Quadricistronic system comprising homing receptor or cytokine and chimeric antigen receptor for stable genetic modification of cellular immunotherapies

Abstract

To provide engineered cells using the cytotoxic activated natural killer cell line as the basis to improve immunotherapies to cancer and tumors.SOLUTION: The present invention provides a recombinant modified natural killer (NK)-92 cell stably transfected with nucleic acids, wherein the recombinant modified NK-92 cells have American Type Culture Collection (ATCC) deposit number CRL-2407; the nucleic acid encodes an anti-cluster of differentiation 123 (CD123) chimeric antigen receptor (CAR), an Fc receptor, and a cytokine; and the recombinant modified cells express the anti-CD123 CAR and the Fc receptor on the cell surface of the recombinant cells.SELECTED DRAWING: Figure 16

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 713,264, filed August 1, 2018; U.S. Provisional Patent Application No. 62 / 713,278, filed August 1, 2018; U.S. Provisional Patent Application No. 62 / 713,310, filed August 1, 2018; and U.S. Provisional Patent Application No. 62 / 713,323, filed August 1, 2018. Each of these applications is incorporated herein by reference in its entirety.

[0002] Sequence Listing The contents of the ASCII text file of the Sequence Listing entitled 104077_0007PCT_Seq_listing_rev004_ST25, 97KB in size, was created on Aug. 1, 2019, was submitted electronically via EFS-Web with the present application, and is incorporated by reference in its entirety.

[0003] The field of the invention is genetically engineered cells using a cytotoxic activated natural killer cell line (NK-92) as a basis for improving immunotherapy against cancer and tumors. [Background technology]

[0004] The Background Description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the present claims, or that any publication specifically or implicitly referenced is prior art.

[0005] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event that a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0006] Cancer immunotherapy based on adoptively transferred tumor-specific cytotoxic lymphocytes shows promise for the treatment of patients with malignant tumors. Despite this initial success in certain cancers, tumor treatment remains a challenge, mainly due to the immunosuppressive nature of the tumor microenvironment. See Swarts et al., “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol., 72, pages 2473-2480, 2012. In addition to engineered T cells, NK cell-based immunotherapy has been explored. Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Natural killer (NK) cells generally account for approximately 10-15% of circulating lymphocytes and bind to and kill target cells, including virus-infected cells and many malignant cells, nonspecifically with respect to antigen and without prior immune sensitization. Herberman et al., Science 214:24 (1981). NK-92 is a cytolytic cancer cell line that was discovered in the blood of subjects suffering from non-Hodgkin's lymphoma and then immortalized ex vivo. NK-92® cells are derived from NK cells, but lack the major inhibitory receptors displayed by normal NK cells and retain most of the activating receptors. However, NK-92® cells do not attack normal cells, nor do they induce unacceptable immune rejection in humans.

[0007] A common driver of lymph node metastasis is the hypoxia-induced upregulation of CCR7, a chemokine receptor found primarily on naive T cells and dendritic cells. It has previously been demonstrated that upregulation of the CCR7 receptor on blood NK cells improves homing of NK cells to lymph nodes, allowing them to follow the same route to the nodal compartment, a common route for metastatic spread, but this has not yet been demonstrated in clinically relevant cell lines.

[0008] Thus, there remains a need to imprive NK cells and NK cell-based therapies, particularly in the homing and regulation of NK cells to the tumor microenvironment. Summary of the Invention [Means for solving the problem]

[0009] Provided herein are modified NK-92® cells that comprise a nucleic acid encoding multiple functional elements. A functional element is typically a protein or polypeptide that provides a specific function that improves the effectiveness of the cell as a cell line for immunotherapy. In one aspect, the NK-92® cells comprise a nucleic acid construct that encodes four functional elements. In some embodiments, the nucleic acid construct comprises a sequence encoding the four functional elements operably linked to a promoter (referred to as a "quadricistronic construct").

[0010] In some embodiments, the first element encoded by the nucleic acid construct is a cytokine that provides for selection of NK-92® cells expressing a cytokine, such as IL-2 or IL-15. Thus, in some embodiments, the nucleic acid encodes a cytokine, such as IL-2 or IL-15. In one embodiment, IL-2 is expressed with a signal sequence that directs IL-2 to endoplasmic reticulum IL-2 ("erIL-2"). In another embodiment, IL-15 is expressed with a signal sequence that directs IL-15 to endoplasmic reticulum IL-15 ("erIL-15").

[0011] In some embodiments, the second element encoded by the nucleic acid construct is an Fc receptor. In some embodiments, the Fc receptor is an Fc gamma receptor (FCγR). In some embodiments, the Fc gamma receptor is FCγRIII-A (also called CD16), which is a low affinity Fc receptor that binds IgG antibodies and activates ADCC. In some embodiments, the CD16 receptor comprises a phenylalanine (F) to valine (V) substitution at amino acid position 158 (F158V) of the mature form of the polypeptide (SEQ ID NO: 12) (corresponding to position 176 of the full-length form of the polypeptide including the signal sequence). In one embodiment, the Fc receptor comprises the nucleic acid sequence of SEQ ID NO: 13 or the amino acid sequence of SEQ ID NO: 12.

[0012] In some embodiments, the first and second elements are present in a nucleic acid construct. Thus, in some embodiments, the nucleic acid construct encodes an Fc receptor (such as CD16) and erIL-2.

[0013] In some embodiments, the third element encoded by the nucleic acid construct is a homing receptor. In some embodiments, the homing receptor is a cytokine receptor, a G protein-coupled receptor, a chemokine receptor, a cell adhesion molecule, a selectin, or an integrin. In some embodiments, the homing receptor is operably linked to a promoter that allows transcription of the nucleic acid. The modified NK-92® cells can migrate toward a source of a chemokine that is a ligand for the receptor. Unlike normal blood-derived NK cells, the modified NK-92® cells can be developed into a cell line relevant for human clinical trials, providing distinct advantages for immunotherapy. Examples of homing receptors include chemokine receptors, including but not limited to CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, or CXCL14 receptors; cytokine receptors; cell adhesion molecules, such as selectins, including L-selectin (CD62L); G-protein coupled receptors, such as integrins, such as α4β7 integrin, LPAM-1, and LFA-1. In some embodiments, the homing receptor is a cell adhesion molecule, such as LFA-1. In some embodiments, the homing receptor is a selectin, such as L-selectin (CD62L). In some embodiments, the homing receptor is an integrin, such as α4β7 integrin, LPAM-1, or VLA-4. In some embodiments, the homing receptor is a CC or CXC chemokine receptor. Thus, in some embodiments, the third element encoded by the nucleic acid is a homing receptor described herein.

[0014] In some embodiments, the third element encoded by the nucleic acid construct is a secreted cytokine, whereby the cytokine increases or improves the function of the NK-92® cells as an immunotherapeutic agent. The cytokine may also modulate the tumor microenvironment. In some embodiments, the secreted cytokine that modulates the tumor microenvironment is IL-12 or IFN-alpha. Thus, in some embodiments, the third element encoded by the nucleic acid construct is a cytokine, such as IL-12 or IFN-alpha.

[0015] Thus, in some embodiments, the third element encoded by the nucleic acid construct is a chemokine, such as XCL1, CCL5, CCL21, or CCL16. In some embodiments, the third element encoded by the nucleic acid construct is a Toll-like receptor (TLR) agonist.

[0016] In one embodiment, the third element encoded by the nucleic acid construct is IL-12.

[0017] It is known that the expression of TGF-β in tumors suppresses the antitumor activity of leukocytes in tumor microenvironment. Thus, in some embodiments, the third element encoded by the nucleic acid construct is a TGF beta inhibitor, e.g., a peptide that inhibits TGF-β. In some embodiments, the third element encoded by the nucleic acid construct is a TGF beta trap. In some embodiments, the TGF beta trap comprises an extracellular domain of a TGFβRII molecule. In some embodiments, the TGF beta trap comprises a single-chain dimer of the extracellular domain of a TGFβRII molecule, most preferably a single-chain dimer of a TGF-β receptor II ectodomain.

[0018] In some embodiments, the NK cells described herein are administered with a TGF-β inhibitor to help block TGF-β and remove immune suppression. In some embodiments, the NK cells described herein are administered with other immunotherapeutic agents to help reduce or eliminate tumors. For example, TGF-β can be inhibited by intratumoral injection of inhibitory peptides in combination with intratumoral injection of poly(I:C) and α-CD40 antibodies. In some embodiments, the TGF-β inhibitor is combined with IL-2.

[0019] In some embodiments, the fourth element encoded by the nucleic acid construct is an antigen binding protein ("ABP"). In some embodiments, the antigen binding protein specifically binds to a tumor associated antigen. In some embodiments, the ABP comprises a fragment of an antibody, such as an scFv. In some embodiments, the antigen binding protein comprises or is part of a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes an ABP or CAR that specifically binds to CD19, CD20, an NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, CD33, B7-H4, or 41BB.

[0020] In another aspect, the modified NK-92® cells comprise a nucleic acid encoding IL-12. In another aspect, the modified NK-92® cells comprise a nucleic acid encoding a TGFbeta trap. In some embodiments, the TGFbeta trap comprises a single-chain dimer of the extracellular domain of a TGFβRII molecule.

[0021] In another aspect, the modified NK-92® cells comprise a nucleic acid encoding a cytokine that provides for selection or enables survival of NK-92® cells expressing the cytokine. In some embodiments, the nucleic acid encodes a cytokine such as IL-2 or IL-15. In one embodiment, IL-2 is expressed with a signal sequence that directs IL-2 to endoplasmic reticulum IL-2 ("erIL-2"). In one embodiment, IL-15 is expressed with a signal sequence that directs IL-15 to endoplasmic reticulum IL-2 ("erIL-15").

[0022] In some embodiments, the engineered NK-92® cells comprise a nucleic acid encoding an Fc receptor. In some embodiments, the Fc receptor is an Fc gamma receptor (FCγR). In some embodiments, the Fc gamma receptor is FcγRIII-A (also called CD16), which is a low affinity Fc receptor that binds IgG antibodies and activates ADCC. In some embodiments, the CD16 receptor comprises a phenylalanine (F) to valine (V) substitution at amino acid position 158 (F158V) of the mature form of the polypeptide (SEQ ID NO: 12) (corresponding to position 176 of the full-length form of the polypeptide including the signal sequence). In one embodiment, the Fc receptor comprises the nucleic acid sequence of SEQ ID NO: 13 or the amino acid sequence of SEQ ID NO: 12.

[0023] In some embodiments, the engineered NK-92® cells comprise a nucleic acid encoding an antigen binding protein ("ABP"). In some embodiments, the antigen binding protein specifically binds to a tumor-associated antigen. In some embodiments, the ABP comprises a fragment of an antibody, such as an scFv. In some embodiments, the antigen binding protein comprises or is a portion of a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes an ABP or CAR that specifically binds to CD19, CD20, an NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, CD33, B7-H4, or 41BB.

[0024] In another aspect, the modified NK-92® cells comprise a nucleic acid encoding a secreted cytokine that modulates the tumor microenvironment. In some embodiments, the cytokine that modulates the tumor microenvironment is a chemokine, such as XCL1, CCL5, CCL21, or CCL16. In some embodiments, the engineered NK-92® cells comprise a nucleic acid encoding a Toll-like receptor (TLR) agonist. In some embodiments, the engineered NK-92® cells comprise a nucleic acid encoding IL-12 or IFN-alpha. In some embodiments, the engineered NK-92® cells comprise a nucleic acid encoding a TGFbeta inhibitor, e.g., a peptide that inhibits TGF-β. In some embodiments, the engineered NK-92® cells comprise a nucleic acid encoding a TGFbeta trap. In some embodiments, the TGFbeta trap comprises the extracellular domain of a TGFβRII molecule, or a single chan dimer of the extracellular domain of a TGFβRII molecule.

[0025] In one aspect, the modified NK-92® cells comprise one or more nucleic acid molecules encoding a homing receptor, an ABP or CAR, an Fc receptor, and / or a cytokine that provides for selection or enables survival of NK-92® cells expressing the cytokine. Thus, in some embodiments, the modified NK-92® cells comprise nucleic acid molecules encoding a chemokine receptor, a CAR, CD16, and erIL-2. In some embodiments, the modified NK-92® cells comprise nucleic acid molecules encoding CCR7 or CXCR2, a CAR, CD16, and erIL-2. In some embodiments, the modified NK-92® cells comprise nucleic acid molecules encoding IL-12 or TGF beta trap, a CAR, CD16, and erIL-2.

[0026] In some embodiments, the CAR comprises an intracellular signaling domain from Fc epsilon receptor gamma (FcεRIγ). In one embodiment, the CAR is transiently expressed by NK-92® cells. In one embodiment, the CAR is stably expressed by NK-92® cells.

[0027] To date, FcεRIγ-containing CARs have not been utilized in NK-92® cells, other NK cell lines, or endogenous NK cells because other signaling domains (e.g., CD3ζ) have been determined to be more efficient, especially when combined with additional signaling domains (second and third generation CARs). Described herein is the unexpected and surprising finding that NK-92® cells expressing a "first generation" CAR that includes the intracellular domain from FcεRIγ have equal or greater cytotoxic activity against cancer cells expressing the antigen recognized by the CAR, compared to NK-92® cells expressing a CAR with the CD3ζ signaling domain alone or in combination with other signaling domains (i.e., second or third generation CARs). In one embodiment, a CD3ζ signaling domain contemplated herein may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:40.

[0028] In one aspect, an NK-92® cell or cell line expressing a chimeric antigen receptor (CAR) on the surface of an NK-92® cell is described, wherein said CAR comprises a cytoplasmic domain of FcεRIγ. In one embodiment, the cytoplasmic domain of FcεRIγ comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:31.

[0029] In some embodiments, the cytoplasmic domain of FcεRIγ is encoded by a nucleic acid having at least 95% sequence identity to SEQ ID NO:32.

[0030] In some embodiments, the CAR comprises a hinge region from CD8. In some embodiments, the CAR comprises a transmembrane domain from CD28.

[0031] In some embodiments, the NK-92® cell or cell line is genetically modified with a nucleic acid construct comprising SEQ ID NO:31 (FcεRIγ intracellular cytoplasmic domain), SEQ ID NO:32 (FcεRIγ intracellular signaling domain minus transmembrane domain), SEQ ID NO:33 (CD8 hinge region), SEQ ID NO:34 (CD8 hinge region DNA), SEQ ID NO:35 (CD28 transmembrane domain), and / or SEQ ID NO:36 (CD28 transmembrane domain, minus ITAM or intracellular sequence). In one embodiment, the CD8 hinge region, CD28 transmembrane, and FceRI gamma signaling domain amino acid sequences may comprise a polypeptide or polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:37 or SEQ ID NO:38. In some embodiments, the nucleic acid construct further comprises a promoter that facilitates transcription of the nucleic acid sequence. In some embodiments, the promoter is an inducible promoter. In some embodiments, the nucleic acid construct is a multicistronic vector that includes one or more internal ribosome entry sites (IRES), allowing initiation of translation from an internal region of the mRNA transcribed from the nucleic acid sequence. In some embodiments, the nucleic acid construct includes a sequence encoding a 2A peptide, such as a T2A, P2A, E2A, or F2A peptide, to generate equimolar levels of polypeptides encoded by the same mRNA. In some embodiments, the nucleic acid construct further includes a nucleic acid sequence encoding an antigen binding protein (ABP). In some embodiments, the ABP is an scFv or a codon-optimized scFv. In some embodiments, the ABP specifically binds to an antigen expressed by a tumor cell. In some embodiments, the ABP is part of a chimeric antigen receptor (CAR). In some embodiments, the construct includes a nucleic acid encoding a cytokine, which provides for selection or allows survival of NK-92® cells that express a cytokine, such as IL-2. In one embodiment, the cytokine is targeted to the endoplasmic reticulum.In one embodiment, the CAR scFv may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:39.

[0032] In some embodiments, the construct comprises the vector shown in Figure 10. In some embodiments, the NK-92® cells or cell lines are genetically modified to express CD16 on the cell surface. In one embodiment, the NK-92® cells or cell lines are genetically modified to express high affinity CD16 (F158V) on the cell surface.

[0033] In one embodiment, the ABP or CAR targets or specifically binds to a tumor-associated antigen. In one embodiment, the tumor-associated antigen is selected from the group consisting of CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, CD33, B7-H4, and 41BB. In one embodiment, the tumor-associated antigen is CD19. In another embodiment, the tumor-associated antigen is CD33.

[0034] In one aspect, the present disclosure relates to an NK-92® cell line transformed with a nucleic acid encoding a chimeric antigen receptor (CAR) having a cytoplasmic domain of FcεRIγ, wherein the CAR is expressed on the surface of the NK-92® cell. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is DNA.

[0035] In some embodiments, the NK-92® cells are further modified to express at least one cytokine or a variant thereof that provides for selection of or allows survival of NK-92® cells expressing the cytokine. In one embodiment, the at least one cytokine is transiently expressed by the NK-92® cells. In one embodiment, the at least one cytokine is stably expressed by the NK-92® cells.

[0036] In some embodiments, the modified NK-92® cells comprise an expression vector comprising one or more or a plurality of nucleic acid molecules described herein. In some embodiments, the nucleic acid molecule is operably linked to a promoter capable of initiating transcription of the nucleic acid molecule. In some embodiments, each nucleic acid molecule of the plurality of nucleic acid molecules is operably linked to a separate, distinct, and / or different promoter. In some embodiments, the one or more nucleic acid molecules are operably linked to the same promoter. In one embodiment, the nucleic acid molecules encoding the homing receptor, the CAR, the Fc receptor, and the cytokine are operably linked to the same promoter or a single promoter. In some embodiments, the promoter is an inducible promoter. In one embodiment, the nucleic acid molecule encoding the cytokine is located downstream or 3' of the nucleic acid molecule encoding the homing receptor, the CAR, and the Fc receptor (e.g., CD16 or high affinity CD16).

[0037] In some embodiments, the NK-92® cells express a protein encoded by a nucleic acid molecule described herein on the cell surface. For example, in some embodiments, the modified NK-92® cells express a homing receptor, ABP or CAR, and an Fc receptor (e.g., CD16 or high affinity CD16) on the cell surface.

[0038] Compositions and kits comprising the modified NK-92® cells are also provided. Methods of making the modified cells and methods of using the cells to treat cancer are provided.

[0039] In another aspect, a method for treating cancer or reducing the size of a tumor is described. In some embodiments, the method for treating cancer or reducing the size of a tumor comprises administering to a subject in need thereof a therapeutically effective amount of modified NK-92® cells as described herein, wherein the administration treats the cancer or reduces the size of a tumor in the subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of modified NK-92® cells comprising a nucleic acid encoding a homing receptor, an ABP or CAR that specifically binds to a target antigen, an Fc receptor such as CD16 or CD16-158V, and / or a cytokine such as erIL-2 or erIL-15. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of modified NK-92® cells comprising a nucleic acid encoding a secreted cytokine, an ABP or CAR that specifically binds to a target antigen, an Fc receptor such as CD16 or CD16-158V, and / or a cytokine such as erIL-2 or erIL-15.

[0040] In some embodiments, the NK cells described herein are administered with a TGF-β inhibitor to help block TGF-β and remove immune suppression. In some embodiments, the NK cells described herein are administered with other immunotherapeutic agents to help reduce or eliminate tumors. For example, TGF-β can be inhibited by intratumoral injection of inhibitory peptides in combination with intratumoral injection of poly(I:C) and α-CD40 antibodies. In some embodiments, the TGF-β inhibitor is combined with IL-2.

[0041] In another aspect, the use of the compositions described herein for treating a disease is provided. In some embodiments, the modified NK-92® cells described herein are provided for use as a medicament for treating a disease. In some embodiments, the modified NK-92® cells described herein are provided for use in treating a disease. In some embodiments, the modified NK-92® cells comprise a nucleic acid encoding a homing receptor, an ABP or CAR that specifically binds to a target antigen, an Fc receptor such as CD16 or CD16-158V, and / or a cytokine such as erIL-2 or erIL-15. In some embodiments, the modified NK-92® cells comprise a nucleic acid encoding a secreted cytokine, an ABP or CAR that specifically binds to a target antigen, an Fc receptor such as CD16 or CD16-158V, and / or a cytokine such as erIL-2 or erIL-15. In some embodiments, the disease is cancer.

[0042] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0043] [Figure 1] FIG. 1 is a schematic diagram showing plasmid pNKAT-CCR7-LP3 containing the CCR7 receptor for insertion into the AAVS1 locus of NK-92® cells. [Diagram 2] FIG. 1 is a schematic diagram showing the plasmid pCRENFAT-CCL21, which contains the NFAT-responsive CCL21 gene. [Diagram 3]Graph showing expression of phenotypic markers associated with NK-92® cells in wild-type NK-92® cells and modified NK-92® cells expressing CCR7. (Lane 1: aNK (wild type); Lane 2: modified NK-92® cells (MA3); Lane 3: modified NK-92® cells (MB4); Lane 4: modified NK-92® cells (MB6); Lane 5: modified NK-92® cells (ME6); Lane 6: modified NK-92® cells (MH3); A: isotype (APC); B: CD54 (ICAM-1); C: NKp30; D: NKG2D [Figure 4] 1 is a graph showing the cytotoxic activity of modified NK-92® cells expressing CCR7 against K562 cells. [Diagram 5] 1 is a graph showing the cytotoxic activity of modified NK-92® cells expressing CCR7 against HL-60 cells. [Figure 6A] Graph showing activation of NFAT-luciferase reporter gene in NK-92 cells as demonstrated in binding to K562 and SUP-B15 cells (NK-92 cells electroporated with CD19-CAR mRNA). [Figure 6B] Graph showing activation of NFAT-luciferase reporter gene in NK-92 cells as demonstrated in binding to K562 and SUP-B15 cells (NK-92 cells electroporated with CD19-CAR mRNA). [Figure 7] Graph showing modified NK-92® cells expressing CCR7 (Mi-aNK) migrated towards chemokines CCL19 and CCL21. [Figure 8]FIG. 1 shows a diagram depicting an exemplary method for in vitro testing of modified NK-92® cells described herein. Activated NK-92® cells (aNK) were modified to express a chemokine receptor (e.g., CCR7) and target cells were modified to express a chemokine that binds to the receptor (e.g., CCL19 or CCL21). The modified NK-92® cells were tested in a modified Boyden chamber transwell assay as shown. [Figure 9]

[0023] Figure 1 shows a representative cytotoxicity assay using modified NK-92® cells described herein. Modified NK-92® cells were tested for cytotoxicity against K562 target cells expressing and secreting one or both chemokine ligands. The ML4 clone had the highest percentage of target cell lysis, with the percentage increasing when K562 target cells expressed both CCL19 and CCL21. [Figure 10] FIG. 1 is a schematic diagram showing the plasmid pNKAT-CCR7-CD19CAR-CD16-ERIL2, designated a "quadricistronic vector," which can be used to stably transfect cells with a single insertion site. [Figure 11] FIG. 11 is a schematic diagram showing the linearized plasmid from FIG. [Figure 12] Figure 1 shows cell surface expression of CCR7, CD16, and CD19 CARs by NK-92® cells. "aNK" is a wild-type NK-92® cell line. "ML4" is an aNK cell line transfected with a nucleic acid construct encoding CCR7 operably linked to a promoter (i.e., Mi-aNK). "P2" is an aNK cell line transfected with a nucleic acid construct encoding CCR7, CD16, ER-IL2, and CD19 CAR (i.e., Mi-T-hanK). [Figure 13]Homing of non-CR vs. Mi-T-haNK cells to parental or CCL19-expressing tumors at the indicated times after NK cell administration. Data are mean ± SEM. - and + symbols indicate expression status of CCR7 receptor (first symbol) and CCL19 ligand (second symbol). *, P<0.05 by one-way ANOVA followed by Tukey's test for multiple comparisons. The last panel shows the time course curves. [Figure 14] Direct comparison of non-CR and Mi-T-haNK cell infiltration into parental or CCL19 expressing tumors in a single animal 24 hours post-treatment. Three of four animals receiving Mi-T-haNK cells showed higher infiltration into CCL19+ tumors, whereas three of four animals receiving non-CR CD19 t-haNK cells showed similar levels of infiltration into both K562 and K-19 tumors. One "outlier" animal in each group is indicated by a dashed line. [Figure 15] Survival curves of Raji-19.5 IV tumor-bearing animals are shown. Survival curves of Raji-19.5 IV tumor-bearing NSG mice treated with vehicle, CD19 t-haNK cells, or R7-19.1 cells. Statistical analysis was performed by log-rank (Mantel-Cox) test. ***, P=0.0002; ****, P<0.0001. [Figure 16] Figure 1 shows body weight change in IV Raji-19.5 tumor model. Body weight change curves (percent change relative to body weight on day 0) of IV Raji-19.5 tumor-bearing animals treated with vehicle, CD19 t-haNK cells, or R7-19.1 cells. Data are mean ± SEM. Red arrows indicate the day of dosing. Body weight measurements taken on the day of dosing were performed prior to dosing. At all time points prior to day 20, curves for NK cell-treated groups reached statistical significance (P<0.05) compared to the vehicle control group by 2-way ANOVA followed by Tukey's multiple comparison test. [Figure 17] The size of SC Raji-19.5 tumors at the time of randomization is shown. Individual tumor sizes at the time of randomization are shown. Black boxes enclose large tumors >200 mm3 in size, and blue boxes enclose small tumors <200 mm3 in size. Group means ± SEM are also shown. [Figure 18]Tumor growth of a large tumor subpopulation in SC Raji-19.5 tumor-bearing mice. (A) Group analysis. Data are mean ± SEM. Statistical analysis was performed by two-way mixed-effects analysis followed by multiple comparisons with Tukey's test. Statistical significance was not achieved. (B) Individual curves. Red arrows indicate dosing days. Tx: treatment. [Figure 19] Tumor growth of a subpopulation of small tumors in SC Raji-19.5-bearing mice. (A) Group analysis. Data are mean ± SEM. Statistical analysis was performed by two-way mixed-effects analysis followed by multiple comparisons with Tukey's test. No statistical significance was detected between the two groups at any time point. (B) Individual curves. Red arrows indicate the days of treatment. Tx: treatment. [Figure 20] Figure 1 shows body weight change in the SC Raji-19.5 tumor model. Body weight change curves (percent change relative to body weight on day 1) of SC Raji-19.5 tumor-bearing animals treated with vehicle, CD19 t-haNK cells, or R7-19.1 cells. Data are mean ± SEM. Red arrows indicate dosing days. Body weight measurements taken on dosing days were performed before dosing. At all time points prior to day 16, the curves for the NK cell-treated groups reached statistical significance compared to the vehicle control group by 2-way mixed-effects analysis followed by multiple comparisons by Tukey's test. [Figure 21] 1 shows one embodiment of a quadricistronic TGFβ trap armored PD-L1 CAR construct. [Figure 22] Expression analysis of PD-L1CAR and CD16 in PD-L1(TGFβ trap) t-haNK clones. [Diagram 23] Figure 2 shows that TGFβTrap is secreted into the culture supernatant of TGFβTrap / PD-L1 t-haNK clones. [Figure 24] Figure 1 shows the cytotoxicity of quadricistronic TGFβ trap contructs against K562 target cells. [Diagram 25] Shows CAR killing of quadricistronic TGFβ trap contruct against PD-L1 expressing SUP-B15 target cells. [Figure 26] Shows CAR killing of quadricistronic TGFβ trap contructs against MDA-MB231 target cells. [Figure 27] 1 shows ADCC of quadricistronic TGFβ trap contructs against SUP-B15CD19-CD20+. [Figure 28] 1 shows that TGFβ / SMAD luciferase reporter HEK293 cells are induced by TGFβ. [Figure 29] We show that secreted TGFβ Trap sequestered TGFβ and inhibited luciferase expression in a HEK293T reporter assay. [Diagram 30] 1 shows IL-12 secretion from NK-92® cell line transduced with IL-12 virus. [Diagram 31] 1 shows one embodiment of a quadricistronic IL-12 / PD-L1 t-hanK construct. [Diagram 32] Cytotoxicity data of CCR7 CD19 t-haNK cells are shown. [Diagram 33] IL-12 secretion from IL-12 / PD-L1 t-haNK™ cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of immunology and immunotherapy.

[0045] In this specification and the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0046] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0047] All numerical designations, including ranges and variations, such as pH, temperature, time, concentration, amount, and molecular weight, are those that are commonly encountered by those skilled in the art. Thus, numerical values ​​can include variations of (+) or (-) increments of 0.1 or 1.0, as appropriate, depending on the relevant significant figures. It should be understood that, although not necessarily stated, all numerical designations can be preceded by the term "about". The term "about" as used herein can also mean that a value can vary by ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0048] It is also to be understood, although not necessarily stated, that the reagents described herein are merely exemplary and that equivalents of such reagents are known in the art.

[0049] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0050] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are of some essential importance to the combination. For example, a composition consisting essentially of elements as defined herein does not exclude other elements that do not materially affect the basic and novel characteristics of the claim. "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and substantial method steps as recited. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0051] The term "homing receptor" refers to a receptor that activates a cellular pathway that directly or indirectly leads to the migration of cells toward a target cell or tissue. For example, homing receptors expressed by leukocytes are used by leukocytes and lymphocytes to enter secondary lymphoid tissues via high endothelial venules. Homing receptors can also be used by cells to migrate toward the source of a chemical gradient, such as a chemokine gradient. Examples of homing receptors include G-protein coupled receptors, such as chemokine receptors, including but not limited to CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, and DARC; cytokine receptors; cell adhesion molecules, such as selectins, including L-selectin (CD62L); integrins, such as α4β7 integrin, LPAM-1, and LFA-1. Homing receptors generally bind to a recognition ligand on a target tissue or cell. In some embodiments, the homing receptor binds to an addressin on the endothelium of venules, such as mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1).

[0052] As used herein, "immunotherapy" refers to the use of modified or unmodified NK-92® cells, naturally occurring or modified NK cells, or T cells, whether alone or in combination, which are capable of inducing cytotoxicity upon contact with target cells.

[0053] As used herein, a "natural killer (NK) cell" is a cell of the immune system that kills target cells in the absence of specific antigenic stimulation and without major histocompatibility complex (MHC) class restriction. NK cells are characterized by the presence of the CD56 surface marker and the expression of CD3. It is characterized by the absence of face markers.

[0054] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), as distinct from the NK-92® cell line. Endogenous NK cells are generally a heterogeneous population of cells in which NK cells are enriched. Endogenous NK cells can be for autologous or allogeneic treatment of the patient.

[0055] The term "NK-92" refers to natural killer cells derived from a highly potent, proprietary cell line described in Gong et al. (1994) and owned by NantKwest® (hereinafter "NK-92® cells"). The immortal NK cell line was originally obtained from a patient with non-Hodgkin's lymphoma. Unless otherwise specified, the term "NK-92®" is intended to refer to the original NK-92® cell line as well as NK-92® cell lines that have been modified (e.g., by the introduction of an exogenous gene). NK-92® cells, as well as exemplary, non-limiting modifications thereof, are described in U.S. Patent Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; 9,150,636 and published as U.S. Patent Application Serial No. 10 / 008,955, all of which are incorporated herein by reference in their entireties, and include wild-type NK-92®, NK-92®-CD16, NK-92®-CD16-γ, NK-92®-CD16-ζ, NK-92®-CD16(F176V), NK-92® MI, and NK-92® CI. NK-92® cells are known to those of skill in the art, and such cells are readily available from NantKwest, Inc.

[0056] The term "aNK" refers to unmodified natural killer cells derived from a highly potent, proprietary cell line described in Gong et al. (1994) and owned by NantKwest (hereinafter "aNK® cells"). The term "haNK" refers to natural killer cells derived from a highly potent, proprietary cell line modified to express CD16 on the cell surface described in Gong et al. (1994) and owned by NantKwest (hereinafter "CD16+NK-92® cells" or "haNK® cells"). In some embodiments, CD16+NK-92® cells contain a high affinity CD16 receptor on the cell surface. The term "taNK" refers to natural killer cells derived from a highly potent, proprietary cell line modified to express a chimeric antigen receptor described in Gong et al. (1994) and owned by NantKwest (hereinafter "CAR-modified NK-92® cells" or "taNK® cells"). The term "t-haNK" refers to natural killer cells derived from a highly potent, proprietary cell line described in Gong et al. (1994) and owned by NantkWest, which has been engineered to express CD16 on the cell surface and to express a chimeric antigen receptor (hereinafter "CAR-modified CD16+NK-92® cells" or "t-haNK cells"). In some embodiments, t-haNK cells express the high affinity CD16 receptor on the cell surface.

[0057] The terms "chemokine-targeted t-haNK" and "Mi-T-haNK" refer to t-haNK cells that have been modified to express a chemokine receptor on the cell surface.

[0058] As used herein, the terms "cytotoxic" and "cytolytic" are intended to be synonymous when used to describe the activity of effector cells, such as NK-92® cells. In general, cytotoxic activity involves killing a target cell by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of an effector to lyse the plasma membrane of a target cell, thereby disrupting its physical integrity. This results in the death of the target cell. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK-92® cells is due to cytolysis.

[0059] The term "killing" in relation to a cell / cell population is intended to include any type of manipulation that leads to the death of that cell / cell population.

[0060] The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the defensive function of immune cells by binding to a portion of an antibody known as the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates the phagocytic or cytotoxic activity of the cell through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fc-gamma receptors (FCγR) bind to antibodies of the IgG class. FcγRIII-A (also called CD16) is a low-affinity Fc receptor that binds to IgG antibodies and activates ADCC. FcγRIII-A is typically found on NK cells. NK-92® cells do not express FcγRIII-A. Fc epsilon receptors (FcεR) bind to the Fc region of IgE antibodies.

[0061] The term "chimeric antigen receptor" (CAR), as used herein, refers to an extracellular antigen-binding domain fused to an intracellular signaling domain. CARs can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen-binding domain is an scFv specific for an antigen found on the cell of interest. CAR-expressing NK-92® cells target cells expressing a particular antigen on the cell surface based on the specificity of the scFv domain. The scFv domain can be engineered to recognize any antigen, including tumor-specific antigens. For example, CD19CAR recognizes CD19, a cell surface marker expressed by some cancers.

[0062] The term "tumor specific antigen" as used herein refers to an antigen that is present on a cancer or neoplastic cell, but is not detectable on a normal cell derived from the same tissue or lineage as the cancer cell. Tumor specific antigen as used herein also refers to a tumor associated antigen, i.e., an antigen that is expressed at a higher level on a cancer cell compared to a normal cell derived from the same tissue or lineage as the cancer cell.

[0063] The terms "polynucleotide", "nucleic acid", and "oligonucleotide" are used interchangeably and refer to a polymeric form of any length of nucleotides, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides may assume any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted with non-nucleotide components. Polynucleotides can be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specifically stated or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0064] A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) instead of thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule.

[0065] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Sequence similarity can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The percentage of sequence similarity between sequences is a function of the number of matching or homologous positions shared by the sequences over a given comparison window. A sequence can be at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences described herein.

[0066] The term identical or percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical (i.e., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm using default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website, etc.). Such sequences are then said to be substantially identical. This definition may also refer to or be applied to the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As explained below, preferred algorithms can account for gaps, etc. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0067] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer; subsequence coordinates are designated if necessary; sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0068] A comparison window as used herein includes reference to any one segment of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, and after two sequences are optimally aligned, the sequence can be compared to a reference sequence of the same number of contiguous positions. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); by search for similarity of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0069] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used to determine percent sequence identity of nucleic acids or proteins with the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information, as known in the art. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of a selected length (W) in the query sequence, which, when aligned with words of the same length in the database sequence, match or meet a portion of a positive threshold score T. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is stopped if: the cumulative alignment score has decreased by a quantity X from the maximum achieved; if the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below zero; or if the end of either sequence has been reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The expectation (E) represents the number of different alignments with scores equal to or greater than that expected to occur by chance in a database search.The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), alignment (B) of 50, expectation (E) of 10, M=5, N=-4.

[0070] The term transformation, as used herein, refers to a process by which an exogenous or heterologous nucleic acid molecule (e.g., a vector or a recombinant nucleic acid molecule) is introduced into a recipient cell. The exogenous or heterologous nucleic acid molecule may or may not be integrated (i.e., covalently linked) into the chromosomal DNA constituting the genome of the host cell. For example, the exogenous or heterologous polynucleotide may be maintained on an episomal element, such as a plasmid. Alternatively, or in addition, the exogenous or heterologous polynucleotide may be integrated into a chromosome so that it is inherited by daughter cells via chromosomal replication. Methods of transformation include, but are not limited to, calcium phosphate precipitation; fusion of the recipient cell with bacterial protoplasts containing the recombinant nucleic acid; treatment of the recipient cell with liposomes containing the recombinant nucleic acid; DEAE dextran; fusion using polyethylene glycol (PEG); electroporation; magnetoporation; gene gun delivery; retroviral infection; lipofection; and microinjection of DNA directly into cells.

[0071] The term transformed, when used with respect to a cell, refers to a cell that has undergone transformation as described herein such that the cell has delivered exogenous or heterologous genetic material (e.g., a recombinant nucleic acid). The term transformed can also, or alternatively, be used to refer to cells, cell types, tissues, organisms, etc. that contain exogenous or heterologous genetic material.

[0072] The term introduction, as used herein with respect to the introduction of a nucleic acid into a cell or organism, for example by transformation methods (e.g., calcium chloride mediated transformation, electroporation, particle bombardment), as well as by other methods including transduction, conjugation, conjugation, etc. Optionally, a construct is used to introduce the nucleic acid into the cell or organism.

[0073] The terms modified and recombinant, when used with respect to cells, nucleic acids, polypeptides, vectors, etc., refer to cells, nucleic acids, polypeptides, vectors, etc. that have been modified by, or are the result of, laboratory methods and do not occur naturally. Thus, for example, modified cells include cells that have been produced or modified by laboratory methods, e.g., transformation methods to introduce nucleic acids into cells. Modified cells may contain nucleic acid sequences that are not found within the native (non-recombinant) form of the cell or may contain nucleic acid sequences that have been modified, e.g., linked to a non-native promoter.

[0074] As used herein, the term exogenous refers to a substance, such as a nucleic acid (e.g., a nucleic acid including a regulatory sequence and / or a gene) or a polypeptide, that is artificially introduced into a cell or organism and / or does not naturally occur in the cell in which it resides. In other words, the substance, such as a nucleic acid or a polypeptide, originates outside the cell or organism into which it is introduced. An exogenous nucleic acid may have a nucleotide sequence identical to that of a nucleic acid that naturally occurs in the cell. For example, an NK-92® cell can be genetically engineered to contain a nucleic acid, such as heparanase, having an NK-92® sequence. Optionally, the endogenous NK-92® heparanase sequence is operably linked to a gene that is not associated with a regulatory sequence under natural conditions. Although the NK-92® heparanase sequence may naturally occur in a host cell, the introduced nucleic acid is exogenous according to the present disclosure. An exogenous nucleic acid may have a nucleotide sequence that is different from the nucleotide sequence of any nucleic acid that naturally occurs in the cell. For example, an exogenous nucleic acid may be a heterologous nucleic acid, i.e., a nucleic acid from a different species or organism. Thus, an exogenous nucleic acid may have a nucleic acid sequence that is identical to a nucleic acid naturally found in a source organism, but different from the cell into which the exogenous nucleic acid is introduced. As used herein, the term endogenous refers to a nucleic acid sequence that is native to the cell. As used herein, the term heterologous refers to a nucleic acid sequence that is not native to the cell, i.e., originates from a different organism than the cell. The terms exogenous and endogenous or heterologous are not mutually exclusive. Thus, a nucleic acid sequence may be exogenous and endogenous. This means that a nucleic acid sequence may be introduced into a cell, but has a sequence that is identical or similar to the sequence of a nucleic acid naturally occurring in the cell. Similarly, a nucleic acid sequence may be exogenous and heterologous. This means that a nucleic acid sequence may be introduced into a cell, but has a sequence that is not native to the cell, e.g., a sequence from a different organism.

[0075] As described herein, a control or standard control refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison with a test sample, measurement, or value. For example, a test cell, e.g., a cell transformed with a nucleic acid sequence encoding a gene for an Fc receptor, can be compared to a known normal (wild type) cell (e.g., a standard control cell). A standard control can also represent an average measurement or value collected from a population of cells that do not express an Fc receptor or have no or minimal levels of Fc receptor activity (e.g., a standard control cell). Those skilled in the art will recognize that a standard control can be designed for the evaluation of any number of parameters (e.g., RNA levels, polypeptide levels, specific cell types, etc.).

[0076] The term "express" refers to the production of a gene product (e.g., a protein). When referring to expression, the term "transient" means that the polynucleotide is not integrated into the genome of the cell. When referring to expression, the term "stable" means that the polynucleotide is integrated into the genome of the cell or that a positive selection marker (i.e., an exogenous gene expressed by the cell that confers an advantage under certain growth conditions) is utilized to maintain expression of the transgene.

[0077] The term "cytokine" refers to a general class of biological molecules that affect cells of the immune system. Exemplary cytokines include, but are not limited to, interferons and interleukins (IL), particularly IL-2, IL-12, IL-15, IL-18, and IL-21. In a preferred embodiment, the cytokine is IL-2.

[0078] The term "cytokines that modulate the tumor microenvironment" refers to molecules expressed by NK-92® cells that function to enhance anti-tumor responses. Certain cytokines may inhibit the endogenous immune system's response to tumors, thereby reducing the effectiveness of immunotherapy in treating cancer. Thus, the term also includes inhibitors of tumor-promoting cytokines, such as peptide inhibitors and / or ligands or receptors that bind to tumor-promoting cytokines, e.g., ligand traps.

[0079] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains an intact replicon such that it can replicate when placed in a permissive cell, for example, by the process of transformation. A vector can replicate in one cell type, such as bacteria, but has limited or no ability to replicate in another cell, such as a mammalian cell. A vector can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include: naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles, including DNA condensed with heterologous polylysine, oligopeptides of a certain length, and cationic polymers such as polyethyleneimine, in some cases contained within liposomes; and the use of ternary complexes including viruses and polylysine-DNA. In one embodiment, the vector is a viral vector, such as an adenovirus. Viral vectors are well known in the art.

[0080] As used herein, the term "targeting", when referring to the expression of a protein, is intended to include, but is not limited to, directing a protein or polypeptide to an appropriate destination within or outside a cell. Targeting is typically achieved through a signal peptide or targeting peptide, which is a stretch of amino acid residues in a polypeptide chain. Such signal peptides can be located anywhere in the polypeptide sequence, but are often located at the N-terminus. Polypeptides can also be engineered to have a signal peptide at the C-terminus. Signal peptides can direct a polypeptide to an extracellular area, to placement in the plasma membrane, to the Golgi, endosomes, endoplasmic reticulum, or other intracellular compartments. For example, a polypeptide with a particular amino acid sequence (e.g., KDEL) at its C-terminus is retained in or sent back to the ER lumen.

[0081] As used herein, the term "targeted," when referring to tumor targeting, refers to the ability of NK-92® cells to recognize and kill tumor cells (i.e., target cells). The term "targeted" in this context refers, for example, to the ability of a CAR expressed by an NK-92® cell to recognize and bind to a cell surface antigen expressed by the tumor.

[0082] As used herein, the term "transfect" refers to the insertion of a nucleic acid into a cell. Transfection can be performed using any means that allows the nucleic acid to enter the cell. DNA and / or mRNA can be transfected into the cell. Preferably, the transfected cell expresses the gene product (i.e., protein) encoded by the nucleic acid.

[0083] Headings or subheadings may be used herein for the convenience of the reader and are not intended to affect the scope of the invention. In addition, some terms used herein are more specifically defined below.

[0084] Provided herein are genetically engineered cells that use cytotoxic activated natural killer cell lines (NK-92) as a basis for improving immunotherapy against cancers and tumors and / or increasing homing (migration) to a target of interest. In some embodiments, the NK-92® cells are genetically engineered to express a homing receptor that, when expressed, is known to direct lymphocytes to lymph nodes. In some embodiments, the NK-92® cells are genetically engineered to express a secreted cytokine that modulates the tumor microenvironment, or an inhibitor that blocks a cytokine that modulates the tumor microenvironment.

[0085] The present disclosure provides the advantage of using quadracistronic vectors to insert multiple genes driven by a single highly active promoter to generate stable immunotherapeutic cell lines for use in clinical immunotherapy. Quadracistronic vectors use one or more approaches including P2A peptides and IRES elements to link four genes under the control of a single promoter, linking them to the expression of the final element (in this case a selective drug whose expression is required for cell survival and / or proliferation).

[0086] In a proof-of-concept embodiment, the four genes used to generate an altered gene expression profile in a therapeutic cell line are CCR7, CD19 chimeric antigen receptor, a high affinity variant of CD16, and endoplasmic reticulum-bound IL-2. IL-2 bound to the ER acts as a selection agent in addition to stimulating the cytotoxic potential of the NK-92®-based cell line in which it is integrated. The IL-2 production gene is located at the end of the quadracistronic vector, furthest from the promoter, and is therefore the element most likely to be lost if the gene is assembled into a fragment (leading to negative selection and self-excision from the pool, since cells require IL-2 for continued survival). However, if all elements are successfully integrated into the genome, only cells that have integrated their own source of IL-2 will survive, and therefore cells are selected for by removing IL-2 from the medium. As the IL-2 element is furthest from the promoter, this favors complete integration of the entire cassette of four elements. This can be further verified by flow cytometric staining analysis of the other components (see Examples).

[0087] The constructs described herein offer the advantage of reducing development time in generating new therapeutic cell lines and reducing the stress and adverse effects on cells from multiple rounds of genome engineering and subsequent selection. Furthermore, by placing the selection agent (in this case, ER-IL-2) at the end of the construct, it is expected that due to the nature of RNA transcription and processing, it will be difficult to silence any given component of the construct without causing cells to starve for IL-2. Thus, stable cell lines constructed in this manner should retain expression of all components contained in the vector as long as they continue to produce their own IL-2.

[0088] To demonstrate proof of concept, four specific goals were addressed by the four components that are part of this quadracistronic construct: IL-2 acts as a selection agent and is a known agonist of the cytotoxic effect of NK-92® cells, making it an effective cancer therapeutic; CCR7 element (CC chemokine receptor type 7) is a chemokine receptor that is responsible for directing immune cells expressing it to migrate in the direction of a chemokine gradient of the ligands CCL19 and CCL21, which are commonly expressed in lymph nodes; In one embodiment, the CCR7 element contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 (CCR7 sequence). In one embodiment, a CCL21 contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 (the CCL21 sequence). In one embodiment, a CCL19 contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 (the CCL19 sequence).

[0089] CD19 CAR (chimeric antigen receptor) is a construct used to enhance the cytotoxicity of cells when they encounter the CD19 differentiation cluster on the surface of the cells they encounter. This is a cluster of differentiation commonly expressed on both normal and malignant B cells, and has shown efficacy in direct immunotherapy trials against B cell lymphoma. The high affinity CD16 receptor allows the modified NK-92® cells to recognize and respond to cells recognized by IgG antibodies, such as those used as monoclonal antibodies in cancer treatment regimens, such as Rituxumab and Herceptin. When immune cells armed with CD16 receptors encounter cells coated with one of these antibodies, they trigger ADCC (antibody-dependent cellular cytotoxicity) and attempt to destroy the cells. In practice, this allows such armed cells to be used in combination therapy regimens with monoclonal antibodies against cancer neoantigens, etc. While each of these components has utility on its own, combining this particular combination is proposed to create a potent therapy for B cell lymphoma that can migrate to common sites of tumor growth (lymph nodes) and recognize the B cell antigen CD19 and initiate CAR-mediated cytotoxicity or work with monoclonal antibodies such as Rituxumab to avoid the possibility of antigen escape. It will be understood that this proof-of-concept example is non-limiting and that NK-92® cells can be modified using the methods described herein to express other homing receptors and / or CARs that target other antigens of interest to produce effective immunotherapeutic cell lines.

[0090] As described herein, modified NK-92® cells have been generated with stable long-term expression of the CCR7 lymph node homing receptor driven by the elongation factor 1a (EF1a) promoter after electroporation with a linearized genetic construct containing a CCR7 expression cassette together with a removable selection cassette containing a selectable marker. After one week of puromycin selection, monoclonal cell lines were established that retained high levels of CCR7 expression after serial dilution cloning. These CCR7-overexpressing NK cells exhibit functional responses to lymph node-associated chemokines CCL21 and CCL19 in migration / invasion assays. In one embodiment, the EF1a promoter contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3 (EF1a promoter sequence).

[0091] In some exemplary embodiments, chemokines and homing receptors contemplated herein are selected from the group consisting of SEQ ID NO:44 (CCR7 aa sequence), or SEQ ID NO:45 (CCL19 aa sequence), or SEQ ID NO:46 (CCL21 aa sequence), or SEQ ID NO:47 (CXCR2 nt sequence), or SEQ ID NO:48 (CXCR2 aa sequence), or SEQ ID NO:49 (CXCL14 nt sequence), or SEQ ID NO:50 (CXCL14 aa sequence), or SEQ ID NO:51 (CD62L nt sequence), or SEQ ID NO:52 (CD62L aa sequence), or SEQ ID NO:53 (IL-8 nt sequence), or SEQ ID NO:54 (IL-8 aa sequence), or SEQ ID NO:55 (CXCL1 nt sequence), or SEQ ID NO:56 (CXCL1 The term "polynucleotide sequence" may include a polypeptide or polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence (aa sequence).

[0092] Target engagement of sensitive cell lines has been shown to be recognized in NK-92® cells by activation of NFAT transcription factors and their nuclear translocation. Target engagement involving FceRIg or CD3zeta pathways (including target recognition via ADCC or CAR) is sufficient to induce NFAT activation in NK-92® cells. This was demonstrated by inserting a reporter cassette containing three stop regions flanking the NFAT binding domain and a minimal promoter driving firefly luciferase. NFAT activation by the CD3zeta pathway via electroporation of CD19 CAR mRNA into this reporter cell line, followed by co-culture with SUP-B15 (CD19+, but resistant to non-specific cytotoxicity), resulted in luciferase expression.

[0093] In one embodiment, the NFAT response element sequence (binding site for activated NFAT) contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4.

[0094] In one embodiment, a minimal promoter contemplated herein (downstream of three NFAT response elements) may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5.

[0095] In one embodiment, the complete NFAT response cassette contemplated herein (a polyA+ pause site followed by three NFAT response elements followed by a minimal promoter) may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6.

[0096] In one embodiment, the complete sequence of the initial insertion contemplated herein (EF1a promoter, CCR7 gene with polyA, and LoxP-flanked puromycin resistance gene driven by the ubiquitin promoter, all wrapped in homology arms targeting the AAVS1 locus) may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7.

[0097] In one embodiment, the complete sequence of the second insertion contemplated herein (including the NFAT response cassette driving CCL21+Poly-A and the FRT embedded blasticidin resistance gene driven by CMV) may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8. The sequence of SEQ ID NO: 8 may replace the LoxP-flanked puromycin resistance cassette from the first insertion, the FRT sequences surrounding the blasticidin gene in this sequence, allowing for the later removal or replacement of that cassette using similar Flp-FRT recombination.

[0098] The NK-92® cell line is a human IL-2-dependent NK cell line established from peripheral blood mononuclear cells (PBMCs) of a 50-year-old man diagnosed with non-Hodgkin's lymphoma (Gong, et al., Leukemia. 8:652-8 (1994)). NK-92® cells express CD56 in the absence of CD3, CD8, and CD16. bright and CD2. bright / CD16 negThe / low phenotype is typical of a minor subset of NK cells in peripheral blood that have immune-regulatory functions as cytokine-producing cells. Unlike normal NK cells, NK-92® lacks expression of most killer cell inhibitor receptors (KIR) (Maki, et al., J Hematother Stem Cell Res. 10: 369-83 (2001)). On the surface of NK-92, only KIR2DL4, a KIR receptor with activating and suppressive functions expressed on all NK cells, was detected. KIR2DL4 is thought to mediate its inhibitory effect via binding to the HLA allele G (Suck, Cancer Immunol. Immunother. 65 (4): 485-92 (2015)). The primary pathway for cytotoxic killing of NK-92® cells is the perforin / esterase pathway; NK-92® expresses high levels of perforin and granzyme B (Maki, et al., J Hematother Stem Cell Res. 10:369-83 (2001)).

[0099] NK-92® cells have a very broad cytotoxic spectrum and are active against cell lines derived from hematological malignancies and solid tumors (Klingemann, Blood, 87(11):4913-4(1996); Swift, Haematologica. 97(7):1020-8(2012); Yan, et al., Clin Cancer Res. 4:2859-68(1998)). Safety evaluations in severe combined immunodeficiency (SCID) mice showed no NK-92® treatment-related effects, such as acute toxicity and long-term carcinogenicity (Tam, et al., J Hematother. 8:281-90(1999), Yan, et al., Clin Cancer Res. 4:2859-68(1998)). Administration of NK-92® cells to mice challenged with human leukemia cells or mouse models of human melanoma resulted in improved survival and inhibition of tumor growth, including complete remission in some mouse tumors (Tam, et al., J Hematother. 8:281-90 (1999), Yan, et al., Clin Cancer Res. 4:2859-68 (1998)). Phase I clinical trials have confirmed its safety profile. The properties of the NK-92® cell line are described in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, which are incorporated herein by reference in their entireties.

[0100] Optionally, modified NK-92® cells may also express the Fc receptor CD16. As used herein, the term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the defensive function of immune cells by binding to a portion of an antibody known as the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates the phagocytic or cytotoxic activity of the cell via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified by the type of antibody they recognize. For example, Fc-gamma receptors (FCγR) bind to antibodies of the IgG class. FCγRIII-A (also called CD16) is a low affinity Fc receptor that binds to IgG antibodies and activates ADCC. FCγRIII-A is typically found on NK cells. A representative amino acid sequence encoding CD16 is shown in SEQ ID NO:12. A representative polynucleotide sequence encoding CD16 is shown in SEQ ID NO:13. The complete sequence of CD16 is available in the SwissProt database entry P08637.

[0101] In some embodiments, the CD16 receptor comprises a phenylalanine (F) to valine (V) substitution at amino acid position 158 (F158V) in the IgG binding domain of the mature CD16 receptor (corresponding to Val at position 176 in the full-length protein), which affects the antibody-dependent cellular cytotoxicity (ADCC) function of NK cells. The CD16 158V variant binds human IgG1 and IgG3 with higher affinity than the 158F variant.

[0102] Optionally, the modified NK-92® cells comprise a nucleic acid sequence having 70%, 80%, 90%, or 95% identity to SEQ ID NO: 13. Optionally, the modified NK-92® cells comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13. Optionally, the modified NK-92® cells comprise a polypeptide having 70%, 80%, 90%, or 95% identity to SEQ ID NO: 12 (having a valine at position 176 of the full length polypeptide). Optionally, the modified NK-92® cells comprise a polypeptide having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

[0103] The cytotoxicity of NK-92® cells is dependent on the presence of cytokines, such as interleukin-2 (IL-2). Thus, optionally, the modified NK-92® cells are further modified to express at least one cytokine. Optionally, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. Optionally, the at least one cytokine is IL-2, IL-15, or a combination thereof. Optionally, the IL-2 and / or IL-15 are expressed with a signal sequence that directs the cytokine to the endoplasmic reticulum. Directing IL-2 to the endoplasmic reticulum allows for expression of IL-2 at levels sufficient for autocrine activation, without releasing significant amounts of IL-2 outside the cell. See Konstantinidis et al "Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92® cells" Exp Hematol. 2005 Feb;33(2):159-64. An exemplary nucleic acid encoding IL-2 is set forth in SEQ ID NO:14, and an exemplary polypeptide of IL-2 is set forth in SEQ ID NO:15.

[0104] Optionally, the modified NK-92® cells comprise a nucleic acid sequence encoding IL-2 having 70%, 80%, 90%, or 95% identity to SEQ ID NO: 14. Optionally, the modified NK-92® cells comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14. Optionally, the modified NK-92® cells comprise an IL-2 polypeptide having 70%, 80%, 90%, or 95% identity to SEQ ID NO: 15. Optionally, the modified NK-92® cells comprise an IL-2 polypeptide having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. The provided modified NK-92® cells advantageously can be maintained in the absence of IL-2 without secreting amounts of IL-2 that would cause adverse clinical effects.

[0105] In one aspect, the subject matter of the present invention includes modified NK-92® cells capable of modulating the tumor microenvironment. The modified NK-92® cells preferably include a quadracistronic vector comprising one or more nucleic acids encoding i) IL-12 or TGF beta trap, ii) an antigen binding protein (ABP) or chimeric antigen receptor (CAR) that specifically binds to a target antigen, iii) an Fc receptor such as CD16 or CD16-158V, and / or iv) a cytokine such as erIL-2 or erIL-15, wherein the nucleic acid sequence is operably linked to a promoter. In one embodiment, the quadracistronic vectors contemplated herein are shown in Figures 21, 30, and 31, respectively. IL-12 contemplated herein may comprise a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:57 (p35 nt sequence) or SEQ ID NO:59 (p40 nt sequence). IL-12 contemplated herein may also comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:58 (p35 aa sequence, isoform 1 precursor) or SEQ ID NO:60 (p40 aa sequence, precursor).

[0106] In an exemplary embodiment, the IL-12 single chain p40_p35 sequence in the IL-2 / PD-L1 quadricistronic vector may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:61, or may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:62.

[0107] TGF beta traps contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 63 (TGFBRII extracellular domain) or SEQ ID NO: 65 (TGFb trap sequence). TGF beta traps contemplated herein may also comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 64 (TGFBRII extracellular domain) or SEQ ID NO: 66 (TGFb trap sequence). Other suitable TGF beta traps include those described in Mol.Canc.THer.2012,Vol 11(7),1477-1487.

[0108] Additionally, the nucleic acid constructs of the subject invention may also include sequences encoding 2A peptides, such as T2A, P2A, E2A, or F2A peptides, to generate equimolar levels of polypeptides encoded by the same mRNA. E2A peptides contemplated herein may include polynucleotide sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 17. T2A peptides contemplated herein may include polynucleotide sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18.

[0109] In one illustrative and non-limiting example, a plasmid disclosed herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:19 (5' homology arm of AAVS1), SEQ ID NO:20 (EF1a promoter), SEQ ID NO:21 (T7 promoter), SEQ ID NO:22 (CCR7 cDNA), SEQ ID NO:23 (P2A element), SEQ ID NO:24 (IgHC leader), SEQ ID NO:25 (CD19 CAR minus signal peptide), SEQ ID NO:26 (high affinity CD16), SEQ ID NO:27 (IRES), SEQ ID NO:28 (SC40 polyA), SEQ ID NO:29 (3' homology arm of AAVS1), and / or SEQ ID NO:30 (homology arm of AAVS1).

[0110] Chimeric Antigen Receptor Optionally, the modified NK-92® cells are further engineered to express a chimeric antigen receptor (CAR) on the cell surface. Optionally, the CAR is specific for a tumor-specific antigen. Tumor-specific antigens are described, by way of non-limiting example, in US Patent Publication No. 2013 / 0189268; WO 1999024566 A1; US ​​Patent No. 7098008; and WO 2000020460 A1, each of which is incorporated herein by reference in its entirety. Tumor-specific antigens include, but are not limited to, NKG2D, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, CD19, CD33, B7-H4, CD20, and 41BB. The CAR can be engineered, for example, as described in WO2014039523; US20140242701; US20140274909; US20130280285; and WO2014099671, each of which is incorporated herein by reference in its entirety. Optionally, the CAR is a CD19 CAR, a CD33 CAR, or a CSPG-4 CAR. In one illustrative and non-limiting example, CD19CAR_CD3a can comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:41.

[0111] Homing receptors Provided herein is an engineered NK-92® cell comprising a nucleic acid encoding a homing receptor. In some embodiments, the homing receptor is operably linked to a promoter. In some embodiments, the homing receptor is a G protein-coupled receptor. In some embodiments, the homing receptor is a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, or CXCL14 receptors. In some embodiments, the nucleic acid encoding CCR7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1. Optionally, the homing receptor is expressed on the cell surface of the modified NK-92® cell. Optionally, the modified NK-92® cell further comprises a CAR. Optionally, the CAR is CD19. Optionally, the modified NK-92® cell further comprises an Fc receptor. Optionally, the Fc receptor is CD16. Optionally, the modified NK-92® cell further comprises a cytokine, such as IL-2. In some embodiments, the IL-2 polypeptide may have a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:42. In some embodiments, the IL-2 polypeptide may have a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:43.

[0112] Expression vector Provided herein is an expression vector comprising a nucleic acid operably linked to a promoter. Each of the nucleic acids encoding different elements of the vector can be operably linked to the same or different promoters. Exemplary promoters include, but are not limited to, CMV promoter, ubiquitin promoter, PGK promoter, and EF1 promoter. Optionally, provided herein is an expression vector comprising a nucleic acid having SEQ ID NO:1, or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1. Optionally, the nucleic acid is operably linked to a promoter. Optionally, the promoter is selected from the group consisting of SEQ ID NO:3, 9, 10 or 11, or a promoter having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3, 9, 10 or 11. Optionally, the nucleic acid is operably linked to a promoter. Optionally, the promoter comprises SEQ ID NO: 4 and / or SEQ ID NO: 5. Optionally, the promoter comprises SEQ ID NO: 6, or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6.

[0113] In some embodiments, the expression vector provided comprises SEQ ID NO:1 or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1; SEQ ID NO:25 (CD19 CAR) or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:13 (CD16 158V), or a nucleic acid or polypeptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:14 (erIL-2 nt sequence); and / or SEQ ID NO:15 (erIL-2 aa sequence), or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14. In some embodiments, the expression vectors provided include SEQ ID NO: 47 (CXCR2) or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 47; SEQ ID NO: 25 (CD19 CAR) or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12; and SEQ ID NO: 13 (CD16 158V), and / or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14; and / or SEQ ID NO: 15 (erIL-2), or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14. Suitable expression vectors are known in the art and can be used. In a further embodiment, the recombinant nucleic acid comprises a segment encoding erIL-15, and the nucleic acid encoding erIL-15 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 67. Optionally, the expression vector is a plasmid.

[0114] Expression analysis of PD-L1 CAR and CD-16 in PD-L1 (TGFβ trap) t-haNK cells is shown in Figure 22. Furthermore, TGFβ trap is secreted into the culture supernatant of TGFβ trap / PD-L1 t-haNK cells, as shown in Figure 23. Similarly, IL-12 secretion from IL-12 virally transduced NK-92® cell line is shown in the right column of Figure 30.

[0115] Methods for Producing Modified NK-92® Cells Provided herein are methods of making modified NK-92® cells comprising a nucleic acid molecule described herein. The methods include transforming NK-92® cells with an expression vector comprising a nucleic acid molecule described herein operably linked to a promoter.

[0116] As used herein, the terms promoter, promoter element, and control sequence refer to a polynucleotide that regulates the expression of a selected polynucleotide sequence operably linked to a promoter and affects the expression of the selected polynucleotide sequence in a cell. In some embodiments, a promoter element is or includes an untranslated region (UTR) at the 5' position of a coding sequence. The 5'UTR forms part of the mRNA transcript and is therefore an essential part of protein expression in eukaryotes. After transcription, the 5'UTR can control protein expression at both the transcriptional and translational levels. Promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, for example, from the genomes of viruses such as polyoma, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus and cytomegalovirus (e.g., SEQ ID NO: 11), or from heterologous mammalian promoters, such as the beta-actin promoter, the eukaryotic translation elongation factor 1 alpha 1 (EF1α) promoter (e.g., SEQ ID NO: 3), the phosphoglycerate kinase (PGK) promoter (e.g., SEQ ID NO: 10), and the ubiquitin promoter (e.g., SEQ ID NO: 9). Provided herein are promoters having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3, 9, 10, or 11.

[0117] The term "selectable marker" as used herein refers to either a nucleotide sequence, e.g., a gene, that encodes a product (polypeptide) that allows for selection, or the gene product (e.g., polypeptide) itself. The term selectable marker is used herein as generally understood in the art and refers to a marker whose presence in a cell or organism confers a significant growth or survival advantage or disadvantage to the cell or organism under certain defined culture conditions (selective conditions). The term selection agent as used herein refers to an agent that introduces selection pressure on a cell or cell population that favors or opposes a cell or cell population that has the selectable marker. For example, the selection agent is an antibiotic and the selectable marker is an antibiotic resistance gene. Examples of selectable markers suitable for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin.

[0118] Nucleic acid, as used herein, refers to deoxyribonucleotides or ribonucleotides and polymers and their complements. The term includes deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, conservatively modified variants of nucleic acid sequences (e.g., degenerate codon substitutions) and complementary sequences can be used in place of the specific nucleic acid sequences described herein. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0119] A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a presequence or secretory leader is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to promote translation. Generally, operably linked means that the DNA sequences being linked are close to each other, contiguous in the case of a secretory leader, and in reading phase. However, enhancers need not be contiguous. For example, a nucleic acid sequence that is operably linked to a second nucleic acid sequence is directly or indirectly covalently linked to such second sequence, although any effective three-dimensional association is permissible. A single nucleic acid sequence can be operably linked to multiple other sequences. For example, a single promoter can direct the transcription of multiple RNA species. Linkage can be achieved by ligation at convenient restriction sites. If no such sites exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0120] Treatment Methods for treating cancer or tumor in a subject are described herein. As used herein, the term "cancer" refers to all types of cancer, neoplasm, or malignant tumor found in a mammal, including leukemia, carcinoma, and sarcoma. Exemplary cancers include cancer of the brain, breast, cervix, colon, head and neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus, and medulloblastoma. Further examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesion, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine and exocrine pancreas, and prostate cancer.

[0121] As used herein, the terms "metastasis", "metastatic" and "metastatic cancer" may be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer from one organ or another non-adjacent organ or part of the body. Cancer begins at a site of origin, e.g., the breast, which is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and invade normal tissues surrounding the local area, and / or penetrate the walls of the lymphatic or vascular system and circulate through the system to reach other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of the primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the breast site will be composed of abnormal lung cells rather than abnormal breast cells. A secondary tumor in the breast is referred to as metastatic lung cancer. Thus, the term metastatic cancer refers to a disease in which a subject has or has had a primary tumor in the past and has one or more secondary tumors. The term "subject with non-metastatic cancer," or cancer that is not metastatic, refers to disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to disease in a subject who has or has a history of a primary lung tumor and has one or more secondary tumors at a second location or locations, such as in the breast.

[0122] As used herein, "treating" or "treatment" of a condition, disease or disorder, or a symptom associated with a condition, disease or disorder, refers to an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of a condition, disorder or disease, stabilization of the condition, disorder or disease status, prevention of the onset of a condition, disorder or disease, prevention of the spread of a condition, disorder or disease, delay or slowing of the progression of a condition, disorder or disease, delay or slowing of the onset of a condition, disorder or disease, improvement or palliation of a condition, disorder or disease, and partial or total remission. "Treating" may also mean extending the survival of a subject beyond that expected in the absence of treatment. "Treatment" may also mean temporarily inhibiting the progression of a condition, disorder or disease, temporarily slowing the progression of a condition, disorder or disease, but in some cases involves permanently halting the progression of a condition, disorder or disease. As used herein, the terms treatment, treat, or treating refer to a method of reducing one or more symptoms of a disease or condition characterized by expression of a protease or the effects of a disease or condition characterized by expression of a protease. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or a symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if one or more symptoms of the disease are reduced by 10% in a subject compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or a symptom of a disease or condition. Furthermore, as used herein, references to decrease, reduction, or inhibition include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control level, and such terms can, but do not necessarily, include a complete elimination.

[0123] Terms such as subject, patient, individual, etc. are not intended to be limiting and may be generally interchangeable. That is, an individual described as a patient may not necessarily have a specific disease, but may simply be seeking medical advice. As used throughout, a subject may be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, or any other animal. The terms do not indicate a particular age or sex. Thus, adult and neonatal subjects are intended to be subjects, whether male or female. As used herein, patient, individual, and subject may be used interchangeably and these terms are not intended to be limiting. That is, an individual described as a patient may not necessarily have a specific disease, but may simply be seeking medical advice. The terms patient or subject include human and animal subjects.

[0124] As used herein, "administration" or "administering" refers to providing, contacting, and / or delivering one or more compounds by any suitable route to achieve a desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ocular, inhalation, and implant. Optionally, the NK-92® cells are administered parenterally. Optionally, the NK-92® cells are administered intravenously. Optionally, the NK-92® cells are administered peritumorally.

[0125] Thus, provided herein is a method of reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of modified NK-92® cells as described herein. Also provided is a method of treating cancer in a subject, comprising selecting a subject having cancer, and administering to the subject a therapeutically effective amount of modified NK-92® cells as described herein, wherein the administering treats the cancer in the subject. Optionally, the method further comprises administering to the subject an additional therapeutic agent.

[0126] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a modified NK-92® cell described herein, wherein the administration treats cancer or reduces the size of a tumor in the subject. In some embodiments, the method comprises administering to the subject a modified NK-92® cell that comprises a nucleic acid encoding i) an IL-12 or TGFb trap, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor such as CD16 or CD16-158V, and / or iv) a cytokine such as erIL-2 or erIL-15. In some embodiments, the method comprises administering to the subject a modified NK-92® cell that comprises a nucleic acid encoding i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor such as CD16 or CD16-158V, and / or iv) a cytokine such as erIL-2 or erIL-15.

[0127] The cytotoxicity of TGFβ trap / PD-L1 against K562 target cells is shown in Figure 24. Furthermore, SUP-B15 PD-L1+ CAR killing of target cells is shown in Figure 25, and CAR killing of MDA-MB231 target cells is shown in Figure 26. SUP-B15 CD19-CD20+ADCC of TGFβ trap / PD-L1 against PD-L1 is shown in Figure 27. Figure 28 shows that TGFβ / SMAD luciferase reporter HEK293 cells are induced by TGFβ. As shown in Figure 29, secreted TGFβ trap sequestered TGFβ and inhibited luciferase expression in a HEK293T reporter assay. Reporter cells were treated with 1 ng / mL TGFβ1 for 19 hours. Among other options, preferred CAR molecules may specifically bind to PD-L1 and have an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:69 (which may be encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:68).

[0128] NK-92® cells may be administered to a subject by a variety of routes. For example, NK-92® cells may be administered to a subject by infusion (e.g., intravenous infusion) over a period of time. Typically, for a single administration of NK-92® cells, the period is 5-130 minutes. Optionally, the period is 90-120 minutes. Optionally, the period is 15-30 minutes.

[0129] The NK-92® cells, and optionally other anti-cancer agents, can be administered once to a patient with cancer, or multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks, or any range including the endpoints between any two numbers, throughout the treatment period. Thus, for example, NK-92® cells can be administered to a subject once a day for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. Optionally, NK-92® cells are administered once a day in a cycle for 2 days. This cycle is then followed by one or more hours, one or more days, or one or more weeks without treatment with NK-92® cells. As used herein, the term "cycle" refers to treatments that are repeated on a regular schedule with periods of rest (e.g., no treatment or treatment with other agents) in between. For example, one week of treatment followed by two weeks of rest is one treatment cycle. Such a treatment cycle can be repeated one or more times. Thus, the NK-92® cells can be administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cycles.

[0130] NK-92® cells can be administered to a subject in absolute numbers of cells, e.g., the subject can be administered from about 1000 cells / infusion up to about 10 billion cells / infusion, e.g., about, at least about, or at most about 1×10 cells / infusion. 10 , 1×10 9 , 1×10 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×104 , 5×10 4 , 1×10 3 , 5×10 3 NK-92® cells (and so forth), or any range including the endpoints between any two numbers. Optionally, 1×10 8 ~1×10 10 The cells are administered to the subject. Optionally, the cells are administered one or more times per week for one or more weeks. Optionally, the cells are administered once or twice per week for one, two, three, four, five, six, seven, eight, nine, ten or more weeks.

[0131] Optionally, subjects receive approximately 1000 cells / injection / ml 2 up to approximately 10 billion cells / injection / m 2 up to, e.g., about, at least about, or at most about 1×10 per injection 10 / m 2 , 1×10 9 / m 2 , 1×10 8 / m 2 , 1×10 7 / m 2 , 5×10 7 / m 2 , 1×10 6 / m 2 , 5×10 6 / m 2 , 1×10 5 / m 2 , 5×10 5 / m 2 , 1×10 4 / m 2 , 5×10 4 / m 2 , 1×10 3 / m 2 , 5×10 3 / m 2 (etc.) of NK-92® cells, or any range including the endpoints between any two numbers. Optionally, 1 m 2 1 x 10 per 3 ~1×10 101 ml of NK-92® cells are administered to the subject. Optionally, 1 ml 2 2 x 10 per 9 NK-92® cells are administered to the subject.

[0132] Optionally, NK-92® cells can be administered to such individuals in relative numbers of cells, e.g., the individual can be administered from about 1000 cells per kilogram of the individual up to about 10 billion cells per kilogram of the individual, e.g., about, at least about, or at most about 1×10 cells per kilogram of the individual. 10 , 1×10 9 , 1×10 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 (and so on) NK-92® cells, or any range including the endpoints between any two numbers.

[0133] Optionally, the total dose is calculated based on m of body surface area. 2 It can be calculated by 1m 2 Approximately 1 x 10 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 , or any range including the endpoints between any two numbers. Optionally, about 1 billion to about 3 billion NK-92® cells are administered to the patient. Optionally, the amount of NK-92® cells infused per dose is about 1000 μg / m2 of body surface area. 2 It can be calculated by 1m 2 1 x 10 per 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6, 1×10 5 , 1×10 4 , 1×10 3 Includes.

[0134] Optionally, the NK-92® cells are administered in a composition comprising the NK-92® cells and a medium, such as human serum or an equivalent thereof. Optionally, the medium comprises human serum albumin. Optionally, the medium comprises human plasma. Optionally, the medium comprises about 1% to about 15% human serum or a human serum equivalent. Optionally, the medium comprises about 1% to about 10% human serum or a human serum equivalent. Optionally, the medium comprises about 1% to about 5% human serum or a human serum equivalent. Optionally, the medium comprises about 2.5% human serum or a human serum equivalent. Optionally, the serum is human AB serum. Optionally, a serum replacement acceptable for use in human therapy is used in place of human serum. Such serum replacements are known in the art. Optionally, the NK-92® cells are administered in a composition comprising the NK-92® cells and an isotonic solution that supports cell viability. Optionally, the NK-92® cells are administered in a composition reconstituted from a cryopreserved sample.

[0135] According to the methods provided herein, a subject is administered an effective amount of one or more agents provided herein. The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiological response (e.g., reduced inflammation). Effective amounts and schedules for administering agents can be empirically determined by one of skill in the art. For administration, the dosage range is large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. In general, dosages vary with age, condition, sex, type of disease, extent of disease or disorder, route of administration, or whether other agents are included in the regimen, and can be determined by one of skill in the art. Dosages can be adjusted by the individual physician if any contraindications arise. Dosages can vary and can be administered in one or more daily doses over one or several days. Guidance regarding appropriate dosages for a given class of pharmaceutical agent can be found in the literature. For example, for a given parameter, an effective amount indicates an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or greater effect over a control. The exact dosage and formulation will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Editor (2012), and Pickar, Dosage Calculations (1999)).

[0136] Pharmaceutically acceptable compositions may include various carriers and excipients. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable matter. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 22nd Edition, Loyd V. Allen et al., editors, Pharmaceutical Press (2012). Pharmaceutically acceptable carriers refer to materials that are not biologically or otherwise undesirable, i.e., materials that are administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which they are contained. When administered to a subject, carriers are optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject. As used herein, the term pharma-ceutically acceptable is used interchangeably with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions generally include agents for buffering and preservation during storage, and may include buffers and carriers for appropriate delivery depending on the route of administration.

[0137] The compositions may contain acceptable auxiliary substances required for appropriate physiological conditions, such as pH adjusting agents and buffers, as well as toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of cells and / or other agents in these formulations may vary and are selected primarily based on fluid volume, viscosity, weight, and the like according to the particular form of administration selected and the needs of the subject.

[0138] Combination therapy Optionally, the NK-92® cells are administered to the subject in combination with one or more other treatments for the cancer being treated. Without being bound by theory, it is believed that simultaneous treatment of the subject with NK-92® cells and another therapy for the cancer allows the NK-92® cells and the alternative therapy to give the endogenous immune system a chance to eliminate the cancer where it had previously been suppressed by endogenous action. Optionally, the two or more other treatments for the cancer being treated include, for example, antibodies, bispecific engagers, radiation, chemotherapy, stem cell transplant, or hormone therapy.

[0139] Optionally, the antibody is administered to the patient in combination with NK-92® cells. Optionally, the NK-92® cells and the antibody can be administered to the subject together, e.g., in the same formulation; separately, e.g., in separate formulations, at the same time, or separately, e.g., on different dosing schedules or at different times of the day. If administered separately, the antibody can be administered by any suitable route, such as intravenously or orally.

[0140] Optionally, antibodies can be used to target cancerous cells or cells expressing cancer-associated markers. Several antibodies have been approved alone for the treatment of cancer.

[0141] The provided method can further be combined with other tumor therapies, such as radiation therapy, surgery, hormone therapy and / or immunotherapy. Thus, the provided method can further include administering one or more additional therapeutic agents to the subject. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, resuscitatives, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antitumor agents, allosteric inhibitors, anabolic steroids, antirheumatics, psychotherapeutic agents, neuroleptics, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinics, antimycobacteria, antiprotozoal agents, antivirals, dopaminergic agents, hemodynamic agents, immune agents, muscarinics, protease inhibitors, vitamins, growth factors, and hormones. The choice of agent and dosage can be easily determined by one skilled in the art based on the given disease being treated. Optionally, the additional therapeutic agent is octreotide acetate, interferon, pembrolizumab, glucopyranosyl lipid A, carboplatin, etoposide, or any combination thereof.

[0142] Optionally, the additional therapeutic agent is a chemotherapeutic agent. The chemotherapy treatment regimen may include administering to the subject one or a combination of chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, taxanes, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based compounds, retinoids, and vinca alkaloids and derivatives. Optionally, the chemotherapeutic agent is carboplatin.

[0143] Combinations of agents or compositions can be administered either concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines) or sequentially (e.g., one agent is administered first, followed by the second agent). Thus, the term combination is used to refer to the combined, simultaneous, or sequential administration of two or more agents or compositions. The course of treatment is best determined on an individual basis, depending on the particular characteristics of the subject and the type of treatment selected. Treatments such as those disclosed herein can be administered to the subject daily, twice daily, biweekly, monthly, or on any applicable basis that is therapeutically effective. Treatments can be administered alone or in combination with other treatments disclosed herein or known in the art. Additional treatments can be administered simultaneously with the first treatment, at different times, or on completely different treatment schedules (e.g., the first treatment daily and the additional treatment weekly).

[0144] kit Provided herein are kits comprising modified NK-92® cells as described herein. In some embodiments, the kits comprise modified NK-92® cells comprising one or more nucleic acid sequences encoding: i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor, such as CD16 or CD16-158V, and / or iv) a cytokine, such as erIL-2, operably linked to a promoter. Optionally, one or more proteins encoded by the nucleic acid sequences are expressed on the cell surface of the modified NK-92® cells. In some embodiments, the kits comprise modified NK-92® cells comprising a nucleic acid encoding a CC chemokine receptor type 7 (CCR7), CXCR2, or a receptor for CXCL14 operably linked to a promoter. Optionally, the nucleic acid encoding CCR7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1. Optionally, the homing receptor is expressed on the cell surface of the modified NK-92® cells. Optionally, the promoter comprises one or more NFAT binding elements and a minimal promoter. Optionally, the promoter has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6. Optionally, one or more proteins encoded by the nucleic acid sequence are expressed on the cell surface of the modified NK-92® cells.

[0145] Optionally, the modified NK-92® cells are provided in a composition that includes a pharma- ceutically acceptable excipient. Optionally, the kit may include additional compounds, such as therapeutically active compounds or drugs, to be administered prior to, simultaneously with, or after administration of the modified NK-92® cells. Optionally, the kit instructions include directions for using the kit components in the treatment of cancer. The instructions may further include information on how to prepare (in the case of lyophilized proteins, dilution and reconstitution) the antibodies and NK-92® cells (e.g., thawing and / or culturing). The instructions may further include guidance regarding dosage and frequency of administration.

[0146] Disclosed are materials, compositions, and components that can be used in, can be used in conjunction with, can be used in the preparation of, or are products of the disclosed methods and compositions. When these and other materials are disclosed herein and combinations, subsets, interactions, groups, etc. of these materials are described, it is understood that each is specifically contemplated and described herein, although specific descriptions of the various individual and collective combinations and permutations of these compounds may not be explicitly described. For example, when a method is disclosed and discussed, and several modifications that can be made to several molecules that comprise the method are discussed, each and every combination and permutation of the method, and possible modifications, are specifically contemplated, unless there is a specific indication to the contrary. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, when there are various additional steps that can be performed, it is understood that each of these additional steps can be performed by any particular method step or combination of method steps of the disclosed methods, and each such combination or subset of combinations should be considered as specifically contemplated and disclosed.

[0147] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entireties. [1] A modified NK-92 cell comprising a nucleic acid encoding a homing receptor operably linked to a promoter. [2] The modified NK-92 cell according to [1] above, wherein the homing receptor is a G protein-coupled receptor (GPCR), a chemokine receptor, a cytokine receptor, a cell adhesion molecule, a selectin, or an integrin. [3] The modified NK-92 cell according to [2] above, wherein the chemokine receptor is selected from a CCR7, CXCR2, or CXCL14 receptor, and the cell adhesion molecule is selected from L-selectin (CD62L), α4β7 integrin, LPAM-1, and LFA-1. [4] The modified NK-92 cell of [3] above, wherein the nucleic acid encoding CCR7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1. [5] The modified NK-92 cell according to any one of [1] to [4] above, further comprising a nucleic acid encoding an antigen-binding protein operably linked to a promoter. [6] The modified NK-92 cell according to [5] above, wherein the antigen-binding protein specifically binds to a tumor-associated antigen. [7] The modified NK-92 cell of [6] above, wherein the tumor-associated antigen is selected from CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4. [8] The modified NK-92 cell of [5], wherein the antigen-binding protein comprises a chimeric antigen receptor (CAR). [9] The modified NK-92 cell of [8], wherein the CAR specifically binds to CD19 or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[10] The modified NK-92 cell according to any one of [1] to [9] above, further comprising a nucleic acid encoding a cytokine operably linked to a promoter.

[11] The modified NK-92 cell according to

[10] above, wherein the cytokine is IL-2, erIL-2, IL-15, erIL-15, or a combination thereof.

[12] The modified NK-92 cell of

[10] , wherein the cytokine is erIL-2 and the nucleic acid encoding erIL-2 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.

[13] The modified NK-92 cell of

[10] , wherein the cytokine is erIL-15 and the nucleic acid encoding erIL-15 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:67.

[14] The modified NK-92 cell according to any one of [1] to

[13] above, further comprising a nucleic acid encoding an Fc receptor operably linked to a promoter.

[15] The modified NK-92 cell of

[14] above, wherein the Fc receptor is CD16 or high affinity CD16 (SEQ ID NO: 12), or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13.

[16] The modified NK-92 cell according to any one of [1] to

[15] above, wherein the homing receptor, antigen-binding protein, CAR, and / or Fc receptor are expressed on the cell surface of the modified NK-92® cell.

[17] A modified NK-92 cell comprising one or more nucleic acids encoding i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor, and / or iv) a cytokine selected from IL2, IL-15, erIL-2, erIL-15, or a combination thereof, wherein the nucleic acid sequence is operably linked to a promoter.

[18] The modified NK-92 cell of

[17] , wherein the homing receptor is a G protein-coupled receptor (GPCR), a chemokine receptor, a cytokine receptor, a cell adhesion molecule, a selectin, or an integrin.

[19] The modified NK-92 cell of

[17] , wherein the Fc receptor is CD16 or high affinity CD16 (SEQ ID NO: 12), or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13.

[20] The modified NK-92® cell according to any one of [1] to

[15] above, wherein the homing receptor, antigen-binding protein, CAR, and / or Fc receptor are expressed on the cell surface of the modified NK-92® cell.

[21] A composition comprising the modified NK-92 cell according to any one of [1] to

[20] above and a pharma- ceutically acceptable excipient.

[22] A kit comprising the NK-92 cell according to any one of [1] to

[21] above and an instruction manual.

[23] A method for treating cancer or tumor in a subject, the method comprising administering to a subject a therapeutically effective amount of the modified NK-92 cell described in any one of [1] to

[20] above or the composition described in

[21] above, wherein the administration treats the cancer or reduces the size of the tumor in the subject.

[24] A method for reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of the modified NK-92 cell described in any one of [1] to

[20] above or the composition described in

[21] above, thereby reducing cancer metastasis in the subject.

[25] 1m 2 1 x 10 per 3 ~1×10 10 The method according to any one of

[23] to

[24] , wherein the NK-92 cells are administered to the subject.

[26] The method according to any one of

[23] to

[25] , wherein the NK-92 cells are administered parenterally, intravenously, peritumorally, or by infusion.

[27] The method according to any one of

[23] to

[26] , further comprising administering an additional therapeutic agent to the subject.

[28] An inducible promoter for selective and targeted secretion by NK cells that contains an NFAT transcription factor response element.

[29] The inducible promoter according to

[28] , wherein the NFAT transcription factor responsive gene is CCL21.

[30] The inducible promoter according to

[28] , wherein NFAT is activated by the FcεRIγ pathway via activation of CD19 CAR.

[31] The inducible promoter according to

[28] above, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13.

[32] A method for generating modified NK92 cell lines involving targeted homologous recombination at specific gene loci.

[33] The method according to

[32] , wherein the locus is AAVS1.

[34] The method according to

[33] , wherein the AAVS1 locus has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:7.

[35] A modified NK-92 cell comprising a nucleic acid encoding a secreted cytokine that regulates the tumor microenvironment operably linked to a promoter.

[36] The modified NK-92 cell according to

[35] , wherein the secreted cytokine is IL-12 and / or TGF-beta trap.

[37] The modified NK-92 cell according to any one of

[35] to

[36] above, further comprising a nucleic acid encoding an antigen-binding protein operably linked to a promoter.

[38] The modified NK-92 cell according to any one of

[35] to

[37] above, wherein the antigen-binding protein specifically binds to a tumor-associated antigen.

[39] The modified NK-92 cell of

[38] , wherein the tumor-associated antigen is selected from CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.

[40] The modified NK-92 cell of

[38] , wherein the antigen-binding protein comprises a chimeric antigen receptor (CAR).

[41] The modified NK-92 cell of

[40] , wherein the CAR specifically binds to PD-L1 or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:69.

[42] The modified NK-92 cell according to any one of

[35] to

[41] above, further comprising a nucleic acid encoding a second cytokine operably linked to a promoter.

[43] The modified NK-92 cell of

[42] , wherein the second cytokine is IL-2, erIL-2, IL-15, erIL-15, or a combination thereof.

[44] The modified NK-92 cell of

[42] , wherein the second cytokine is erIL-2 or the nucleic acid encoding erIL-2 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.

[45] The modified NK-92 cell according to any one of

[35] to

[44] above, further comprising a nucleic acid encoding an Fc receptor operably linked to a promoter.

[46] The modified NK-92 cell of

[45] , wherein the Fc receptor is CD16 or high affinity CD16 (SEQ ID NO: 12), or a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13.

[47] The modified NK-92 cell according to any one of

[35] to

[46] , wherein the IL-12 and / or TGF beta trap is secreted, and the antigen-binding protein, CAR, and / or Fc receptor are expressed on the cell surface of the modified NK-92 cell.

[48] ​​A modified NK-92 cell comprising one or more nucleic acids encoding i) IL-12 and / or TGF beta trap, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor, and / or iv) a second cytokine selected from erIL-2, erIL-15, IL-2, IL-15, or a combination thereof, wherein the nucleic acid sequence is operably linked to a promoter.

[49] A composition comprising the modified NK-92 cell according to any one of

[35] to

[48] above and a pharma- ceutically acceptable excipient.

[50] A kit comprising the NK-92 cell according to any one of

[35] to

[49] above and an instruction manual for use.

[51] A method for treating cancer or tumor in a subject, comprising administering to the subject a therapeutically effective amount of the modified NK-92 cell according to any one of

[35] to

[48] above or the composition according to

[49] above, wherein the administration treats the cancer or reduces the size of the tumor in the subject.

[52] A method for reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of the modified NK-92 cell described in any one of

[35] to

[48] above or the composition described in

[49] above, thereby reducing cancer metastasis in the subject.

[53] 1m 2 1 x 10 per 3 ~1×10 10 The method according to

[51] or

[52] , wherein the NK-92 cells are administered to the subject.

[54] The method according to any one of

[51] to

[53] , wherein the NK-92 cells are administered parenterally, intravenously, peritumorally, or by infusion.

[55] The method according to any one of

[51] to

[54] , further comprising administering an additional therapeutic agent to the subject.

[56] A method for administering NK cells to an individual, the method comprising administering a first composition of the modified NK-92 cells described in any one of

[35] to

[48] above, and a second composition comprising primary NK cells.

[57] The method of

[56] , wherein the first composition comprises modified NK-92 cells comprising a TGF-beta trap operably linked to a nucleic acid and / or promoter encoding IL-12.

[0148] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims. EXAMPLES

[0149] Example 1. Engineered NK cell lines expressing CCR7, a cytokine that regulates the tumor microenvironment. Modified NK-92® cells were generated by electroporation with the linearized pNKAT-CCR7-LP3 plasmid (Figure 1) using the NEON transfection system (Thermo Fisher Scientific, Waltham, MA) on NK-92® cells. After one week of puromycin selection, the resulting polyclonal population was tested for CCR7 expression and monoclonal cell lines were derived by serial dilution in growth medium supplemented with 5% human serum and IL-2. Modified NK-92® cells contained the EF1α promoter, a polyA-tailed CCR7 gene, and a LoxP-flanked puromycin resistance gene driven by the ubiquitin promoter, all wrapped in homology arms targeting the AAVS1 locus (SEQ ID NO: 7).

[0150] To confirm that expression of CCR7 does not affect NK-92® cells, expression of markers on NK-92® cells was measured. Results are shown in Figure 3. All data in Figure 3 was generated using the Intellicyt iQue Screener Plus. Cells were incubated for 30 minutes at 4°C with either APC-conjugated antibodies against the phenotypic markers listed or the appropriate isotype as a negative control. Cells were then rinsed in PBS+1% BSA, pelleted, and resuspended in 30 uL of PBS+1% BSA. The readout was then gated to remove cell debris from the readout as shown in the top left quadrant, and the percentage of cells above the fluorescence threshold as shown in the top right quadrant was then displayed as two separate heat maps, with the percentage above the "positive" and "highly positive" thresholds in the bottom left and right quadrants, respectively. Figure 3 shows that driving expression of CCR7 does not significantly affect key phenotypic markers associated with the cell line. Specifically, expression of CCR7 does not appear to affect expression of CD54, NKp30, or NKG2D.

[0151] To measure the cytotoxicity of modified NK-92® cells, effector cells (NK-92® cells and modified NK-92® cell clones) were serially diluted in 96-well V-bottom plates, leaving 100k of effector in the highest concentration well, and seven further two-fold dilutions were made across the eight rows of the plate. Stained target cells (K562 (Figure 4), HL-60 (Figure 5)) were then seeded at 10k / well into all wells containing effectors, along with target-only control wells to measure background death. Plates were then spun down briefly and incubated at 37°C, 5% CO2 for 4 hours. Plates were then spun down, supernatants aspirated, and cells were resuspended in PBS with propidium iodide to measure cell death. Cells were then passed through an Intellicyt iQue Screener Plus to measure the percentage of target cells that were also positive for PI staining (distinguished from effectors by that staining). The percentage of dead cells was then compared to the number of naturally dead cells in control wells, and the percentage of cells specifically killed by the effector was calculated. The results are shown in Figures 4 and 5. Figure 4 shows comparable cytotoxicity in the CCR7-upregulated clones compared to the parental cell line versus K562 cells, and Figure 5 shows comparable cytotoxicity of the CCR7-upregulated clones compared to the parental cell line versus HL-60 cells.

[0152] In vitro testing consisted of Boyden chamber assays and using a matrigel layer to block migration. Modified cells expressing CCR7 showed migration towards CCL21 and CCL19 (alternative CCR7 ligands) in these assays. Cells were placed in the upper wells and separated from the lower chamber by a thin layer of matrigel (an ECM-like substrate) coated with 8uM pores. Cells (25k / well) were placed in the upper chamber in reduced serum medium (supplemented with 1% human serum and 500U / mL IL-2) and the same reduced serum medium was used in the lower chamber, either by itself or with the chemokine of interest. In this case, CCL21 was used at 15ng / mL, CCL19 was used at 15ng / mL and SDF-1a was used at 20ng / mL. Each test was performed in triplicate on either NK-92® cells or modified NK-92® cells expressing CCR7. The plates were then placed in an incubator overnight for an 18-hour invasion assay, after which the upper chamber was removed and mixed throughout before 150 μL (out of a total volume of 750 μL) was sampled from the lower wells and read on a MacsQuant FACS analyzer. Live cells in the lower chamber were counted and cell numbers were compared to wells without chemokine to generate an invasiveness index. These numbers were averaged and statistical associations were calculated using a two-tailed t-test. Because the lower wells were sampled without detachment of cells from the lower membrane, cells still attached to the bottom of the ECM are not represented in these numbers, which may lead to differences between CCL19 and CCL21. The results are shown in Figure 7. Specifically, Figure 7 shows a statistically significant increase in invasiveness of modified NK-92® cells expressing CCR7 against CCL19, a CCR7 chemokine. The lack of a statistically significant response to CCL21 may be due to the nature of the assay performed. The assay measures both invasion and subsequent detachment into the ECM, behavior consistent with CCL19 gradient migration. CCL21 induces migration but not detachment from the matrix, requiring an additional step to demonstrate statistically significant invasion potential.

[0153] Example 2. Generation of an NFAT-responsive construct for regulated expression of CCL21. To identify NFAT response elements, a cell line stably expressing an NFAT-based luciferase expression cassette (NR2.2) was electroporated with a linearized construct into NK-92® cells containing a stop region followed by three NFAT response elements (SEQ ID NO:4) and a minimal promoter (SEQ ID NO:5) (which drives the production of firefly luciferase in the presence of activated NFAT). A subset of these cells was then electroporated with mRNA containing an anti-CD19 CAR (an antigen present on Sup-B15 cells, an antigen that is otherwise resistant to killing by NK-92® cells). These cells are represented in the graph on the left as ENR2.2. Cells were then plated in triplicate in the absence or presence of target cells and incubated for periods ranging from 2.5 hours to 24 hours. At the end of the incubation period, the Step 1 reagent of the Promega DualGlo system was added to the wells to activate the luciferase (by providing the substrate luciferin). Results were then read on a SpectraMax i3x plate reader and displayed as the mean with standard deviation calculated in Microsoft Excel. The results are shown in Figures 6A and 6B. NFAT activation was demonstrated in targets that bound K562 in a time-dependent manner, and in targets that bound Sup-B15 only when electroporated with mRNA for CD19-CAR.

[0154] Example 3. Engineered NK cell lines expressing CCR7 and CCL21. The pCRENFAT-CCL21 plasmid is integrated into NK-92® cells containing CCR7 using the LoxP sites embedded in the pNKAT-CCR7-LP3 construct in a recombinase-mediated cassette exchange. Following electroporation of the circular plasmid (pCRENFAT-CCL21), Cre recombinase is transiently expressed, which mediates the exchange of the new LoxP-flanked cassette for the old selection cassette. Selection with blasticidin is used to promote the integration of the new cassette, and monoclonal cell lines are subcloned from the resulting population in the same manner as described above in Example 1 to obtain modified NK-92® cells expressing CCR7 and CCL21.

[0155] To evaluate modified NK-92® cells expressing CCR7 and CCL21, unstained modified NK-92® cells are co-cultured with cells known to cause NFAT activation (K562 or other cell lines) in the lower well of a Boyden chamber, and stained modified NK-92® cells are placed in the upper chamber. If co-culture with a sensitive cell line demonstrates induction of migration, the system is functional.

[0156] Example 4. In vitro cytotoxicity assay using modified NK cell lines expressing CCR7 FIG. 9 shows that NK-92® cells modified to express CCR7 maintain cytotoxicity against target cells following migration in a modified Boyden chamber transwell assay as described in Example 1.

[0157] Example 5. Cell surface expression of CCR7, CD16, and CD19 CARs in NK-92® cells transfected with nucleic acid constructs FIG. 12 shows that modified NK-92® cells transfected with nucleic acid constructs encoding CCR7, CD16, and CD19 CARs express high levels of each protein on the cell surface.

[0158] Example 6. Biodistribution of CCR7-expressing chemokine-responsive NK cells in NSG mice bearing CCL19-positive subcutaneous K562 tumors This study shows that chemokine-responsive aNK cells home to chemokine-expressing target tissues after intravenous administration. Because mouse and human chemokines do not cross-react, we developed a localized ligand-expressing tumor model as a surrogate model.

[0159] Experimental method (Table 1): A. Animals: i. Animal type: NSG mouse (JAX), female, 7-8 weeks old ii. Number of animals: 30 (28 animals that received NK cell injections [24 + 4 extra]; 2 additional mice did not receive NK treatment and were used as negative controls for flow cytometry) b. Tumor model: i. Cell lines: K562, parent and CCL19 expressing subline, K-19 ii. Inoculation route: subcutaneous; parental K562 in the left flank and K-19 in the right flank iii. Inoculum: 1E6 cells in 100 μL serum-free medium / Matrigel (v / v 1:1) iv. Tumor burden at the start of treatment: average of 107 mm for K-19 3 ; Parent K562 average 135mm 3 v. Randomization: Animals were primarily randomized based on K-19 tumor volume. c. Test substance: i.NK cells: 1.CD19 t-haNK(Non-CR)(NantKwest Torrey Pines) 2.Quandracistronic Mi-aNK R7-19.1(NantKwest Woburn) ii. Fluorescent labeling: 1. Both types of NK cells were labeled with CFSE according to the manufacturer's manual immediately prior to in vivo administration. 2. At each time point, cultured CFSE-labeled cells were harvested and used as a positive control for flow cytometry. iii. Method of administration: Intravenous iv.Dosage: 1.1E7 cells / mouse v. Dosage frequency: Single dose d.Tumor collection: i. Time points: 3, 24 and 48 hours (± 2 hours) after administration ii. N=4 mice / group / time point iii. Tumor processing: Harvested tumors were dissociated into single cell suspensions according to our in-house protocol (appended) and subjected to flow cytometric enumeration of CFSE-positive NK cells. e. Formulas and statistical analysis: Tumor volume = length x width 2 / 2 (length and width are the longest and shortest diameters of the tumor, respectively) ii. Statistical analysis was performed by one-way ANOVA followed by multiple comparisons with Tukey's test using GraphPad Prism version 7.0. P<0.05 was considered statistically significant.

[0160] [Table 1]

[0161] result: a. Safety: iii. Animals receiving both types of NK cells showed mild to moderate acute reactions immediately after cell infusion (G1-G2, mild to marked depression, lethargy, sluggish, or unresponsive). Two (out of 14) animals in the iv.R7-19.1 group were found dead within 24 hours of injection, whereas there were no deaths in the CD19 t-haNK group. After 24 hours, animals in the CD19 t-haNK group were able to recover, whereas animals in the R7-19.1 group continued to appear mildly depressed and less responsive to stimuli (G1). They became more responsive at 48 hours (G0), but still appeared to have ruffled fur and tachypnea. b.NK cell homing: vi. The number of tumor-infiltrating NK cells at each time point is shown in Table 2 and graphically depicted in FIG. 1. The four CCR7 receptor-CCL19 ligand combinations are as follows: a.CD19 t-haNK®, K562 tumor: no receptor - no ligand [--]; b. CD19 t-haNK®, K-19 tumor: no receptor - ligand present [-+]; c. R7-19.1 cells, K562 tumor: receptor present - no ligand [+-]; and d.R7-19.1 cells, K-19 tumor: Receptor present - Ligand present [++] vii.After 3 hours, homing of R7-19.1 cells to K-19 tumors [++] was significantly greater than that of non-CR CD19 t-haNK cells to CCL19- or CCL19+ tumors ([--] and [-+], respectively; P<0.05). However, we were unable to recover many cells from two of the four K562 tumors in group B, so we could not directly compare the homing of R7-19.1 cells to CCL19-negative and -positive tumors in the same animals. viii.After 24 hours, there were no statistically significant differences in NK cell homing between any of the four CCR7 receptor-CCL19 ligand combinations. However, when comparing homing of each NK cell line to CCL19+ and -tumors within the same animals, three of four animals receiving R7-19.1 cells showed improved infiltration into CCL19+ tumors, while the majority of animals receiving CD19 t-haNK® showed similar levels of NK infiltration to both tumors, regardless of CCL19 expression (Figure 14). After 48 hours, the total number of tumor-infiltrating NK cells was reduced in all combinations, with no difference between any of the groups.

[0162] [Table 2]

[0163] This example shows that at 3 hours post-administration, coexistence of the CCR7 receptor and CCL19 ligand resulted in more efficient NK cell infiltration. At 24 hours post-administration, in animals receiving non-CR aNK cells, NK cells showed similar levels of tumor homing, regardless of CCL19 expression status, in 3 out of 4 animals. In contrast, in animals receiving R7-19.1 cells, NK cells were able to home more efficiently to CCL19-positive tumors, in 3 out of 4 animals, compared to the parental non-ligand-expressing control. The number of tumor-infiltrating NK cells decreased at 48 hours, regardless of receptor or ligand expression. There was a tendency for R7-19.1 cells to home to CCL19 tumors, especially at early times (<48 hours post-administration), suggesting higher exposure and potentially stronger cytotoxicity when the cells are used in a therapeutic setting.

[0164] Example 7. Comparative efficacy evaluation of CCR7-expressing R7-19.1 cells in intravenous CCL19-positive RAJI tumor-bearing NSG mice CCR7 is a chemokine receptor that induces cell migration towards a gradient of the chemokines CCL19 and CCL21 and is typically expressed in lymph nodes and other lymphoid organs, which are the main disease manifestation sites of B-cell lymphoma. Based on the t-haNK platform, chemokine-responsive NK-92® cells (i.e., R7-19.1 cells) expressing CD19-CAR, CCR7, CD16.158V, and ERIL-2 were generated. In addition to CCR7 expression, these cells carry cancer-targeting chimeric antigen receptors (CARs) for CD19 cancer antigen, CD16 variant, and ER-IL-2. Previous distribution studies have shown preferential homing of R7-19.1 cells to CCL19-expressing subcutaneous (SC) tumors compared to their parental counterparts.

[0165] In this study, the antitumor efficacy of repeated intravenous (IV) administration of R7-19.1 cells was evaluated in an IV xenograft model of Raji-19.5, Raji human Burkitt's lymphoma cells genetically engineered to express CCL19, in NSG mice. Non-CCR7-expressing CD19 t-haNK cells (NK-92® [anti-CD19-CAR, CD16.158V, ERIL-2]) were used as a control NK cell line. A vehicle control group was also included.

[0166] While both NK cell lines demonstrated significant treatment efficacy in extending survival of IV Raji-19.5 tumor-bearing animals when compared to vehicle controls, treatment with R7-19.1 cells provided a significantly greater survival benefit than CD19 t-haNK cells. Although clear treatment-related reactions were observed with both NK cell lines, these reactions are speculated to be mouse-specific issues related to the administration of relatively high doses of human-derived cells.

[0167] Rationale and objectives of the study: In vivo distribution data showed that IV-administered R7-19.1 cells exhibited increased homing to CCL19-expressing SC tumors. In this study, the antitumor efficacy of repeated IV administration of R7-19.1 cells was evaluated in an IV xenograft model of Raji-19.5 (a CCL19-expressing subline of Raji) in NSG mice. Note that the original study protocol included additional animal groups (groups A-C) that are not included in this report. These are not relevant to determining the efficacy of R7-19.1 cells in this tumor model (see Table 3 for a simplified experimental design).

[0168] Research materials: Test Articles. The test articles were R7-19.1 cells and CD19 t-haNK cells (non-CCR7 expressing control NK cells; clone 6). R7-19.1 cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68. For CD19 t-haNK cells (non-CCR7 expressing control NK cells; clone 6), CD19 t-haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68. Serum-free growth medium was used as a vehicle control.

[0169] Test system: Test animals: NOD.Cg-Prkdc mice, aged 10-11 weeks at the start of the study (after quarantine and acclimation) and weighing 20-27 grams at randomization. scid Il2rg tm1Wjl / SzJ (NSG) female mice were used. The number of animals used in the study was 30, and the source was The Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US). The animals were identified by ear tag, cage number, and tail mark number.

[0170] Raji-19.5 tumor model (cancer cell line) Cell culture medium: Raji-19.5 cancer cells were grown in ATCC formulated modified RPMI-1640 medium supplemented with 10% fetal bovine serum.

[0171] Cell harvest: Log-phase Raji-19.5 cells (passage 16) were harvested by centrifugation according to NantKwest's SOP_Suspension Cancer Cell Collection for In Vivo Studies. The cells were then washed and diluted with 5 × 10 5 The cells were resuspended in serum-free medium at a concentration of 1000 cells / mL and stored on ice before inoculation into animals. Cells used in the in vivo studies had a viability of 97%.

[0172] Inoculation: 1 × 10 in a volume of 0.2 mL for 30 mice 5 Inoculate cells via the lateral tail vein This was defined as day 0.

[0173] Experimental procedure Body weight: Animals were weighed prior to enrollment (after quarantine / acclimation), prior to randomization, on each dosing day (but not prior to dosing), on each post-dosing day, and prior to euthanasia. Animals exhibiting greater than 20% weight loss compared to baseline (day 0) weight were euthanized in accordance with institutional IACUC policy and subsequently necropsied at the discretion of the principal investigator.

[0174] Clinical Observations: Animals were observed daily for mortality / morbidity (G0–G4; see Table 2 of the study protocol in Appendix 1). Moribund and paralyzed animals were euthanized and subsequently necropsied at the discretion of the principal investigator.

[0175] Randomization: On day 3 (3 days after tumor cell inoculation), 30 tumor-bearing mice were pseudo-randomized into 3 study groups of 10 based on the animals' weights.

[0176] Test article administration: Twice weekly (days 3, 6, 10, 13, 17, 20, 24, and 27) for four consecutive weeks, log-phase expanded R7-19.1 and control CD19 t-haNK cells were harvested by centrifugation and cultured at 5 × 10 7 At a concentration of cells / mL, 1 × 10 per mouse in an injection volume of 200 μL. 7 The cells were formulated for IV administration at doses of 100-200 mg / mL. All cell handling and compounding procedures were performed at room temperature. Cell viability was greater than 80% for all dose preparations. As shown in Table 3, group D received vehicle control, and groups E and F received CD19 t-haNK cells and R7-19.1 cells, respectively.

[0177] Endpoints: Animals were euthanized when they became paralyzed, moribund, or met other endpoint criteria defined by the institution's IACUC. The experiment was terminated on day 30 when the last surviving animal died of disease. Euthanasia was performed by CO2 inhalation followed by cervical dislocation. Within the discretion of the principal investigator, some of the euthanized animals were necropsied to identify visible tumor nodules in internal organs. A complete record of mortality and death events, as well as necropsy findings, can be found in Appendix 5.

[0178] [Table 3]

[0179] Data Analysis: Weight curves: Weight curves were analyzed by two-way ANOVA (or mixed-effects analysis if there were missing values; see correction 2 in Appendix 1) followed by multiple comparisons with Tukey's test.

[0180] Survival curves: Survival curves were analyzed by the log-rank (Mantel-Cox) test.

[0181] Statistical Analysis: All statistical analyses were performed using GraphPad Prism version 8. P<0.05 was considered statistically significant.

[0182] result Efficacy: The primary measure of efficacy was the survival rate of animals. Mortality events were counted if animals were euthanized due to morbidity, paralysis, or weight loss of more than 20%. No dead animals were observed. As shown in Figure 15 and Table 4, both treatment with R7-19.1 and treatment with control CD19 t-haNK cells could significantly prolong the survival of Raji-19.5 IV tumor-bearing animals when compared to vehicle control (P<0.0001 for R7-19.1; P=0.0002 for CD19 t-haNK by log-rank test), with median survival times increased by 6.5 and 2.5 days, respectively. This corresponds to a 30% and 12% increase, respectively, over the vehicle control. More importantly, expression of the CCR7 chemokine-responsive receptor on R7-19.1 cells increased median survival by an additional 4 days (17% improvement) compared to CD19 t-haNK cells (P<0.0001).

[0183] [Table 4]

[0184] safety As shown in Figure 16, animals treated with NK cells consistently showed a 5-10% weight loss after each treatment dose. In most cases, animals were able to recover from cell injection and showed weight recovery, resulting in fluctuating weight change curves between doses. However, the first dose appeared to cause the most severe reaction and was associated with the longest recovery time in both clinical symptoms and weight change. Such reactions are not uncommon in animals receiving NK intravenous infusions and are naturally not specific to R7-19.1 cells. Weight recovery suggests that the weight loss was temporary and reversible.

[0185] Of note, towards the end of the study, animals showed a rapid loss of body weight, likely due to disease progression (Figure 16). Necropsy revealed tumor nodules in the liver, ovaries, and occasionally the spleen in almost all animals examined. One exception was the first mouse euthanized in the CD19 t-haNK cell group, which reached a weight loss of more than 20% the day after the sixth dose. No visible tumor nodules were identified during necropsy of this animal. Thus, the exact cause of weight loss justifying euthanasia could not be determined.

[0186] conclusion Freshly prepared R7-19.1 cells at 1 × 10 7 At this dose level of cells / dose, IV administration twice weekly for 4 weeks demonstrated striking and statistically significant antitumor efficacy in the IV Raji-19.5 xenograft model. This treatment resulted in an increase in median survival of 6.5 days (30%) compared to the vehicle control group and 4 days (17%) compared to the CD19 t-haNK-treated group.

[0187] Although clear treatment-related reactions were observed, these reactions were transient and surviving animals showed signs of recovery. These reactions are speculated to be mouse-specific issues related to the administration of relatively high doses of human-derived NK cells and are therefore unlikely to translate to humans.

[0188] Overall, CCR7-expressing R7-19.1 cells demonstrated significant therapeutic efficacy in this IV model of Raji-19.5 compared to vehicle and their non-chemokine responsive counterparts.

[0189] Example 8. Comparative efficacy evaluation of CCR7-expressing R7-19.1 cells in NSG mice bearing subcutaneous CCL19-positive RAJI tumors CCR7 is a chemokine receptor that induces cell migration towards a gradient of the chemokines CCL19 and CCL21 and is typically expressed in lymph nodes and other lymphoid organs, which are the main disease manifestation sites of B-cell lymphoma. Based on the t-haNK platform, chemokine-responsive NK-92® cells (i.e., R7-19.1 cells) expressing aCD19-CAR, CCR7, CD16.158V, and ERIL-2 were generated. In addition to CCR7 expression, these cells carry cancer-targeting chimeric antigen receptors (CARs) for CD19 cancer antigen, CD16 variant, and ER-IL-2. Previous distribution studies have shown preferential homing of R7-19.1 cells to CCL19-expressing subcutaneous (SC) tumors compared to their parental counterparts.

[0190] In this study, the antitumor efficacy of repeated intravenous (IV) administration of R7-19.1 cells was evaluated in a SC xenograft model of Raji-19.5, Raji human Burkitt's lymphoma cells genetically engineered to express CCL19, in NSG mice. Non-CCR7-expressing CD19 t-haNK cells (NK-92® [anti-CD19-CAR, CD16.158V, ERIL-2]) were used as a control NK cell line. A vehicle control group was also included.

[0191] In a subpopulation of tumor-bearing animals, both NK cell treatments were able to demonstrate clear effects in suppressing tumor growth, with R7-19.1 treatment demonstrating stronger inhibition than control CD19 t-haNK cells. Although clear treatment-related reactions were observed with both NK cell lines, these reactions are speculated to be mouse-specific issues related to the administration of relatively high doses of human-derived cells.

[0192] Rationale and Objective of the Study: In vivo distribution data demonstrated that IV-administered R7-19.1 cells exhibited increased homing to CCL19-expressing SC tumors. In this study, the antitumor efficacy of repeated IV administration of R7-19.1 cells was evaluated in IV and SC xenograft models of Raji-19.5 (a CCL19-expressing subline of Raji) in NSG mice.

[0193] research materials Test Articles: R7-19.1 cells (clone: ​​and CD19 t-haNK cells (non-CCR7 expressing control NK cells; clone 6) were used as test articles and growth medium was used as vehicle control.

[0194] R7-19.1 cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68.

[0195] CD19 t-haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68.

[0196] Test system: NOD.Cg-Prkdc mice, 10-11 weeks old at the start of the study (after quarantine and acclimation) and weighing 19-28 grams on the day of tumor implantation. scid Il2rg tm1Wjl Thirty 10 / SzJ(NSG) female mice were used in the study. They were supplied by The Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US). Animals were identified using ear tags, cage numbers, and tail mark numbers.

[0197] Raji-19.5 tumor model (cancer cell line) Cell culture medium: Raji-19.5 cancer cells were cultured in ATCC formulated modified RPMI-1640 medium supplemented with 10% fetal bovine serum.

[0198] Cell harvest: Log-phase Raji-19.5 cells (passage 16) were harvested by centrifugation according to NantKwest's SOP_Suspension Cancer Cell Collection for In Vivo Studies. The cells were then washed and resuspended in serum-free medium, then mixed with an equal volume of Matrigel and cultured at 2.5 × 10 6 A final concentration of 1000 cells / mL was reached. Cells were stored on ice before inoculation into animals. Cells used in in vivo studies had a viability of 97%.

[0199] Inoculation: 30 animals, 2.5 × 10 in a volume of 100 μL 5 Cancer cells were inoculated unilaterally on the right flank. The skin was shaved prior to injection.

[0200] Experimental procedure Tumor volume measurements: After SC tumor implantation, animals were examined at least twice a week for tumor establishment. When tumors became measurable, tumor volumes (TV) were measured twice a week with digital calipers and calculated using the following formula: TV=length×width. 2 / 2[length is the maximum diameter of the tumor and width is the minimum diameter of the tumor]. 2000mm 3 Animals with tumor volumes exceeding 100 μg / kg or with ulcerated tumors were euthanized according to the institution's IACUC policy and subsequently necropsied. Tumor growth inhibition (TGI) was calculated as follows: TGI=(T C -T t ) / ΔT C ×100% (in the formula, T C and T t are the mean tumor volumes of the control and treated groups at a particular time point, respectively; ΔT C is the change in mean tumor volume in the control group).

[0201] Body weight: Animals were weighed prior to enrollment (after quarantine / acclimation), prior to randomization, on each dosing day (but not prior to dosing), on each post-dosing day, and prior to euthanasia. Animals that showed more than 20% weight loss compared to baseline (day 1) weight were euthanized in accordance with institutional IACUC policy and subsequently necropsied.

[0202] Clinical Observations: Animals were observed daily for mortality / morbidity (G0-G4; see Table 5). Moribund or paralyzed animals were euthanized and subsequently necropsied.

[0203] Randomized: Mean tumor volume was 190 mm 3When tumor volume reached 100 mm, 30 mice were pseudorandomized into three study groups with 10 mice per group to achieve similar tumor volumes between groups, which was defined as day 1. Of note, due to variability in tumor size due to randomization, each group contained two subpopulations: 4–5 animals had large (>200 mm) tumors; 3 ) tumors, 5–6 had small (<200 mm 3 ) tumors (see FIG. 17).

[0204] Test article administration: Twice weekly (days 1, 4, 9, 12, and 15) for 4 consecutive weeks, log-phase grown R7-19.1 and control CD19 t-haNK cells were harvested by centrifugation and cultured at 5×10 7 At a concentration of cells / mL, 1 × 10 per mouse in an injection volume of 200 μL. 7 The cells were formulated for IV administration at doses of 100-200 mg / mL. All cell handling and compounding procedures were performed at room temperature. Cell viability was greater than 80% for all dose preparations. As shown in Table 5, Group A received vehicle control, while Groups B and C received CD19 t-haNK cells and R7-19.1 cells, respectively.

[0205] Endpoints: Animals were euthanized when they reached any of the above endpoints defined by the institution's IACUC. Euthanasia was performed by CO2 inhalation followed by cervical dislocation. At the discretion of the principal investigator, some of the euthanized animals were necropsied to identify visible tumor nodules in internal organs.

[0206] [Table 5]

[0207] 4. Data Analysis Calculate tumor volume: tumor volume = length x width 2 / 2 (length and width are the longest and shortest diameters of the tumor, respectively).

[0208] Calculation of tumor growth inhibition (TGI): TGI = (T C -Tt ) / ΔT C ×100% (in the formula, T C and T t are the mean tumor volumes of the control and treated groups at a particular time point, respectively, and T C is the change in mean tumor volume in the control group).

[0209] Statistical analysis of tumor growth and body weight curves: Tumor growth and body weight curves were analyzed by two-way ANOVA (or mixed-effects analysis in case of missing values; see correction 2 in Appendix 1) followed by multiple comparisons with Tukey's test. All statistical analyses were performed using GraphPad Prism version 8. P<0.05 was considered statistically significant.

[0210] result Efficacy: The primary measurement of this study is tumor growth inhibition. SC tumors with different initial tumor volumes may have differently developed vasculature and CCL19 gradients, which may affect the distribution of test substances to the tumor. Furthermore, small and large tumors may respond to NK cell treatment in different ways. For these reasons, the two subpopulations (i.e., large and small tumors) are analyzed separately.

[0211] As shown in FIG. 18, in the subpopulation of animals bearing large primary tumors, treatment with both NK cell lines significantly improved survival compared to vehicle controls (tumor volumes of 2000 mm 3 The R7-19.1 group showed clear tumor growth inhibition when compared to the non-CCR7 expressing R7-19.1 group (three of four animals had to be euthanized on day 9 due to tumor size exceeding 100 μg / kg or the presence of ulcerated tumors). In contrast, in the NK cell treated groups, the majority of animals survived until days 12 or 15. More importantly, at day 15, there was a superficial enhancement of tumor growth inhibition in the R7-19.1 group when compared to their counterparts not expressing CCR7, achieving a TGI of 35%. This difference did not reach statistical significance, possibly due to the small size of the cohort (N of 2 and 4 for the CD19 t-haNK and R7-19.1 groups, respectively).

[0212] However, for animals that bore small tumors at the start of treatment, neither NK cell therapy was effective in suppressing tumor growth when compared to vehicle controls or compared to each other (Figure 19). This may be due to three factors: 1) small tumors had underdeveloped vasculature, which may have led to low exposure / accessibility to test articles, resulting in the failure of both NK cell therapies; 2) CCR7-mediated chemotaxis requires a gradient of chemokines, rather than responding to the mere presence of chemokines. Such a gradient of CCL19 may have been understated in small tumors, thereby lacking distinguishability between R7-19.1 cells and CD19 t-haNK controls; and, 3) there were several "outliers" in tumor growth in these already small cohorts, further hindering statistical analysis.

[0213] As shown in Figure 20, animals treated with NK cells typically showed a 5-10% weight loss after each treatment dose. In most cases, animals were able to recover from cell injection and showed weight recovery, resulting in fluctuating weight change curves between doses. However, the first dose appeared to cause the most acute and severe reaction and was associated with the longest recovery time in both clinical symptoms and weight change. Such reactions are not uncommon in animals receiving NK intravenous infusions and are of course not specific to R7-19.1 cells. Weight recovery suggests that the weight loss was temporary and reversible.

[0214] conclusion Freshly prepared chemokine-responsive R7-19.1 cells were cultured at 1 × 10 7At the cell / dose dose level, IV administration twice weekly for 4 weeks produced clear tumor growth inhibition in animals bearing large SCRaji-19.5 tumors, although such effects did not reach statistical significance, likely due to the small size of the cohort. This treatment regimen did not demonstrate therapeutic efficacy in animals with small starting tumor volumes. This may be due to underdeveloped tumor vasculature and / or poor establishment of chemokine gradients in small tumors. Clear treatment-related responses were observed. However, these responses were transient, and animals showed signs of recovery. These responses are speculated to be mouse-specific issues associated with administration of relatively high doses of human-derived NK cells, and therefore are unlikely to translate to humans.

[0215] Example 9. Generation of aTGFβ / PD-L1 CAR-modified NK-92® cells A TGFβ trap, composed of a single-chain dimer of the extracellular domain of TGFβRII, was cloned into a quadricistronic plasmid vector that also contained the PD-L1 CAR, CD16, and erIL-2 transgenes (Figure 21). The quadricistronic plasmid was electroporated into aNK cells to generate modified NK-92 cells. The modified NK-92® cells were selected by IL-2 depletion medium, as non-transformed aNK cells, which are IL-2 dependent, could not survive in IL-2 depletion medium. Figure 22 shows that aTGFβ / PD-L1 CAR modified NK-92® cells co-express high levels of PD-L1 CAR and CD16.

[0216] Limiting dilution cloning An aliquot of the polyclonal aTGFβ / PD-L1 t-haNK™ pool culture was diluted to a density of 3 cells / ml in growth medium without the addition of IL-2. The cell suspension was dispensed into 96-well plates in a volume of 200 μl per well, corresponding to an average of 0.6 cells per well. Plates were incubated at 37° C. for 10 days and then visually checked for cell growth. Clones were picked and transferred to larger vessels for expansion and characterization.

[0217] bioanalytical method Cell culture: Polyclonal and clonal aTGFβ / PD-L1 t-haNK™ cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum without IL-2.

[0218] aNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 500 IU / ml recombinant human IL-2.

[0219] haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum without IL-2.

[0220] K562 and MDA-MB-231 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic / antimycotic cocktail. K562 cells were passaged every 2–5 days or whenever the cultures appeared yellow.

[0221] SUP-B15 PD-L1+ and SUP-B15 CD19KO / CD20+ Cells were cultured in RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum, 55 uM beta-mercaptoethanol, and an antibiotic / antimycotic cocktail, otherwise cells were passaged as K562 cells above.

[0222] Antibody staining for flow cytometry analysis: Cells were harvested by centrifugation, washed twice with FACS buffer (5% FBS in 1XD-PBS) and resuspended in 1 ml of FACS buffer. For direct fluorophore-conjugated antibody staining of surface proteins, cells were incubated with the appropriate conjugated antibody (or isotype control) for 20 minutes at 4°C in the dark and then washed twice with FACS buffer. To detect CAR protein, cells were incubated with biotinylated anti-F(ab')2 fragment antibody followed by streptavidin-APC antibody. Samples were analyzed on a MACSQuant flow cytometer.

[0223] Cytotoxicity: Suspension grown cell lines were resuspended by pipetting the cell culture. Cell viability was determined by automated counting (trypan blue exclusion method). Target cells were labeled with CFSE dye and target and effector cells were diluted to the required cell concentration in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotic / antimycotic. Effector and target cells were mixed in 96-well plates at different effector-to-target ratios (E:T of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, 0.31:1, 0.15:1) and co-cultured for 4 h in a 37°C incubator under a 5% CO2 atmosphere. PI was then added for fluorescent labeling of dead cells and the assay was analyzed on a MACSquant flow cytometry instrument.

[0224] ADCC: Suspension-grown cell lines were resuspended by pipetting the cell culture. Cell viability was determined by automated counting (trypan blue exclusion method). Target cells were labeled with PKH67-GL dye, and target and effector cells were diluted to the required cell concentration in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotic / antimycotic. Target cells were preincubated for 30 min at room temperature with monoclonal antibodies trastuzumab, rituximab, or no antibody. Antibody-labeled target cells (and no antibody control) were then mixed with effector cells at different effector-to-target ratios (E:T of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, 0.31:1, 0.15:1) in 96-well plates and co-cultured for 4 h in a 37°C incubator under a 5% CO2 atmosphere. PI was then added for fluorescent labeling of dead cells and the assay was analyzed on a MACSquant flow cytometry instrument.

[0225] Quantification of TGFβ trap secreted from aTGFβ / PD-L1 t-haNK cells Sample supernatants for analysis were prepared with a first centrifugation step at 500 × g for 5 min to remove cells, followed by a second centrifugation at 2000 × g for 5 min to remove cell debris. Sample supernatants were frozen at -80 °C until analysis. Cell pellets from the 500 × g centrifugation step were resuspended, triplicates pooled, and cell density recorded. The concentration of TGFβ TRAP in sample supernatants was measured using a human TGFβ RII ELISA detection kit according to the manufacturer's instructions and compared to the provided standard. TGFβ TRAP concentrations were normalized to cell number and expressed as pg / ml / 10 6 Figure 23 shows that all aTGFβ PD-L1 t-haNK clones expressed large amounts of TGFβ trap (approximately 6 to 13 ng / ml / 10 6 This indicates that the antibody secreted the IgG1-dependent agonist (IgG1-dependent agonist) from the cells.

[0226] Cytotoxicity of aTGFβ / PD-L1 t-haNK™ cells against target cell lines The cytotoxicity of aTGFβ / PD-L1 t-haNK™ cells was evaluated by comparing the target cells K562 cells, SUP-B15 PD-L1+ The aTGFβ / PD-L1 t-haNK™ cells were analyzed by incubating with K562 cells, MDA-MB-231 cells, and MDA-MB-231 cells. Figure 24 shows that aTGFβ / PD-L1 t-haNK™ cells maintained comparable cytotoxicity to parental aNK cells in killing K562 cells (target cells).

[0227] Figure 25 shows that aTGFβ / PD-L1 t-haNK™ cells exhibited enhanced specific killing of the aNK™-resistant, PD-L1 positive SUP-B15 cell line. Approximately 70% of the cells were killed by aTGFβ / PD-L1 t-haNK™ cells compared to only approximately 10% of the cells killed by aNK cells at an effector to target ratio of 8.

[0228] Figure 26 shows that aTGFβ / PD-L1 t-haNK™ cells exhibited enhanced specific killing of the MDA-MB-231 cell line. Approximately 90% of the cells were killed by aTGFβ / PD-L1 t-haNK™ cells compared to only approximately 40% of the cells killed by aNK cells at an effector to target ratio of 8.

[0229] FIG. 27 shows the results of SUP-B15 in combination with anti-CD20 rituximab monoclonal antibody or with anti-Her2-neu trastuzumab monoclonal antibody. CD19KO / CD20+ Figure 1 shows the ADCC activity of aTGFβ / PD-L1 t-haNK™ cells against resistant SUP-B15 cells (CD19-, CD20+, Her2-neu-, NK resistant) in a 4-hour cytotoxicity assay. CD19KO / CD20+ Both the haNK® and aTGFβ / PD-L1 t-haNK™ clones were able to effectively target and kill the target SUP-B15 when combined with the anti-Her2 / neu control antibody trastuzumab. CD19KO / CD20+ Could not kill the cells.

[0230] Example 10. TGFβ trap secreted from modified NK-92® cells inhibits TGFβ activity HEK293 cells engineered with a TGFβ response element (SMAD-linked promoter) to direct expression of a luciferase reporter gene show a dose-dependent increase in luciferase activity when treated with TGFβ (FIG. 28).

[0231] Figure 29 shows that TGFβ induction of luciferase activity in HEK293 reporter cells can be inhibited by co-culture with culture supernatants of aTGFβ / PD-L1 t-haNK cells, whereas culture supernatants from haNK control cells have limited effect on luciferase activity.

[0232] Example 11. Production of IL-12 by modified NK-92® cells NK-92 cells were transduced with lentiviral constructs encoding functional IL-12 p70 dimers as single polypeptide chains in either p35-p40 or p40-p35 orientation (Figure 30). After neomycin selection, transduced NK-92 cells were able to secrete detectable levels of p70 IL-12. Addition of the 2A peptide to the C-terminus of the IL-12 dimer did not affect protein secretion.

[0233] Generation of IL-12 / PD-L1 CAR-modified NK-92® cells The single chain dimer of IL-12 (scIL-12 p70) was cloned into a quadricistronic plasmid vector that also contained the PD-L1 CAR, CD16, and erIL-2 transgenes. The quadricistronic plasmid (Figure 31) was electroporated into aNK cells to generate modified NK-92 cells. The modified NK-92® cells were selected by IL-2 depletion medium because non-transformed aNK cells, which are IL-2 dependent, cannot survive in IL-2 depletion medium. Figure 33 shows that the IL-12 / PD-L1 CAR modified NK-92® cells were able to secrete significant amounts of scIL-12p70 cytokine.

[0234] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0235] It will be apparent to those skilled in the art that many more modifications beyond those already described are possible without departing from the inventive concept herein. Thus, the subject matter of the present invention should not be limited except as set forth in the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not expressly referenced. If a claim of the specification refers to at least one selected from the group consisting of A, B, C... and N, the sentence should be interpreted as requiring only one element from the group, not A and N, or B and N, etc.

Claims

1. a recombinant modified natural killer (NK)-92 cell stably transfected with a nucleic acid; the recombinant modified NK-92 cells have American Type Culture Collection (ATCC) deposit number CRL-2407; the nucleic acid encodes an anti-cluster of differentiation 123 (CD123) chimeric antigen receptor (CAR), an Fc receptor, and a cytokine; The recombinant modified cell expresses the anti-CD123 CAR and the Fc receptor on the cell surface of the recombinant cell. Recombinant modified NK-92 cells.

2. The recombinant modified NK-92 cell of claim 1, wherein the Fc receptor has the amino acid sequence of SEQ ID NO:

12.

3. 3. The recombinant modified NK-92 cell of claim 2, wherein the amino acid sequence of SEQ ID NO: 12 has the F158V mutation.

4. The recombinant modified NK-92 cell of claim 1, wherein said cytokine has IL-12 activity.

5. The recombinant modified NK-92 cell of claim 4, wherein said cytokine is a single-chain IL-12 having the amino acid sequence of SEQ ID NO:

61.

6. The recombinant modified NK-92 cell of claim 4, wherein said cytokine is a heterodimeric IL-12 having a p35 component having the amino acid sequence of SEQ ID NO:58 and a p40 component having the amino acid sequence of SEQ ID NO:

60.

7. The recombinant modified NK-92 cell of claim 1, wherein said anti-CD123 CAR comprises an intracellular signaling domain derived from FcεRIγ.

8. The recombinant modified NK-92 cell of claim 7, wherein said anti-CD123 CAR comprises a CD8 hinge region and a CD28 transmembrane domain linked to an intracellular signaling domain from FcεRIγ.

9. 8. The recombinant modified NK-92 cell of claim 7, wherein said anti-CD123 CAR comprises an intracellular signaling domain derived from FcεRIγ having the amino acid sequence of SEQ ID NO: 31, a CD8 hinge region having the amino acid sequence of SEQ ID NO: 33, and a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO:

35.

10. 10. The recombinant modified NK-92 cell of claim 9, wherein said anti-CD123 CAR comprises an anti-CD123 scFv portion followed by a CD8 hinge region having the amino acid sequence of SEQ ID NO:33 followed by a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO:35 followed by an intracellular signaling domain from FcεRIγ having the amino acid sequence of SEQ ID NO:31 in a single polypeptide chain.

11. A recombinant modified NK-92 cell stably transfected with a recombinant nucleic acid and expressing an anti-CD123 chimeric antigen receptor (CAR), an Fc receptor, and a cytokine from said recombinant nucleic acid, the anti-CD123 CAR and the Fc receptor are expressed on the cell surface of the recombinant modified NK-92; The recombinant modified NK-92 cells have ATCC deposit number CRL-2407. Recombinant modified NK-92 cells.

12. The recombinant modified NK-92 cell of claim 11, wherein said Fc receptor has the amino acid sequence of SEQ ID NO: 12 and optionally has an F158V mutation.

13. The recombinant modified NK-92 cell of claim 11, wherein said cytokine is a single-chain IL-12 having the amino acid sequence of SEQ ID NO:

61.

14. The recombinant modified NK-92 cell of claim 11, wherein said cytokine is a heterodimeric IL-12 having a p35 component having the amino acid sequence of SEQ ID NO:58 and a p40 component having the amino acid sequence of SEQ ID NO:

60.

15. 12. The recombinant modified NK-92 cell of claim 11, wherein said anti-CD123 CAR comprises an intracellular signaling domain derived from FcεRIγ having the amino acid sequence of SEQ ID NO:31, a CD8 hinge region having the amino acid sequence of SEQ ID NO:33, and a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO:

35.

16. 12. The recombinant modified NK-92 cell of claim 11, wherein said anti-CD123 CAR comprises an anti-CD123 scFv portion followed by a CD8 hinge region having the amino acid sequence of SEQ ID NO:33 followed by a CD28 transmembrane domain having the amino acid sequence of SEQ ID NO:35 followed by an intracellular signaling domain from FcεRIγ having the amino acid sequence of SEQ ID NO:31 in a single polypeptide chain.

17. 1. A method of treating cancer in a subject, comprising: administering to the subject a therapeutically effective amount of a composition; 13. The method of claim 1, wherein the composition comprises a plurality of recombinant modified NK-92 cells of claim 1, and the cancer has cancer cells that express CD123.

18. 1m ２ 1 x 10 per ３ ~1×10 １０ 18. The method of claim 17, wherein said recombinant modified NK-92 cells are administered to said subject.

19. 18. The method of claim 17, wherein the recombinant modified NK-92 cells are administered parenterally, intravenously, peritumorally, intratumorally, or by injection.

20. 20. The method of claim 17, wherein the subject undergoes another therapy selected from the group consisting of radiation therapy, surgery, hormone therapy, and immunotherapy.

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