaactaaa gtcagcaaat 240 acaggaaaca atgaaaggat aatcaatgta tcaattaaaa agctgaagag gaaaccacct 300 tccacaaatg cagggagaag acagaaacac agactaacat gcccttcatg tgattctttat 360 gagaaaaaac cacccaaaga attcctagaa agattcaaat cacttctcca aaagatgatt 420 catcagcatc tgtcctctag aacacacgga agtgaagatt cc 462 <210> 2 <211> 154 <212> PRT <213> Artificial sequence <220> <223> Exemplary amino acid sequences from the extracellular domain of IL-21 <400> 2 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Val Asp Gln Gly Gln Asp Arg His Met Ile Arg Met Arg 20 25 30 Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu 35 40 45 Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu 50 55 60 Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn 65 70 75 80 Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys 85 90 95 Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu 100 105 110 Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe 115 120 125 Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu 130 135 140 Ser Ser Arg Thr His Gly Ser Glu Asp Ser 145 150 <210> 3 <211> 1881 <212> DNA <213> Artificial sequence <220> <223> Exemplary nucleic acid sequences of constructs for transducing cells with membrane-bound (m) IL-21 <400> 3 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgcgtc 60 gaccaaggtc aagatcgcca catgattaga atgcgtcaac ttatagatat tgttgatcag 120 ctgaaaaatt atgtgaatga cttggtccct gaatttctgc cagctccaga agatgtagag 180 acaaactgtg agtggtcagc tttttcctgc tttcagaagg cccaactaaa gtcagcaaat 240 acaggaaaca atgaaaggat aatcaatgta tcaattaaaa agctgaagag gaaaccacct 300 tccacaaatg cagggagaag acagaaacac agactaacat gcccttcatg tgattcttat 360 gagaaaaaac cacccaaaga attcctagaa agattcaaat cacttctcca aaagatgatt 420 catcagcatc tgtcctctag aacacacgga agtgaagatt ccctgcggcc gccgtacgtc 480 accgtctctt cacaggatcc cgccgagccc aaatctcctg acaaaactca cacatgccca 540 ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 600 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 660 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 720 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 780 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 840 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 900 gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 960 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcaaccg 1020 gagacaacact acagaccac gcctcccgtg ctggactccg acggctcctt cttccctac 1080 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtctctc atgctccgtg 1140 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 1200 aagatccca aattttgggt gctggtggtg gttggtggag tcctggctg ctatagcttg 1260 ctagtacag tggcctttat tattttgg gtgaggagta agaggagcag gctcctgcac 1320 agtgactaca tgaacatgac tcccgccgc cccgggccca cccgcaagca tcccccc 1380 1440 tatgccccac cacgcgactt cgcagcctat cgctccaac tatatattca aaaaccatt tatgagacca gtashaacta ctcagagga agatggctgt 1500 agctgccgat ttccagaga agagaagga ggatgtgaac tgagtgaa gttcagcagg 1560 agcgcagacg cccccgcgta ccagcaggc cagaaccagc tctataacga gctcaatcta 1620 ggacgaagg aggagtacga tgttttggac agagacgtg gccgggaccc tgagatgggg 1680 ggaaagccga gaggagaa ccctcaggaa ggcctgtaca atgaacctgca gaagataag 1740 atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1800 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1860 caggccctgc cccctcgcta a 1881 <210> 4 <211> 1566 <212> DNA <213> Artificial Sequence <220> <223> Exemplary nucleic acid sequence of IL-15Ralpha <400> 4 ctgggcagcg ctcgcccggg gagtccagcg gtgtcctgtg gagctgccgc catggccccg 60 cggcgggcgc gcggctgccg gaccctcggt ctcccggcgc tgctactgct gctgctgctc 120 cggccgccgg cgacgcgggg catcacgtgc cctcccccca tgtccgtgga acacgcagac 180 atctgggtca agagctacag cttgtactcc agggagcggt acatttgtaa ctctggtttc 240 aagcgtaaag ccggcacgtc cagcctgacg gagtgcgtgt tgaacaaggc cacgaatgtc 300 gcccactgga caacccccag tctcaaatgc attagagacc ctgccctggt tcaccaaagg 360 ccagcgccac cctccacagt aacgacggca ggggtgaccc cacagccaga gagcctctcc 420 ccttctggaa aagagcccgc agcttcatct cccagctcaa acaacacagc ggccacaaca 480 gcagctattg tcccgggctc ccagctgatg ccttcaaaat caccttccac aggaaccaca 540 gagataagca gtcatgagtc ctcccacggc accccctctc agacaacagc caagaactgg 600 gaactcacag catccgcctc ccaccagccg ccaggtgtgt atccacaggg ccacagcgac 660 accactgtgg ctatctccac gtccactgtc ctgctgtgtg ggctgagcgc tgtgtctctc 720 ctggcatgct acctcaagtc aaggcaaact cccccgctgg ccagcgttga aatggaagcc 780 atggaggctc tgccggtgac ttgggggacc agcagcagag atgaagactt ggaaaactgc 840 tctcaccacc tatgaaactc ggggaaacca gcccagctaa gtccggagtg aaggagcctc 900 tctgctttag ctaaagacga ctgagaagag gtgcaaggaa gcgggctcca ggagcaagct 960 caccaggcct ctcagaagtc ccagcaggat ctcacggact gccgggtcgg cgcctcctgc 1020 gcgagggagc aggttctccg cattcccatg ggcaccacct gcctgcctgt cgtgccttgg 1080 acccagggcc cagcttccca ggagagacca aaggcttctg agcaggattt ttatttcatt 1140 acagtgtgag ctgcctggaa tacatgtggt aatgaaataa aaaccctgcc ccgaatcttc 1200 cgtccctcat cctaactttc agttcacaga gaaaagtgac atacccaaag ctctctgtca 1260 attacaaggc ttctcctggc gtgggagacg tctacaggga agacaccagc gtttgggctt 1320 ctaaccaccc tgtctccagc tgctctgcac acatggacag ggacctggga aaggtgggag 1380 agatgctgag cccagcgaat cctctccatt gaaggattca ggaagaagaa aactcaactc 1440 agtgccattt tacgaatata tgcgtttata tttatacttc cttgtctatt atatctatac 1500 attatatatt atttgtattt tgacattgta ccttgtataa acaaaataaa acatctattt 1560 tcaata 1566 <210> 5 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Exemplary Amino Acid Sequence of IL-15Ralpha <400> 5 Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr 20 25 30 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 35 40 45 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 50 55 60 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 65 70 75 80 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 85 90 95 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr 100 105 110 Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu 115 120 125 Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala 130 135 140 Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr 145 150 155 160 Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser 165 170 175 Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln 180 185 190 Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile 195 200 205 Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu 210 215 220 Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu 225 230 235 240 Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg 245 250 255 Asp Glu Asp Leu Glu Asn Cys Ser His His Leu 260 265 <210> 6 <211> 2333 <212> DNA <213> Artificial Sequence <220> <223> Exemplary Nucleic Acid Sequence of IL-15 <400> 6 gttgggactc cgggtggcag gcgcccgggg gaatcccagc tgactcgctc actgccttcg 60 aagtccggcg ccccccggga gggaactggg tggccgcacc ctcccggctg cggtggctgt 120 cgccccccac cctgcagcca ggactcgatg gagaatccat tccaatatat ggccatgtgg 180 ctctttggag caatgttcca tcatgttcca tgctgctgac gtcacatgga gcacagaaat 240 caatgttagc agatagccag cccatacaag atcgttttca actagtggcc ccactgtgtc 300 cggaattgat gggttcttgg tctcactgac ttcaagaatg aagccgcgga ccctcgcggt 360 gagtgttaca gctcttaagg tggcgcatct ggagtttgtt ccttctgatg ttcggatgtg 420 ttcggagttt cttccttctg gtgggttcgt ggtctcgctg gctcaggagt gaagctacag 480 accttcgcgg aggcattgtg gatggatggc tgctggaaac cccttgccat agccagctct 540 tcttcaatac ttaaggattt accgtggctt tgagtaatga gaatttcgaa accacatttg 600 agaagtattt ccatccagtg ctacttgtgt ttacttctaa acagtcattt tctaactgaa 660 gctggcattc atgtcttcat tttgggatgc agctaatata cccagttggc ccaaagcacc 720 taacctatag ttatataatc tgactctcag ttcagtttta ctctactaat gccttcatgg 780 tattgggaac catagatttg tgcagctgtt tcagtgcagg gcttcctaaa acagaagcca 840 actgggtgaa tgtaataagt gatttgaaaa aaattgaaga tcttattcaa tctatgcata 900 ttgatgctac tttatatacg gaaagtgatg ttcaccccag ttgcaaagta acagcaatga 960 agtgctttct cttggagtta caagttatt cacttgagtc cggagatgca agtattcatg 1020 atacagtaga aaatctgatc atcctagcaa acaacagttt gtcttctaat gggaatgtaa 1080 cagaatctgg atgcaaagaa tgtgaggaac tggagaaaa aaatattaaa gaattttgc 1140 agagttttgt acatattgtc caaatgttca tcaacacttc ttgattgcaa ttgattcttt 1200 ttaaagtgtt tctgttatta acaaacatca ctctgctgct tagacataac aaaacactcg 1260 gcatttcaaa tgtgctgtca aaacaagttt ttctgtcaag aagatgatca gaccttggat 1320 cagatgaact cttagaaatg aaggcagaaa aatgtcattg agtaatatag tgactatgaa 1380 cttctctcag acttacttta ctcattttt taatttatta ttgaaattgt acatatttgt 1440 ggaataatgt aaaatgttga ataaaaatat gtacaagtgt tgttttttaa gttgcactga 1500 tattttacct cttattgcaa aatagcattt gtttaagggt gatagtcaaa ttatgtattg 1560 gtggggctgg gtaccaatgc tgcaggtcaa cagctatgct ggtaggctcc tgccagtgtg 1620 gaaccactga ctactggctc tcattgactt ccttactaag catagcaaac agaggaagaa 1680 tttgttatca gtaagaaaaa gaagaactat atgtgaatcc tcttctttat actgtaattt 1740 agttattgat gtataaagca actgttatga aataaagaaa ttgcaataac tggcatataa 1800 tgtccatcag taaatcttgg tggtggtggc aataataaac ttctactgat aggtagaatg 1860 gtgtgcaagc ttgtccaatc acggattgca ggccacatgc ggcccaggac aactttgaat 1920 gtggcccaac acaaattcat aaactttcat acatctcgtt tttagctcat cagctatcat 1980 tagcggtagt gtatttaaag tgtggcccaa gacaattctt cttattccaa tgtggcccag 2040 ggaaatcaaa agattggatg cccctggtat agaaaactaa tagtgacagt gttcatattt 2100 catgctttcc caaatacagg tattttattt tcacattctt tttgccatgt ttatataata 2160 ataaagaaaa accctgttga tttgttggag ccattgttat ctgacagaaa ataattgttt 2220 atattttttg cactacactg tctaaaatta gcaagctctc ttctaatgga actgtaagaa 2280 agatgaaata tttttgtttt attataaatt tatttcacct taaaaaaaaa aaa 2333 <210> 7 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> Example amino acid sequence of IL-15 <400> 7 Met Val Leu Gly Thr Ile Asp Leu Cys Ser Cys Phe Ser Ala Gly Leu 1 5 10 15 Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys 20 25 30 Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr 35 40 45 Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe 50 55 60 Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile 65 70 75 80 His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser 85 90 95 Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu 100 105 110 Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val 115 120 125 Gln Met Phe Ile Asn Thr Ser 130 135 <210> 8 <211> 822 <212> Ms <213> artificial sequence <220> <223> IL-2 receptors are also present <400> 8 agttccctat cactctttt aatcactact cacagtaacc tcaactcctg ccacaatgta 60 caggatgcaa ctcctgtctt gcattgcact aagtcttgca cttgtcacaa acagtgcacc 120 tacttcaagt tctacaaaga aaacacagct acaactggag catttactgc tggatttaca 180 gatgatttg aatggaatta ataattacaa gaatcccaaa ctcaccagga tgctcacatt 240 taagttttac atgcccaaga aggccacaga actgaaacat cttcagtgtc tagaagaaga 300 actcaaacct ctggaggaag tgctaaattt agctcaaagc aaaactttc acttaagacc 360 cagggactta atcagcaata tcaacgtaat agttctggaa ctaaagggat ctgaaacaac 420 attcatgtgt gaatatgctg atgagacagc aaccattgta gaatttctga acagatggat 480 taccttttgt caaagcatca tctcaacact gacttgataa ttaagtgctt cccacttaaa 540 acatatcagg ccttctattt atttaaatat ttaaatttta tatttattgt tgaatgtatg 600 gtttgctacc tattgtaact attattctta atcttaaaac tataaatatg gatcttttat 660 gattcttttt gtaagcccta ggggctctaa aatggtttca cttatttatc ccaaaatatt 720 tattattatg ttgaatgtta aatatagtat ctatgtagat tggttagtaa aactatttaa 780 taaatttgat aaatataaaa aaaaaaaaaa aaaaaaaaaa aa 822 <210> 9 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of IL-2 <400> 9 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 10 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> Exemplary Nucleic Acid Sequence of IL-27 <400> 10 gaccaaagag gctgggcccc gccatgggcc agacggcagg cgaccttggc tggcggctca 60 gcctgttgct gcttcccttg ctcctggttc aagctggtgt ctggggattc ccaaggcccc 120 cagggaggcc ccagctgagc ctgcaggagc tgcggaggga gttcacagtc agcctgcatc 180 tcgccaggaa gctgctctcc gaggttcggg gccaggccca ccgctttgcg gaatctcacc 240 tgccaggagt gaacctgtac ctcctgcccc tgggagagca gctccctgat gtttccctga 300 ccttccaggc ctggcgccgc ctctctgacc cggagcgtct ctgcttcatc tccaccacgc 360 ttcagccctt ccatgccctg ctgggagggc tggggaccca gggccgctgg accaacatgg 420 agaggatgca gctgtgggcc atgaggctgg acctccgcga tctgcagcgg cacctccgct 480 tccaggtgct ggctgcagga ttcaacctcc cggaggagga ggaggaggaa gaggaggagg 540 aggaggagga gaggaagggg ctgctcccag gggcactggg cagcgcctta cagggcccgg 600 cccaggtgtc ctggccccag ctcctctcca cctaccgcct gctgcactcc ttggagctcg 660 tcttatctcg ggccgtgcgg gagttgctgc tgctgtccaa ggctgggcac tcagtctggc 720 ccttggggtt cccaacattg agcccccagc cctgatcggt ggcttcttag ccccctgccc 780 cccacccttt agaactttag gactggagtc ttggcatcag ggcagccttc gcatcatcag 840 ccttggacaa gggagggctc ttccagcccc ctgccccagg ctctacccag taactgaaag 900 cccctctggt cctcgccagc tatttatttc ttggatattt atttattgtt tagggagatg 960 atggtttatt tattgtcttg gggcccgatg gtcctcctcg ggccaagccc ccatgctggg 1020 tgcccaataa agcactctca tcca 1044 <210> 11 <211> 243 <212> PRT <213> artificial sequence <220> <223> Example amino acid sequence of IL-27 <400> 11 Met Gly Gln Thr Ala Gly Asp Leu Gly Trp Arg Leu Ser Leu Leu Leu 1 5 10 15 Leu Pro Leu Leu Leu Val Gln Ala Gly Val Trp Gly Phe Pro Arg Pro 20 25 30 Pro Gly Arg Pro Gln Leu Ser Leu Gln Glu Leu Arg Arg Glu Phe Thr 35 40 45 Val Ser Leu His Leu Ala Arg Lys Leu Leu Ser Glu Val Arg Gly Gln 50 55 60 Ala His Arg Phe Ala Glu Ser His Leu Pro Gly Val Asn Leu Tyr Leu 65 70 75 80 Leu Pro Leu Gly Glu Gln Leu Pro Asp Val Ser Leu Thr Phe Gln Ala 85 90 95 Trp Arg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe Ile Ser Thr Thr 100 105 110 Leu Gln Pro Phe His Ala Leu Leu Gly Gly Leu Gly Thr Gln Gly Arg 115 120 125 Trp Thr Asn Met Glu Arg Met Gln Leu Trp Ala Met Arg Leu Asp Leu 130 135 140 Arg Asp Leu Gln Arg His Leu Arg Phe Gln Val Leu Ala Ala Gly Phe 145 150 155 160 Asn Leu Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 165 170 175 Arg Lys Gly Leu Leu Pro Gly Ala Leu Gly Ser Ala Leu Gln Gly Pro 180 185 190 Ala Gln Val Ser Trp Pro Gln Leu Leu Ser Thr Tyr Arg Leu Leu His 195 200 205 Ser Leu Glu Leu Val Leu Ser Arg Ala Val Arg Glu Leu Leu Leu Leu 210 215 220 Ser Lys Ala Gly His Ser Val Trp Pro Leu Gly Phe Pro Thr Leu Ser 225 230 235 240 Pro Gln Pro <210> 12 <211> 2347 <212> Ms <213> artificial sequence <220> <223> Example nucleic acid sequence of IL-12B <400> 12 ctgtttcagg gccattggac tctccgtcct gcccagagca agatgtgtca ccagcagttg 60 gtcatctctt ggttttccct ggtttttctg gcatctcccc tcgtggccat atgggaactg 120 aagaaagatg tttatgtcgt agaattggat tggtatccgg atgcccctgg agaaatggtg 180 gtcctcacct gtgacacccc tgaagaagat ggtatcacct ggaccttgga ccagagcagt 240 gaggtcttag gctctggcaa aaccctgacc atccaagtca aagagtttgg agatgctggc 300 footcacct gtcacaaagg aggcgaggtt ctaagccatt cgctcctgct gcttcacaaa 360 aaagaagtg gaatttggtc cactgatatt ttaaaggacc agaagaacc caaaaataag 420 acctttctaa gatgcgaggc caagaattat tctggacgtt tcacctgctg gtggctgacg 480 aaatcagta ctgatttgac attcagtgtc aaaagcagca gaggctcttc tgacccccaa 540 ggggtgacgt gcggagctgc tacactctct gcagagagag tcagaggga caacaaggag 600 tatgagtact cagtggagtg ccaggaggac agtgcctgcc cagctgctga ggagagtctg 660 cccattgagg tcatggtgga tgccgttcac aagctcaagt atgaaacta caccagcagc 720 ttcttcatca gggacatcat caaacctgac ccacccaaga acttgcagct gaagccatta 780 aagaattctc ggcaggtgga ggtcagctgg gagtaccctg acacctggag tactccacat 840 tcctacttct ccctgacatt ctgcgttcag gtccagggca agagcaagag agaaaagaaa 900 gatagagtct tcacggacaa gacctcagcc acggtcatct gccgcaaaaa tgccagcatt 960 agcgtgcggg cccaggaccg ctactataggc tcatcttgga gcgaatgggc atctgtgccc 1020 tgcagttagg ttctgatcca ggatgaaaat ttggaggaaa agtggaagat attaagcaaa 1080 atgtttaaag acacaacgga atagacccaa aaagataatt tctatctgat ttgctttaaa 1140 acgttttttt aggatcacaa tgatatcttt gctgtatttg tatagttaga tgctaaatgc 1200 tcattgaaac aatcagctaa tttatgtata gattttccag ctctcaagtt gccatgggcc 1260 ttcatgctat ttaaatattt aagtaattta tgtatttatt agtatattac tgttatttaa 1320 cgtttgtctg ccaggatgta tggaatgttt catactctta tgacctgatc catcaggatc 1380 agtccctatt atgcaaaatg tgaatttaat tttatttgta ctgacaactt ttcaagcaag 1440 gctgcaagta catcagtttt atgacaatca ggaagaatgc agtgttctga taccagtgcc 1500 atcatacact tgtgatggat gggaacgcaa gagatactta catggaaacc tgacaatgca 1560 aacctgttga gaagatccag gagaacaaga tgctagttcc catgtctgtg aagacttcct 1620 ggagatggtg ttgataaagc aatttagggc cacttacact tctaagcaag tttaatcttt 1680 ggatgcctga atttaaaag ggctagaaaa aaatgattga ccagcctggg aaacataaca 1740 agaccccgtc tctacaaaaa aaatttaaaa ttagccaggc gtggtggctc atgcttgtgg 1800 tcccagctgt tcaggaggat gaggcaggag gatctcttga gcccaggagg tcaaggctat 1860 ggtgagccgt gattgtgcca ctgcatacca gcctaggtga cagaatgaga ccctgtctca 1920 aaaaaaaaaa tgattgaaat taaaattcag ctttagcttc catggcagtc ctcacccccca 1980 cctctctaaa agacacagga ggatgacaca gaaacaccgt aagtgtctgg aaggcaaaaa 2040 gatcttaaga ttcaagagag aggacaagta gttatggcta aggacatgaa attgtcagaa 2100 tggcaggtgg cttcttaaca gccctgtgag aagcagacag atgcaaagaa aatctggaat 2160 cccttctca ttagcatgaa tgaacctgat acacaattat gaccagaaaa tatggctcca 2220 tgaaggtgct acttttaagt aatgtatgtg cgctctgtaa agtgattaca tttgtttcct 2280 gtttgtttat ttatttattt atttttgcat tctgaggctg aactaataaa aactcttctt 2340 tgtaatc 2347 <210> 13 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Exemplary amino acid sequences of IL-12B <400> 13 Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser 325 <210> 14 <211> 823 <212> DNA <213> Artificial Sequence <220> <223> Exemplary Nucleic Acid Sequence of IL-12 p35 <400> 14 agaaagcaag agaccagagt cccgggaaag tcctgccgcg cctcgggaca attataaaaa 60 tgtggccccc tgggtcagcc tcccagccac cgccctcacc tgccgcggcc acaggtctgc 120 atccagcggc tcgccctgtg tccctgcagt gccggctcag catgtgtcca gcgcgcagcc 180 tcctccttgt ggctaccctg gtcctcctgg accacctcag tttggccaga aacctccccg 240 tggccactcc agacccagga atgttcccat gccttcacca ctcccaaaac ctgctgaggg 300 ccgtcagcaa catgctccag aaggccagac aaactctaga attttaccct tgcacttctg 360 aagagattga tcatgaagat atcacaaaag ataaaaccag cacagtggag gcctgtttac 420 cattggaatt aaccaagaat gagagttgcc taaattccag agagacctct ttcataacta 480 atgggagttg cctggcctcc agaaagacct cttttatgat ggccctgtgc cttagtagta 540 tttatgaaga cttgaagatg taccaggtgg agttcaagac catgaatgca aagcttctga 600 tggatcctaa gaggcagatc tttctagatc aaaacatgct ggcagttatt gatgagctga 660 tgcaggccct gaatttcaac agtgagactg tgccacaaaa atcctccctt gaagaaccgg 720 atttttataa aactaaaatc aagctctgca tacttcttca tgctttcaga attcgggcag 780 tgactattga tagagtgatg agctatctga atgcttccta aaa 823 <210> 15 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> Exemplary Amino Acid Sequence of IL-12 p35 <400> 15 Met Trp Pro Pro Gly Ser Ala Ser Gln Pro Pro Pro Ser Pro Ala Ala 1 5 10 15 Ala Thr Gly Leu His Pro Ala Ala Arg Pro Val Ser Leu Gln Cys Arg 20 25 30 Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val 35 40 45 Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro 50 55 60 Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg 65 70 75 80 Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr 85 90 95 Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys 100 105 110 Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu 115 120 125 Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys 130 135 140 Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser 145 150 155 160 Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn 165 170 175 Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn 180 185 190 Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser 195 200 205 Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys 210 215 220 Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala 225 230 235 240 Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 245 250 <210> 16 <211> 1007 <212> DNA <213> Artificial Sequence <220> <223> Exemplary Nucleic Acid Sequence of IL-12 p40 <400> 16 agcaagatgt gtcaccagca gttggtcatc tcttggtttt ccctggtttt tctggcatct 60 cccctcgtgg ccatatggga actgaagaaa gatgtttatg tcgtagaatt ggattggtat 120 ccggatgccc ctggagaaat ggtggtcctc acctgtgaca cccctgaaga agatggtatc 180 acctggacct tggaccagag cagtgaggtc ttaggctctg gcaaaaccct gaccatccaa 240 gtcaaagagt ttggagatgc tggccagtac acctgtcaca aaagggcga ggttctaagc 300 cattcgctcc tgctgcttca caaaaaggaa gatggaattt ggtccactga tattttaaag 360 gaccagaaag aacccaaaaa taagaccttt ctaagatgcg aggccaagaa ttatctgga 420 cgtttcacct gctggtggct gacgacaatc agtactgatt tgacattcag tgtcaaaagc 480 agcagaggct cttctgaccc ccaaggggtg acgtgcggag ctgctacact ctctgcagag 540 600 tgcccagctg ctgaggagag tctgcccatt gaggtcatgg tggatgccgt tcacaagctc 660 aagtgaaa actacaccag cagcttcttc atcagggaca tcatcaaacc tgacccaccc 720 aagaacttgc agctgaagcc attaaagaat tctcggcagg tggaggtcag ctgggagtac 780 cctgacacct ggagtactcc acattcctac ttctccctga cattctgcgt tcaggtccag 840 ggcaagagca agagaaaa gaagataga gtcttcacgg acaagacctc agccacggtc 900 atctgccgca aaaatgccag cattagcgtg cgggcccagg accgctacta tagctcatct 960 tggagcgaat gggcatctgt gccctgcagt taggttctga tccagga 1007 <210> 17 <211> 328 <212> PRT <213> Artificial sequence <220> <223> Exemplary amino acid sequence of IL-12 p40 <400> 17 Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser 325 <210> 18 <211> 1151 <212> DNA <213> Artificial Sequence <220> <223> Exemplary Nucleic Acid Sequence of IL-18 <400> 18 attctctccc cagcttgctg agccctttgc tcccctggcg actgcctgga cagtcagcaa 60 ggaattgtct cccagtgcat tttgccctcc tggctgccaa ctctggctgc taaagcggct 120 gccacctgct gcagtctaca cagcttcggg aagaggaaag gaacctcaga ccttccagat 180 cgcttcctct cgcaacaaac tatttgtcgc aggaataaag atggctgctg aaccagtaga 240 agacaattgc atcaactttg tggcaatgaa atttattgac aatacgcttt actttataga 300 aaacctggaa tcagattact ttggcaagct tgaatctaaa ttatcagtca taagaaattt 360 gaatgaccaa gttctcttca ttgaccaagg aaatcggcct ctatttgaag atatgactga 420 480 ccagcctaga ggtatggctg taactatctc tgtgaagtgt gagaaaattt caactctctc 540 ctgtgagaac aaaattattt cctttaagga aatgaatcct cctgataaca tcaaggatac 600 aaaaagtgac atcatattct ttcagagaag tgtcccagga catgataata agatgcaatt 660 tgaatcttca tcatacgaag gatactttct agcttgtgaa aaagagagag accttttaa 720 actcattttg aaaaaagagg atgaattggg ggatagatct ataatgttca ctgttcaaaa 780 cgaagactag ctattaaaat ttcatgccgg gcgcagtggc tcacgcctgt aatcccagcc 840 ctttgggagg ctgaggcggg cagatcacca gaggtcaggt gttcaagacc agcctgacca 900 acatggtgaa acctcatctc tactaaaaat acaaaaaatt agctgagtgt agtgacgcat 960 gccctcaatc ccagctactc aagaggctga ggcaggagaa tcacttgcac tccggaggta 1020 gaggttgtgg tgagccgaga ttgcaccatt gcgctctagc ctgggcaaca acagcaaaac 1080 tccatctcaa aaaataaaat aaataaataa acaaataaaa aattcataat gtgaaaaaaa 1140 aaaaaaaaaaa 1151 <210> 19 <211> 189 <212> PRT <213> Artificial sequence <220> <223> Exemplary amino acid sequence of IL-18 <400> 19 Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met 1 5 10 15 Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Glu Asn Leu Glu Ser Asp 20 25 30 Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn 35 40 45 Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp 50 55 60 Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile 65 70 75 80 Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile 85 90 95 Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile 100 105 110 Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys 115 120 125 Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys 130 135 140 Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu 145 150 155 160 Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu 165 170 175 Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp 180 185 <210> 20 <211> 1115 <212> DNA <213> Artificial Sequence <220> <223> Exemplary Nucleic Acid Sequence of IL-18 <400> 20 cctttgctcc cctggcgact gcctggacag tcagcaagga attgtctccc agtgcatttt 60 gccctcctgg ctgccaactc tggctgctaa agcggctgcc acctgctgca gtctacacag 120 cttcgggaag aggaaaggaa cctcagacct tccagatcgc ttcctctcgc aacaaactat 180 ttgtcgcagg aaagatg gctgctgaac cagtagaaga caatgcatc aactttgtgg 240 caatgaatt tattgacaat acgctttact tttagctga agatgatga aacctggaat 300 cagattactt tgcaagctt gatctaatt tatcagtcat aagaatttg atgaccaag 360 ttctcttcat tgaccaagga aatcggcctc tatttgaga tatgactgat tctgactgta 420 gagataatgc accccgacc attattatta taagtagta taaagatagc cagcctagag 480 gtatggctgt aactatctct gtgaagtgtg agaaatttc aactctctc tgtgagaaca 540 aaattatttc ctttaaggaa atgaatcctc ctgataacat caaggataca aaagtgaca 600 tcatattctt tcagagaagt gtcccaggac atgataataa gatgcaattt gatcttcat 660 catacgaagg atactttcta gcttgtgaaa aagagaga cctttttaaa ctcattttga 720 aaaagagga tgaattgggg gatagatcta taatgttcac tgttcaaac gagactagc 780 tattaaaatt tcatgccggg cgcagtggct cacgcctgta atcccagccc tttgggaggc 840 tgaggcgggc agatcaccag aggtcaggtg ttcaagacca gcctgaccaa catggtgaaa 900 cctcatctct actaaaaata caaaaaatta gctgagtgta gtgacgcatg ccctcaatcc 960 cagctactca agaggctgag gcaggagaat cacttgcact ccggaggtag aggttgtggt 1020 gagccgagat tgcaccattg cgctctagcc tgggcaacaa cagcaaaact ccatctcaaa 1080 aaataaaata aataaataaa caaataaaaa attca 1115 <210> twenty one <211> 193 <212> PRT <213> Artificial sequence <220> <223> Exemplary amino acid sequence of IL-18 <400> twenty one Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met 1 5 10 15 Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn 20 25 30 Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile 35 40 45 Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro 50 55 60 Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg 65 70 75 80 Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met 85 90 95 Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys 100 105 110 Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile 115 120 125 Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly 130 135 140 His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe 145 150 155 160 Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys 165 170 175 Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu 180 185 190 Asp <210> 22 <211> 2612 <212> DNA <213> Artificial Sequence <220> <223> Exemplary Nucleic Acid Sequence of IL-33 <400> 22 ctgctgcctt ccaccatcag atgatcctca ccagatgcca gcataatatt cttggatttg 60 cctgcctcta gaagtaatcc caacagaagg ccaaagaagt ttgccccatg tactttatga 120 agctccgctc tggccttatg ataaaaaaagg aggcctgtta ctttaggaga gaaaccacca 180 aaaggccttc actgaaaaca ggtagaaagc acaaaagaca tctggtactc gctgcctgtc 240 aacagcagtc tactgtggag tgctttgcct ttggtatatc aggggtccag aaatatacta 300 gagcacttca tgattcaagt atcacaggaa tttcacctat tacagagtat cttgcttctc 360 taagcacata caatgatcaa tccattactt ttgctttgga ggatgaaagt tatgagatat 420 atgttgaaga cttgaaaaaa gatgaaaaga aagataaggt gttactgagt tactatgagt 480 ctcaacaccc ctcaaatgaa tcaggtgacg gtgttgatgg taagatgtta atggtaaccc 540 tgagtcctac aaaagacttc tggttgcatg ccaacaacaa ggaacactct gtggagctcc 600 ataagtgtga aaaaccactg ccagaccagg ccttctttgt ccttcataat atgcactcca 660 actgtgtttc atttgaatgc aagactgatc ctggagtgtt tataggtgta aaggataatc 720 atcttgctct gattaaagta gactcttctg agaatttgtg tactgaaaat atcttgttta 780 agctctctga aacttagttg atggaaacct gtgagtcttg ggttgagtac ccaaatgcta 840 ccactggaga aggaatgaga gataaagaaa gagacaggtg acatctaagg gaaatgaaga 900 gtgcttagca tgtgtggaat gttttccata ttatgtataa aaatattttt tctaatcctc 960 cagttatct tttatttccc tctgtataac tgcatcttca atacaagtat 1020 aatagggtat tggtaaagaa acggtcaaca ttctaaagag atacagtctg acctttactt 1080 ttctctagtt tcagtccaga aagaacttca tatttagagc taaggccact gaggaaagag 1140 ccatagctta agtctctatg tagacaggga tccattttaa agagctactt agagaataa 1200 ttttccacag ttccaaacga taggctcaaa cactagagct gctagtaaaa agaagaccag 1260 atgcttcaca gaattatcat tttttcaact ggaataaaac accaggtttg tttgtagatg 1320 tcttaggcaa cactcagagc agatctccct tactgtcagg ggatatggaa cttcaaaggc 1380 ccacatggca agccaggtaa cataaatgtg tgaaaaagta aagataacta aaaaatttag 1440 aaaaataaat ccagtatttg taaagtgaat aacttcattt ctaattgttt aatttttaaa 1500 attctgattt ttatatattg agtttaagca aggcattctt acacgaggaa gtgaagtaaa 1560 ttttagttca gacataaaat ttcacttatt aggaatatgt aacatgctaa aacttttttt 1620 ttttaaaga gtactgagtc acaacatgtt ttagagcatc caagtaccat ataatccaac 1680 tatcatggta aggccagaaa tcttctaacc taccagagcc tagatgagac accgaattaa 1740 cattaaaatt tcagtaactg actgtccctc atgtccatgg cctaccatcc cttctgaccc 1800 tggcttccag ggacctatgt cttttaatac tcactgtcac attgggcaaa gttgcttcta 1860 atccttattt cccatgtgca caagtctttt tgtattccag cttcctgata acactgctta 1920 ctgtggaata ttcatttgac atctgtctct ttcatttct tttaactacc atgcccttga 1980 tatatcttt gcacctgctg aacttcattt ctgtatcacc tgacctctgg atgccaaaac 2040 gtttattctg ctttgtctgt tgtagaattt tagataaagc tattaatggc aatatttttt 2100 tgctaaacgt ttttgttttt tactgtcact agggcaataa aatttatact caaccatata 2160 ataacattt ttaactacta aaggagtagt ttttatttta aagtcttagc aatttctatt 2220 acaacttttc ttagacttaa cacttatgat aaatgactaa catagtaaca gaatctttat 2280 gaaatatgac cttttctgaa aatacatact tttacatttc tactttattg agacctatta 2340 gatgtaagtg ctagtagaat ataagataaa agaggctgag aattaccata caagggtatt 2400 acaactgtaa aacaatttat ctttgtttca ttgttctgtc aataattgtt accaaagaga 2460 taaaaataaa agcagaatgt atatcatccc atctgaaaaa cactaattat tgacatgtgc 2520 atctgtacaa taaacttaaa atgattatta aataatcaaa tatatctact acattgttta 2580 tattattgaa taaagtatat tttccaaatg ta 2612 <210> 23 <211> 229 <212> PRT <213> Artificial sequence <220> <223> Exemplary amino acid sequence of IL-33 <400> 23 Met Tyr Phe Met Lys Leu Arg Ser Gly Leu Met Ile Lys Lys Glu Ala 1 5 10 15 Cys Tyr Phe Arg Arg Glu Thr Thr Lys Arg Pro Ser Leu Lys Thr Gly 20 25 30 Arg Lys His Lys Arg His Leu Val Leu Ala Ala Cys Gln Gln Gln Ser 35 40 45 Thr Val Glu Cys Phe Ala Phe Gly Ile Ser Gly Val Gln Lys Tyr Thr 50 55 60 Arg Ala Leu His Asp Ser Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu 65 70 75 80 Tyr Leu Ala Ser Leu Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala 85 90 95 Leu Glu Asp Glu Ser Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp 100 105 110 Glu Lys Lys Asp Lys Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro 115 120 125 Ser Asn Glu Ser Gly Asp Gly Val Asp Gly Lys Met Leu Met Val Thr 130 135 140 Leu Ser Pro Thr Lys Asp Phe Trp Leu His Ala Asn Asn Lys Glu His 145 150 155 160 Ser Val Glu Leu His Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe 165 170 175 Phe Val Leu His Asn Met His Ser Asn Cys Val Ser Phe Glu Cys Lys 180 185 190 Thr Asp Pro Gly Val Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu 195 200 205 Ile Lys Val Asp Ser Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe 210 215 220 Lys Leu Ser Glu Thr 225 <210> 24 <211> 2016 <212> DNA <213> Artificial sequence <220> <223> Exemplary nucleic acid sequence of IL-7 <400> 24 acacttgtgg cttccgtgca cacattaaca actcatggtt ctagctccca gtcgccaagc 60 gttgccaagg cgttgagaga tcatctggga agtcttttac ccagaattgc tttgattcag 120 gccagctggt ttttcctgcg gtgattcgga aattcgcgaa ttcctctggt cctcatccag 180 gtgcgcggga agcaggtgcc caggagagag gggataatga agattccatg ctgatgatcc 240 caaagattga acctgcagac caagcgcaaa gtagaaactg aaagtacact gctggcggat 300 cctacggaag ttatggaaaa ggcaaagcgc agagccacgc cgtagtgtgt gccgcccccc 360 ttgggatgga tgaaactgca gtcgcggcgt gggtaagagg aaccagctgc agagatcacc 420 ctgcccaaca cagactcggc aactccgcgg aagaccaggg tcctgggagt gactatgggc 480 ggtgagagct tgctcctgct ccagttgcgg tcatcatgac tacgcccgcc tcccgcagac 540 catgttccat gtttctttta ggtatatctt tggacttcct cccctgatcc ttgttctgtt 600 gccagtagca tcatctgatt gtgatattga aggtaaagat ggcaaacaat atgagagtgt 660 tctaatggtc agcatcgatc aattattgga cagcatgaaa gaaattggta gcaattgcct 720 gaataatgaa tttaactttt ttaaaagaca tatctgtgat gctaataagg aaggtatgtt 780 tttattccgt gctgctcgca agttgaggca atttcttaaa atgaatagca ctggtgattt 840 tgatctccac ttattaaaag tttcagaagg cacaacaata ctgttgaact gcactggcca 900 ggttaaagga agaaaaccag ctgccctggg tgaagcccaa ccaacaaaga gtttggaaga 960 aaataaatct ttaaaggaac agaaaaaact gaatgacttg tgtttcctaa agagactatt 1020 acaagagata aaaacttgtt ggaataaaat tttgatgggc actaaagaac actgaaaaat 1080 atggagtggc aatatagaaa cacgaacttt agctgcatcc tccaagaatc tatctgctta 1140 tgcagttttt cagagtggaa tgcttcctag aagttactga atgcaccatg gtcaaaacgg 1200 attaggcat ttgagaatg catattgtat tactgaga tgaatacaa caatggaaac 1260 tgaatgctcc agtcacaa ctatttctta tatatgtgaa catttatca tcagtataat 1320 tctgtactga tttgtaag acaatccatg taggtatca gttgcaataa tactctcaa 1380 acctgtttaa atatttcaag acattaatc tatgagtat atatggtttt caagattca 1440 aaattgacat tgctttactg tcaaataat ttatggctc actatgaatc tattatactg 1500 tattaagagt gaaattgtc ttctctgtg ctggagatgt tttagagtta acatgaat 1560 atggataatg ccggtgagaa tagagagtc aaacctta agtaagcac agcatacaa 1620 ggtccaagat acctaaaga gatttcaaga gatttaatta atcatgaatg tgtacacag 1680 tgccttcaat aaatgtata gcaatgttt tgacatgaa aaggacaat ttcaaaaaaa 1740 taaaaaaa taaaaaaaaaaaaaaaaacttag tactgatta 1800 cattgtcatt tatatagatt ataacttgtc taaatagtt tgcaatttgg gagatatatt 1860 Tttaagaata Taatatgt Tacctttta Attaatgaa Tatctgtatt Taattttgac 1920 actatatctg tatataaaat attttcatac agcattacaa attgcttact ttggaataca 1980 tttctccttt gataaaataa atgagctatg tattaa 2016 <210> 25 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> Exemplary Amino Acid Sequence of IL-7 <400> 25 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys 20 25 30 Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu 35 40 45 Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe 50 55 60 Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe 65 70 75 80 Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser 85 90 95 Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr 100 105 110 Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala 115 120 125 Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu 130 135 140 Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu 145 150 155 160 Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu 165 170 175 His <210> 26 <211> 12846 <212> DNA <213> Artificial sequence <220> <223> Exemplary nucleic acid sequence of MICA <400> 26 tggtctgagc ggttctactg cggcaggtgc ttctgagagg cagaggtggc tgagatctgg 60 aaacaggtct gcaaatctgg tcactggtct cattgccagt aacgctgtgc gcggttgagg 120 gagtgtgttg ggagaatagc cacgcgttgt ctgtcctgga aggaacaagc cagtgagagc 180 cggtttaatg gggcggccgg cgaaaggggc ttggtgaggc ccgcgctcct cggggtgggg 240 gcgcggggat gggtggtcgc gatgccggga gggcaggcag ggccctgccc gtgcttatga 300 agttggagct gtactctcag ctactcgaag ctggtccctg ctttaggctg cgctcccgcg 360 tgctccccat tttctgggcc ccaggtcccg ccttctaaat ctccccaggt ctccagccca 420 ctggaatttt ctcttccaag cgtggccccg ccctctccgc tcgtgattgg ccctaagttc 480 cgggccccag tttcattgga tgagcggtcg ggggaccggg ccaggtgact aagtttccgc 540 ggcgccttct ccccggccac tgcttgagcc gctgagaggg tggcgacgtc ggggccatgg 600 ggctgggccc ggtcttcctg cttctggctg gcatcttccc ttttgcacct ccgggagctg 660 ctgctggtga gtggcgttcc tggcggtcct cggcggagcg ggagcagtgg gacgtttccg 720 ggggtcgggt gggtagcggc gagcgctgtg cggtcagggc gggcctcctg tgcgctgtcg 780 gtggcgcagg gagctggacg cggcccgtta ccgccacact tcagccctgc ttccccgtca 840 cttttcagtc ctcctcggga tcgcgcatca cctgcacttt ctggtctcct cctgctcttt 900 ctctcctcgc gtctcctccg cttcctctca cttttcggac aaaccagtcc ttctgaggcc 960 catgggttcc cgggctgcct ccggggctgc tcctgtgaat ggcattcgag tgcccttcca 1020 gcgcggccac tgaagcagcc acaacccccg gtgctcgggg cggctctcag gtccctgaag 1080 tcctgtcctc tcccggagcc gacgtgttct cagctcctgg gccgcagctc ctggagtagg 1140 ggccctcctt tctcgggacc cggagctggt gcttcctgct gctgtgggga ctgtgggggg 1200 tcctgactct caagctgagg ggttggagtc tgcaggctcc gggcagagga ttcttcctgc 1260 gacttctctc atccccagct cattctcccc tcgcctctgg ctccgagggt cctctcctct 1320 ctctcatccc acccctacta atgaccagtg atctaaggac accagattcc ctctcacctc 1380 ctccctgccc atctcagggc ccgctgagtc cttttgccct cccagctccc tgctacccct 1440 tcctgtgtgc tgttctctga tccatttcta gggtgtcctc tgccctcatc ccctgtcccc 1500 gccaccgaag gtccctcctg caccccttat gggcctttcc tacaagcagc cttcacccag 1560 tgctgcccct atgcctcccc gttcccaaat gtccctgact ctaactttct ggtgctgcct 1620 tttatccggg ggggtcttcc ctccatccca ctcccctcca cacccccaag gggaaccctg 1680 atgctaatgg cagttgggcc ttaggcaggg cgcagggcag cgcagatgcc ccctcccctc 1740 cagtgcagat gcctgctctg gaccctgcct cattgtggcc ccttccccac tccttcatcc 1800 tcagcctcac cctcttgagg accccaccct ccagcccaca ggtgctggac catccctccc 1860 tggtccctcc gcccctctcc accttgggac cttgtgctgc tcctatctct tgcccagctg 1920 ccttgggccc tcagcacgtt ctcatctttc agtgggaaag tgggagtgct ggagcatatg 1980 acagtgctga gcatctttcc caagccccac cctcccccag agcaccctcc cctcctgtcc 2040 tcaccctacc ccaagttctc ccacagtcac tcctgcccca tgctcatgcc gccctccagt 2100 tcttgctctg cccatctccc ctccccaacc cagacctaaa acaggctgtt gggccagctg 2160 ttccttgacc ttccttcttt tcttttggtt ccttgacccc agtgggctct cactccccac 2220 accgcatatc taaaatctgt tttgcctgct cttggggtgc cactgctccc cctccagcat 2280 tactcctttt ggcaggtcct tcctcaggct gagaatctcc ccctctacct tggttttctc 2340 tctctggcca gcacccccac cccttgcttt gtttttaatt tttaactttt gtttgggtac 2400 gtagtagata tatatgtata tatttatggg gtacatggga tattttcaca caggcctaca 2460 atatgtaata atcacatcag ggtaaatggg ttatatcaca acaagcattt atcctttctt 2520 tgtgctacaa acaatcccat tatgctcttt cagttatttt taaatgtaca ataaattatt 2580 gttgactgta ctcaccctgc tgtgctatct actagatctt attcattcta attatatttt 2640 tgtacccatt attaaccatc cctgctcccc cactccccac tacccttctc agcctctggt 2700 aatcatcatt ctattgtctc tccccatgag gtccattgtt ttaaattttg gctgccacaa 2760 ataagtgaga acatgcaaag tttgtctgtc tgggcctggg gcttatttca cttcacagga 2820 tgacctccag ttctttgcaa atgacacgat ggctgaatag ttctccacat acacatgtac 2880 accacatttt ctttatccat gcgtctgttg atggacactt agattgcttg cagatcttgg 2940 ctactttgaa tagtgctgca ataaacatgg aaaagtagat agctctttaa tataccgatt 3000 tcctttcttt ggagtatatg cctaacagtg ggagtgctgg agcatatgac agctctattg 3060 tatttttagt ttttggaaga acctccacat tgtttcccat agtggttgta ctagtttacg 3120 ttcccaccaa cagtgtacat cctcaccagc attccttatt tctacatcct cgccagcatt 3180 ccttattgcc tgtcttctgg ataaaagcca gtttatctgg ggtgggatgt tatctcgtag 3240 gagttttgat ttgccttcat ctgttgacga atgatgttga gcaccttttc atatacctgt 3300 ttgccattta tatgtcttct tttgagaaat gactattcag atcttttctc atttttaaat 3360 tggattatta tattttttt tcctatagtt gttcgagctc cttatatgtt tcagttactg 3420 atcctttgtc agatgaatag tttgaaaata ttttctccca ttcttggatg gtctcttcat 3480 tttgtttatt gtttcctttg ctgtgcagaa gccttttac ttgatatgat cccatttatg 3540 caattttact ttggttacct gtgcttgtgg ggtattactt taaaaatctt tgcccagtcc 3600 aatatcctag agagtttccc caatgttttc ttgtatagtt tcatagtttg aggtcataga 3660 tttacatctt taatccactt tgatttgatt tttgtatatg gtgaaagaca gggtctagtt 3720 tcattcttct gcataaggat atctagtttc cccagcacca tttttgaaga gactctcctt 3780 tgccaatgtg tgttcttggt acctttgttg gaaatgagtt tactgtagat gtatggaatt 3840 gtttctgggt tctctattct gtttcattgg tctgtgtgtc tgtttttatg ccagtatcat 3900 gctgttttgg ttactgtagc tctgtagtat aatttgaagt cagataatgt gattcctcta 3960 gttttgttca ttttgctcag gatagcttta tctattctgg tttttttgtg gttccatatg 4020 cattttaggga ttatttttat tatttctgtg aagaatgtca ttagtgtttt gatagggatt 4080 gcattgaatc tgtagattac tttgggtagt atggaatttt caacaaaact gattcttcca 4140 atccatgaac gtggactatc ttttccattt tttgtgtcct tcaatttttt gcatcagtgt 4200 tttttgtttt tggtttttga gatggagttt cactcttgtt gcccaggcta gaatgcaagg 4260 gtgtgatctt ggctcaccgc aacctccgcc tcccaggttc aagcgattct tctgcctcag 4320 cctcccaagt agctgggatt acaggcatgt gccactgtgc ctggctaatt ttctattttt 4380 attagagatg gggtttctct atgttggcca ggctagtctt gaactcctga cctcaggtga 4440 tccacctgcc tcggcctccc aaagtgctgg gattacaggc atgagccacc acgcccagcc 4500 acatcactgt tttatagttt ttatggaga ggtctttcac ttcttcagtt aggtttattc 4560 ctcagtattt tatttattt gtagctattg taaatgggat tcgtttcttg atttcttttt 4620 cagattatt gctgttagca ctgattttg catgttgatt ttgtatcctg caactttact gaatttgttc ttcagttcta atggtttttt ggtggagtct ttaggttttt ccaaatatca gaccacatga tctgcaaaca aggataattt gacttcttct tttccagttt taatgccctt tctttctttc tcctgtctga ttgctctagt taggatctgc agtactgtgt tgcataactg 4860 tggtaaaatt agtcatcctt gtcttattcc agatcttag gaaaaggctt tcagttttcc cccattcagt atgttactag ctgtgagttt gtcatatatg gcttttatta tattgaggtc 4980. tgttccttgt atacttagtt ttttgagagt ttttatcatg aagggatgtt gaatttatca aatgcttttt cagtatcaat tgaatgatac tggcttttgt cctttattct gttgatatga cgtattacat tgattgattt gtgtatgtta aatcatcctt gcatacctgg aatacattcc acttgctcat aaagaatgat cttttttaat gtattgttga atgtggtttg ctagtatttc cttgacgatt tttgcatcgg tgttcatcag ggatataggc ctgtagtttt cttttttatg atgtgtcttt gcctggtttt tgtatcagga tattcctggc tttgtaaaat gagtttgga gtattccctc ctcctctatt tttcagaaca gtttgaatag gactgacata tgttgttctt 5400 taaaagttta attgtggtaa attatacatt acataaattt tactgtttta accactttta 5460 agtgtatact cggtggcatt agatacattc acatttttgt gcaacccaaa actctgtgcc 5520 cattaatcgg taactcccca ttcctcccta cctctggccc ctggtaacca ccattctact 5580 ttttgtttct atgaatttga ccactctagg tacctcattt aagcagaatc atgtaatgtt 5640 tgtctttttg tttctggctt atttcactta tatattttt gaggttcggt gggcacagtg 5700 gctcacgcct ggatttccag cactttggga ggctgaagca ggtggatcac ctgagtttcg 5760 gagtcgaaa ccagcctggc caacatggtg aaaccccatc tctactaaaa ataataaaag 5820 ttagccgggc gtgatggcgg gtgcctgtaa tcccaactac ttgggaggct gaggcaggag 5880 aatcgcttga atccgggaag tggaggttgc agtgagctga gatcaggcca ctgcactcca 5940 gcctgggcaa caagagtgaa attccatctc caaaaaaaa aataaaaaa tataataat 6000 aatatttttg aggttcatcc aagttgtagt atgggtcaga atttcattcc ttttaaggat 6060 ggataatact cattatatgt atgtaccaca tcttggttat ccatccctca gacaacggac 6120 acttgggtta cttctacctt ttggatattg gcaaatattt catttccttt gggtatatat 6180 ttatttcctt tgggtatttc ttttgggtat atatccagaa atagaagcag tacatagggg 6240 cttcattttc tctgtctctt tgccaacctt gctctgtgtg tgtgtgtgta tgtgtgtgtg 6300 taggtgtgtg ataacagcca tcctgattgg tttcaggtgg catctcattg tggtttggat 6360 ttgcattttc ctaatgagtg ctgatattga gcatcttttc atgtgtttgt tgatcatttg 6420 taattttctt tgaagaattg gccatttaag tcttttgccc attttttccc ccacatagct 6480 tctcttatca gatatatgac ttgcaatatt tatttcattt cggggttgat tgctttttca 6540 ctctgattgt gccctttgat gcatagatgt tttgaatttt catcagtcta ctttgtcagt 6600 tctttctatt ctatctgtgc tttggtgtca tatccatgaa agcactgtca aatcctatgt 6660 catgaacatt atccccaatg tttgcttcta agaaattttt aggttttagt tcttgagtgt 6720 agagtttagg tctttgattc attttgagtt aatttttgta tatagtgcaa attaagggtc 6780 caattttatt ttaacacccc ctgcccccag aactatttgc tgaaaagatc aactgactct 6840 ttgtcacctg ctcaccccag tggacactag ctgttccatc caattgctgt cctggggcct 6900 tgtcatgcca ctcttccact ttgaacccaa gcccacaccg ttgctcccct ctgggatact 6960 gaccccacta taaacttcac tggggctaca accttcctac cctttgtgcc tcatgaccac 7020 cccctccctt gtccccacca tgcccatgat gagtctcttc tcgaggcagc tccccttgcc 7080 tccatctcac cctcacctat gcaccacagc cacactggac atgggtccct ctgagcctga 7140 gtcccttccc attcccacca tcccctctgg caagaccttc cttccaccac cttcatgctc 7200 ctcccttgcc cctgcagggc agcctctccc cttggcccct attcccttag ggggcttgtg 7260 gccacccagt ccttgcacct ggcctacaag tttgccatct tcattccccc ttcttctgtt 7320 catcagcccc ctcctctatc ctcccaccct cacagttttc tttgtatatg aaatcctcgt 7380 tcttgtccct ttgcccgtgt gcatttcctg cctcctcagg aaggttggga cagcagacct 7440 gtgtgttaaa catcaatgtg aagttacttc caggaagaag tttcacctgt gatttcctct 7500 tccccagagc cccacagtct tcgttataac ctcacggtgc tgtccgggga tggatctgtg 7560 cagtcagggt ttctcgctga ggtacatctg gatggtcagc ccttcctgcg ctgtgacagg 7620 cagaaatgca gggcaaagcc ccagggacag tgggcagaag atgtcctggg aaataagaca 7680 tgggacagag agaccaggga cttgacaggg aacggaaagg acctcaggat gaccctggct 7740 catatcaagg accagaaaga aggtgagagt cggcaggggc aagagtgact ggagaggcct 7800 tttccagaaa agttaggggc agagagcagg gacctgtatc tacccactgg atctggctca 7860 ggctgggggt gaggaatggg ggtcagtgga actcagcagg gaggtgagcc ggcactcagc 7920 ccacacaggg aggcatggag gagggccagg gaggcgtacc ccctgggctg agttcctcac 7980 ttgggtggaa aggtgatggg ttcgggaatg gagaagtcac tgctgggtgg gggcaggctt 8040 gcattccctc caggagatta gggtctgtga gatccatgaa gacaacagca ccaggagctc 8100 ccagcatttc tactacgatg gggagctctt cctctcccaa aacctggaga ctgaggaatg 8160 gacaatgccc cagtcctcca gagctcagac cttggccatg aacgtcagga atttcttgaa 8220 ggaagatgcc atgaagacca agacacacta tcacgctatg catgcagact gcctgcagga 8280 actacggcga tatctaaaat ccggcgtagt cctgaggaga acaggtaccg acgctggcca 8340 ggggctctcc tctccttcca attctgctag agttgcctca cctcccagat gtgtccaggg 8400 aaaccctccc tgtgctatgg atgaaggcat ttcctgttgg cacatcgtgt cctgattttc 8460 ctctattgtt agagccactg gataaagaca gtgggtcagg gactggacca tccagtgttg 8520 taatcagggc aagtagagga ccctccgaca gaatcctgag cctggggtgg gtgtcaggca 8580 ggagaggaag gcttcagggc cagggctgcc ccctctgcct cccagcctgc ccatcctgga 8640 gagttccctc ctggccccac aacccaggag tccacccctg acatccccct cctcagcatc 8700 aatgtgggga tcccagagcc tgaggccaca gtcccatggc ccatcctcct gccagcctgg 8760 aagaactggg ccccagagtg aggacagact tgcaggtcag gggtcccaga gggcttcagc 8820 cagagtgaga acagtgaaga gaaacagccc tgttcctctc ccctccttag aggggagcag 8880 ggcttcactg gctctgccct ttcttctcca gtgcccccca tggtgaatgt cacccgcagc 8940 gaggcctcag agggcaacat taccgtgaca tgcagggctt ctggcttcta tccctggaat 9000 atcacactga gctggcgtca ggatggggta tctttgagcc acgacaccca gcagtggggg 9060 gatgtcctgc ctgatgggaa tggaacctac cagacctggg tggccaccag gatttgccaa 9120 ggagaggagc agaggttcac ctgctacatg gaacacagcg ggaatcacag cactcaccct 9180 gtgccctctg gtgagcctag ggtgaccctg gagagggtca ggccagggta gggacagcag 9240 ggatggctgt ggctctctgc ccagtgtata acaagtccct tttttttcag ggaaagtgct 9300 ggtgcttcag agtcattggc agacattcca tgtttctgct gttgctgctg ctgctgctgc 9360 tgctgctgct atttttgtta ttattatttt ctacgtctgt tgttgtaaga agaaaacatc 9420 agctgcagag ggtccaggtg agaaaagcgg gcagtttctg gagatggtaa ggcccctgtc 9480 tgggcagtag ggtcccctca ttgctcctgc aaagataggc atgttggtga caaggcttct 9540 gtaacagggg atgaaagttg gggaatttgg gaagggaatg ggggcaacat ctccatctac 9600 acccataagt gctgcccaag cgagggtcaa acgcccagct gtggcatctt cctgctgcag 9660 gtgaggagtg ggcagcaggg agggctgcgg cgcctgctct gtccccatcc cggtctctgt 9720 gtctcttgga ctcactaggg cgcatccagg tggggtgagc tgggaatcac gtgctgaatg 9780 ctgaggacct ggatgatcac ggcctcagag ggagcaaata gtaaaggcag ctgtgatctg 9840 gggagggcca gaaactggag aggaatctga ggagaggcgg tgcccctatt cccttcctct 9900 ctgcatccct ctcccctatt tctccagcca ttggggcgga caccaagaaa aagacctatg 9960 aggcccagcc tgggggccct gcctgtgtag ccctttggag accccttgta acagggaggg 10020 tcctgagcac acatggccat ctctgtccac ttttcagctc cccatgcacc tcctccagga 10080 gctttcttgg ggttgtcgtg tcctctgcac cattcgaggc cctactcttt ccaggttccc 10140 acagcctggt ctccctgagt ttcttgcaga tgagatggat gagtagataa gcagatgtcc 10200 ctgggccatt tgaggagtgg ggcccagccc ctcatcaggg cagctgtggt ccctgttttc 10260 atcctacctc cgagtgtttt cttctccagt ccctgaggga cacagtcctc agggcccatg 10320 tttttggggc tttaatctgt gctctgtggc ctcaccttgc cctccctgag ccaatttccc 10380 tttctaaagg tggtcactgc ctggtaagtt tggagtaagg gacggtcaga atcatttccc 10440 ctacagtcag gttgtttgat gggggatgaa aagacagc aggaagttt gtgtttctgc 10500 aaaagagaa gcagttcagg gcagagaag aggctgggggt gtccaggagg gtgtgtctgg 10560 footgggtc gctggtttct catccttgaa cctaattgca ctgtcagtcg gcccctcagg 10620 cctgagcaga tgggaaggtt tgtcccctgc cctgcagtaa gagggccctg tccaggaggc 10680 accataaca ggggcagtgc aggtctgtgg tcgctcctgc tctcacctgt ggcgtctccc 10740 gtagagggat tatcagttct ggttccctgt gggcaggaat ggtttcctca taggtcactg 10800 gagttttggc caggaaaga gtatgaagtt catgtgccag tttctcaaaa ttcctgcttt 10860 caatgttgat gtcaagcaaa gatattcgta atttcagctc tataatctta ataggatttc 10920 ccctaatatt gtaaagcatc ttatatgaaa caggaacaca aatttctcaa aattcctgcg 10980 atgtccaata aagattttca taatttcggc tctccaatct taataggatt tcctaatact 11040 gtaaagcata ttaaatgaaa caggaaccca aatttggagc cccctctcca ggaggttctg 11100 tgtggagatg gtggctgtgg cagtggcagt tcccaggtgc agagggtggg cagaggcagc 11160 ctcaggctaa ggggtctccc ctactccaca tggagaaaat cccttgtagg ttgcaagggc 11220 agtggccggg tggaatccct gctagggaca gagcaggaag gcctcgcagc ctcaccaagc 11280 agcagccctg gggtggagct gcgtttccag ggttaagcgg accaggcagg agcagtggtt 11340 actcaacagc aggtcacagg cttgggttgt gagggtcagg agaggccagg cctcctcgag 11400 caaggtgggg gtcccagggt caggtcaggt gcagatcctg tggcagccac gtctttccat 11460 gctgggcctg ctgggccccc caggctttct gatggggtcc ccagttagga gctgcctgct 11520 cagggctggg aggggaggag cgctgagctg cagatagagg gcagagccca cagtgggcag 11580 ggcctgccct ggtgtgtagg tgcctctgca ggagaggagg gcctggggac tgagagcaag 11640 ggtcagggcc tctctttgag gaggcctctc actgtaacag gactggtcag gcctgagagg 11700 aggtcactgg gttccctctt gggtcttgtc ctttagtctt ggggcccttt ccctccctgc 11760 acgatgagtg gtgggcacag ggcaggggct gatgttgatg gagtgatggg agggaactgg 11820 taggggctgg gaaaagcaag gagggaggaa gaaaaaagtg ggggcctcat cttctctcag 11880 agaaagggcg aatctggttt tggagcaact gaagagagaa aagtccccag ggaataaaca 11940 caacactgca cccagtggag catttacccg tttccctctt ttctccagag ctcgtgagcc 12000 tgcaggtcct ggatcaacac ccagttggga cgagtgacca cagggatgcc acacagctcg 12060 gatttcagcc tctgatgtca gatcttgggt ccactggctc cactgagggc gcctagactc 12120 tacagccagg cagctgggat tcaattccct gcctggatct cacgagcact ttccctcttg 12180 gtgcctcagt ttcatgacct atgaaacaga gaaaataaaa gcacttattt attgttgttg 12240 gaggctgcaa aatgttagta gatatgaggc gtttgcagct gtaccatatt aattggtgtc 12300 attgtttctg ttgttttcgt attattattt ttttttttta agacagagtc tcaggccagg 12360 cacggtggct cacgcctgta atcccagcac tttgggaggc cgaggcgggc ggatcacaag 12420 gtcaagagat cgagaccacc ctgatcaaca tggtgaaacc ctgtctctac taaaaataca 12480 aaaaattagc tgggcctggt ggcatgtgcc tgtagtccca gctactcagg agctgaggca 12540 ggaaaatcac ttgaacctgg gaggtggaga ttgcagtgag ctgagatcgc gccactgcac 12600 tccagcctgg cgacagagcg agactctgtc tcaaaaaaaa aaaaaaaaaa aaaaaaagac 12660 agagtctcac tctgtcaccc aggctgcagt tcagtgacat gatctcagct cgttgcagcc 12720 tccgcctccc gggttcaagc acttcttgtg cctcagcctc ccgagtagct ggggttacag 12780 acatgcacca ccatacccgg ctaatttttg catttttcat agagacagga ttttgccatg 12840 ttggcc 12846 <210> 27 <211> 388 <212> PRT <213> Artificial Sequence <220> <223> Exemplary Amino Acid Sequence of MICA <400> 27 Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe 1 5 10 15 Ala Pro Pro Gly Ala Ala Ala Glu Pro His Ser Leu Arg Tyr Asn Leu 20 25 30 Thr Val Leu Ser Gly Asp Gly Ser Val Gln Ser Gly Phe Leu Ala Glu 35 40 45 Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys 50 55 60 Arg Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys 65 70 75 80 Thr Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu 85 90 95 Arg Met Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser 100 105 110 Leu Gln Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg 115 120 125 Ser Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn 130 135 140 Leu Glu Thr Glu Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr 145 150 155 160 Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Lys Thr 165 170 175 Lys Thr His Tyr His Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg 180 185 190 Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg Thr Val Pro Pro Met 195 200 205 Val Asn Val Thr Arg Ser Glu Ala Ser Glu Gly Asn Ile Thr Val Thr 210 215 220 Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg 225 230 235 240 Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val 245 250 255 Leu Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile 260 265 270 Cys Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly 275 280 285 Asn His Ser Thr His Pro Val Pro Ser Gly Lys Val Leu Val Leu Gln 290 295 300 Ser His Trp Gln Thr Phe His Val Ser Ala Val Ala Ala Ala Ala Ala 305 310 315 320 Ala Ala Ala Ala Ile Phe Val Ile Ile Ile Phe Tyr Val Cys Cys Cys 325 330 335 Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro Glu Leu Val Ser Leu Gln 340 345 350 Val Leu Asp Gln His Pro Val Gly Thr Ser Asp His Arg Asp Ala Thr 355 360 365 Gln Leu Gly Phe Gln Pro Leu Met Ser Asp Leu Gly Ser Thr Gly Ser 370 375 380 Thr Glu Gly Ala 385

Claims

1. A modified 721.221 cell expressing membrane-bound IL-21 (mIL-21), wherein the mIL-21 consists of the amino acid sequence of SEQ ID NO: 2 and / or is encoded by the nucleic acid sequence of SEQ ID NO:

1.

2. The modified 721.221 cells of claim 1, wherein the mIL-21 is present in a construct that further comprises an extracellular fragment from an IgG sequence, a transmembrane fragment from a CD28 sequence, an intracellular fragment from a CD28 sequence, an intracellular fragment from a 4-1BB sequence, and an intracellular fragment from a CD3ζ sequence.

3. The modified 721.221 cells of claim 1 or claim 2, wherein the mIL-21 is expressed in the 721.221 cells using a viral vector.

4. The modified 721.221 cell of claim 3, wherein the viral vector is a retroviral vector.

5. The modified 721.221 cell of claim 4, wherein the retroviral vector is a Moloney murine leukemia virus (MoMLV) vector.

6. The modified 721.221 cell of claim 5, wherein the MoMLV vector is a SFG retroviral vector.

7. The modified 721.221 cell of claim 1 or 2, wherein the mIL-21 is present in a construct encoded by SEQ ID NO:

3.

8. The modified 721.221 cells of claim 1 or 2, wherein the cells further express at least one additional heterologous interleukin and / or interleukin receptor.

9. The modified 721.221 cells of claim 1, further expressing at least one of IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, membrane-bound IL-15 (mIL-15), toll-like receptor (TRL) ligand, UL16 membrane-bound protein (ULBP)-1, ULPB-2 and / or major histocompatibility complex (MHC) class I chain-associated protein A (MIC-A).

10. The modified 721.221 cells of claim 9, wherein the cells comprise a heterologous nucleic acid encoding at least one of IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, mIL-15, TLR ligands, ULBP-1, ULPB-2, and / or MIC-A.

11. The modified 721.221 cell of claim 9 or claim 10, wherein the cell further expresses at least one additional heterologous cytokine, activating receptor ligand, TRL ligand, or a receptor thereof.

12. The modified 721.221 cell of claim 11, wherein the at least one additional heterologous cytokine, activating receptor ligand, TRL ligand, or receptor thereof comprises IL-15 receptor alpha (IL-15Rα).

13. The modified 721.221 cells of claim 12, wherein the cells comprise a heterologous nucleic acid encoding IL-15Rα.

14. A method for producing a modified 721.221 cell, comprising: transducing or transfecting a population of 721.221 cells with a nucleic acid encoding mIL-21; Isolation of cells expressing mIL-21; as well as Irradiate the isolated cells. Thereby generating modified 721.221 cells; The mIL-21 consists of the amino acid sequence of SEQ ID NO: 2 and / or is encoded by the nucleic acid sequence of SEQ ID NO:

1.

15. The method of claim 14, wherein the population of 721.221 cells is transduced with a virus comprising a nucleic acid encoding the mIL-21.

16. A method according to claim 14 or claim 15, wherein: The mIL-21 is present in a construct that also includes an extracellular fragment from an IgG sequence, a transmembrane fragment from a CD28 sequence, an intracellular fragment from a CD28 sequence, an intracellular fragment from a 4-1BB sequence, and an intracellular fragment from a CD3ζ sequence.

17. The method of claim 15, wherein the 721.221 cell population is transduced with a retroviral vector.

18. The method of claim 17, wherein the retroviral vector is a Moloney murine leukemia virus (MoMLV) vector.

19. The method of claim 18, wherein the MoMLV vector is a SFG retroviral vector.

20. The method of claim 14 or 15, wherein the mIL-21 is present in a construct encoded by SEQ ID NO:

3.

21. A method of producing the modified 721.221 cell according to claim 9, comprising: transducing or transfecting a population of 721.221 cells with a nucleic acid encoding at least one of IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, membrane-bound IL-15 (mIL-15), a TLR ligand, ULBP-1, ULPB-2, and / or MIC-A and said mIL-21; isolating cells expressing at least one of IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, membrane-bound IL-15 (mIL-15), TLR ligands, ULBP-1, ULPB-2 and / or MIC-A and the mIL-21; as well as Irradiate the isolated cells. Modified 721.221 cells are thereby generated.

22. The method of claim 21, wherein the 721.221 cell population is transduced with a virus comprising nucleic acid encoding at least one of IL-2, IL-12, IL-33, IL-27, IL-18, IL-7, mIL-7, IL-15, membrane-bound IL-15 (mIL-15), TLR ligands, ULBP-1, ULPB-2 and / or MIC-A and the mIL-21.

23. The method of claim 21 or claim 22, wherein the virus is a retrovirus or a lentivirus.

24. The method of any one of claims 14-15, 17-19 and 21-22, wherein the 721.221 cell population is further transduced or transfected with a nucleic acid encoding an additional heterologous cytokine, activating receptor ligand, TRL ligand or receptor thereof.

25. The method of claim 24, wherein the additional exogenous heterologous cytokine, activating receptor ligand, TRL ligand, or receptor thereof is membrane bound.

26. The method of claim 24, wherein the additional heterologous cytokine, activating receptor ligand, TRL ligand, or receptor thereof comprises IL-15α.

27. A method of expanding a population of natural killer (NK) cells or T cells, comprising contacting the population of cells with the modified 721.221 cells of any one of claims 1-2 and 9-10 and at least one cytokine for 1-40 days under conditions sufficient for cell expansion.

28. The method of claim 27, wherein the cell population is from peripheral blood, umbilical cord blood, ascites, menstrual blood or bone marrow.

29. The method of claim 27, wherein the cell population comprises peripheral blood mononuclear cells (PBMCs).

30. The method of claim 27, wherein the cell population comprises chimeric antigen receptor (CAR) modified cells.

31. The method of claim 30, wherein the CAR-modified cells comprise CAR-modified T cells or CAR-modified natural killer (NK) cells.

32. The method of claim 30, wherein the CAR-modified cells comprise CD19 CAR-modified NK cells.

33. The method of claim 27, wherein the at least one cytokine comprises at least one interleukin.

34. The method of claim 33, wherein the interleukin is interleukin-15 (IL-15) and / or interleukin-2 (IL-2).

35. The method of claim 27, wherein the population of cells is contacted with the modified 721.221 cells for at least 14-21 days.

36. The method of claim 35, wherein the natural killer (NK) cell population or T cell population increases at least 5,000 to 90,000 fold after contacting the cell population for at least 14-21 days.

37. Use of natural killer (NK) cells or T cells produced by the method according to claim 27 in the preparation of a medicament for treating cancer or an infectious disease or an immune disease in a subject.

38. Use of natural killer (NK) cells or T cells in the preparation of a medicament for treating cancer or an infectious disease or an immune disease in a subject, wherein: The natural killer (NK) cells or T cells are produced by contacting a cell population with a modified 721.221 cell population according to any one of claims 1-2 and 9-10 and at least one cytokine for at least 14-21 days.

39. The use according to claim 37, wherein the cancer or immune disease comprises an autoimmune disease, transplant rejection, a solid tumor or a blood cancer.

40. The use according to claim 37 or 39, wherein the cell population is from a subject suffering from cancer or immune disease.

41. The use according to claim 37, wherein the infectious disease comprises human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), tuberculosis (TB) or malaria.

42. The use according to claim 39, wherein the blood cancer comprises multiple myeloma, leukemia or lymphoma.

43. The use according to claim 39, wherein the solid tumors include lymphoma, breast cancer, hepatocellular carcinoma (HCC) and pancreatic cancer.

44. The use according to claim 42, wherein the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

45. Use according to claim 42 or claim 43, wherein the lymphoma is non-Hodgkin's lymphoma.

46. ​​The use according to claim 38, wherein the cell population comprises CAR-modified lymphocytes.

47. The use according to claim 46, wherein the CAR-modified lymphocytes comprise CD19 CAR-modified natural killer cells.

48. The use according to claim 37, wherein the cancer comprises a sarcoma or a neuroblastoma.

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