Quadricistronic system containing homing receptors or cytokines and chimeric antigen receptors for stable gene modification in cell immunotherapy

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 on August 1, 2018; U.S. Provisional Patent Application No. 62 / 713,278, filed on August 1, 2018; U.S. Provisional Patent Application No. 62 / 713,310, filed on August 1, 2018; and U.S. Provisional Patent Application No. 62 / 713,323, filed on August 1, 2018. Each of these applications is incorporated herein by reference in its entirety.

[0002] Sequence List The contents of the sequence listing ASCII text file named 104077_0007PCT_Seq_listing_rev004_ST25, with a size of 97KB, were created on August 1, 2019, and submitted electronically via EFS-Web with this application, and are incorporated in their entirety by reference.

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

[0004] The background information includes information that may be helpful in understanding the present invention. This does not constitute an admission that any information provided herein is prior art, or is related to the claims thereof, or that any publications specifically or implicitly referenced are prior art.

[0005] All publications and patent applications herein are incorporated by reference to the same extent as individual publications or patent applications are specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference conflicts with or contradicts a definition of that term provided herein, the definition provided herein shall apply, and the definition of that term in the reference shall not apply.

[0006] Cancer immunotherapy based on adopted tumor-specific cytotoxic lymphocytes shows promise in treating patients with malignant tumors. Despite this initial success in certain cancers, tumor treatment remains challenging, primarily 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 modified T cells, NK cell-based immunotherapy is being investigated. Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Natural killer (NK) cells generally account for about 10-15% of circulating lymphocytes and bind to and kill target cells, including virus-infected cells and many malignant cells, nonspecifically with respect to antigens and without prior immunosensitization. Herberman et al., Science 214:24 (1981). NK-92 is a cytolytic cancer cell line discovered in the blood of subjects with non-Hodgkin lymphoma and subsequently immortalized ex vivo. NK-92® cells are derived from NK cells but lack the main inhibitory receptors exhibited by normal NK cells, while retaining most of the activating receptors. However, NK-92® cells do not attack normal cells and do not induce unacceptable immune rejection in humans.

[0007] A common driver of lymph node metastasis is the hypoxic upregulation of CCR7, a chemokine receptor primarily found on naive T cells and dendritic cells. Upregulation of the CCR7 receptor on hematopoietic NK cells has been previously demonstrated to improve NK cell homing to lymph nodes and allow them to follow the same pathway to lymph node compartments, a common route of metastatic diffusion, but this has not yet been demonstrated in clinically relevant cell lines.

[0008] Therefore, there remains a need to improve NK cells and NK cell-based therapies, particularly in the homing and regulation of NK cells within the tumor microenvironment. [Overview of the Initiative] [Means for solving the problem]

[0009] Modified NK-92® cells containing nucleic acids encoding multiple functional elements are provided herein. Functional elements are typically proteins or polypeptides that provide specific functions that improve the efficacy of the cells as a cell line for immunotherapy. In one embodiment, NK-92® cells include a nucleic acid construct encoding four functional elements. In some embodiments, the nucleic acid construct includes a sequence encoding four functional elements (referred to as a “quadricistronic construct”) operably linked to a promoter.

[0010] In some embodiments, the first element encoded by the nucleic acid construct is a cytokine that provides 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 known as CD16), which is a low-affinity Fc receptor that binds to an IgG antibody to activate ADCC. In some embodiments, the CD16 receptor includes a substitution of phenylalanine (F) to valine (V) 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 containing the signal sequence). In one embodiment, the Fc receptor includes 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 the 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, a 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 enables the transcription of the nucleic acid. Modified NK-92® cells can migrate toward a source of chemokines, which are ligands for the receptor. Unlike conventional blood-derived NK cells, modified NK-92® cells can be developed into cell lines relevant to human clinical trials, offering clear advantages in immunotherapy. Examples of homing receptors include, but are not limited to, chemokine receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, or CXCL14; cytokine receptors; cell adhesion molecules such as selectins including L-selectin (CD62L); and 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 nucleic acid is the homing receptor described herein.

[0014] In some embodiments, the third element encoded by the nucleic acid construct is a secreted cytokine, thereby increasing or improving the function of NK-92® cells as an immunotherapy agent. Cytokines can 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] Therefore, 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] The expression of TGF-β within tumors is known to suppress the antitumor activity of leukocytes in the tumor microenvironment. Therefore, in some embodiments, the third element encoded by the nucleic acid construct is a TGF-β inhibitor, such as a peptide that inhibits TGF-β. In some embodiments, the third element encoded by the nucleic acid construct is a TGF-β trap. In some embodiments, the TGF-β trap comprises the extracellular domain of the TGFβRII molecule. In some embodiments, the TGF-β trap comprises a single-stranded dimer of the extracellular domain of the TGFβRII molecule, most preferably a single-stranded dimer of the TGF-β receptor II external domain.

[0018] In some embodiments, the NK cells described herein are administered together with a TGF-β inhibitor to block TGF-β and help eliminate immunosuppression. In some embodiments, the NK cells described herein are administered together with other immunotherapeutic agents to help reduce or eliminate tumors. For example, TGF-β can be inhibited by intratumoral injection of inhibitory peptides combined 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 an antibody fragment such as 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 ABP or CAR that specifically binds to 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, or 41BB.

[0020] In another embodiment, modified NK-92® cells contain a nucleic acid encoding IL-12. In another embodiment, modified NK-92® cells contain a nucleic acid encoding a TGF beta trap. In some embodiments, the TGF beta trap contains a single-stranded dimer of the extracellular domain of the TGFβRII molecule.

[0021] In another embodiment, modified NK-92® cells include nucleic acids encoding cytokines that provide selection of NK-92® cells expressing cytokines or enable their survival. In some embodiments, the nucleic acids encode cytokines such as IL-2 or IL-15. In one embodiment, IL-2 is expressed with a signaling sequence that directs IL-2 to endoplasmic reticulum IL-2 ("erIL-2"). In one embodiment, IL-15 is expressed with a signaling sequence that directs IL-15 to endoplasmic reticulum IL-2 ("erIL-15").

[0022] In some embodiments, modified NK-92® cells contain 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 known as CD16), which is a low-affinity Fc receptor that binds to an IgG antibody to activate ADCC. In some embodiments, the CD16 receptor contains a substitution of phenylalanine (F) to valine (V) 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 containing the signal sequence). In one embodiment, the Fc receptor contains the nucleic acid sequence of SEQ ID NO: 13 or the amino acid sequence of SEQ ID NO: 12.

[0023] In some embodiments, modified NK-92® cells contain nucleic acids encoding antigen-binding proteins ("ABP"). In some embodiments, the antigen-binding proteins specifically bind to tumor-associated antigens. In some embodiments, the ABP contains an antibody fragment such as scFv. In some embodiments, the antigen-binding proteins contain or are part of a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes ABP or CAR that specifically binds to 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, or 41BB.

[0024] In another embodiment, modified NK-92® cells contain nucleic acids encoding secretory cytokines that modulate the tumor microenvironment. In some embodiments, the cytokines that modulate the tumor microenvironment are chemokines such as XCL1, CCL5, CCL21, or CCL16. In some embodiments, modified NK-92® cells contain nucleic acids encoding a Toll-like receptor (TLR) agonist. In some embodiments, modified NK-92® cells contain nucleic acids encoding IL-12 or IFN-alpha. In some embodiments, modified NK-92® cells contain nucleic acids encoding a TGF-beta inhibitor, such as a TGF-β inhibitory peptide. In some embodiments, modified NK-92® cells contain nucleic acids encoding a TGF-beta trap. In some embodiments, the TGF-beta trap contains the extracellular domain of the TGFβRII molecule, or a single-chan dimer of the extracellular domain of the TGFβRII molecule.

[0025] In one embodiment, modified NK-92® cells include one or more nucleic acid molecules encoding a homing receptor, ABP or CAR, an Fc receptor, and / or cytokines that provide selection of NK-92® cells expressing the cytokine or enable their survival. Accordingly, in some embodiments, modified NK-92® cells include nucleic acid molecules encoding a chemokine receptor, CAR, CD16, and erIL-2. In some embodiments, modified NK-92® cells include nucleic acid molecules encoding CCR7 or CXCR2, CAR, CD16, and erIL-2. In some embodiments, modified NK-92® cells include nucleic acid molecules encoding IL-12 or TGF beta trap, CAR, CD16, and erIL-2.

[0026] In some embodiments, CAR contains an intracellular signaling domain from Fc epsilon receptor gamma (FcεRIγ). In one embodiment, CAR is transiently expressed by NK-92® cells. In one embodiment, 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. This is because other signaling domains (e.g., CD3ζ) were deemed more efficient, particularly when combined with additional signaling domains (second- and third-generation CARs). This specification presents the unexpected and surprising finding that NK-92® cells expressing “first-generation” CARs containing an intracellular domain from FcεRIγ exhibit equivalent or greater cytotoxic activity against cancer cells expressing antigens recognized by the CARs compared to NK-92® cells expressing the CAR with the CD3ζ signaling domain alone or in combination with other signaling domains (i.e., second- or third-generation CARs). In one embodiment, the CD3ζ signaling domain intended herein may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40.

[0028] In one embodiment, NK-92® cells, or a cell line expressing a chimeric antigen receptor (CAR) on the surface of NK-92® cells, is described, wherein the CAR comprises the cytoplasmic domain of FcεRIγ. In one embodiment, the cytoplasmic domain of FcεRIγ comprises an amino acid sequence having at least 95% sequence identity with 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 with SEQ ID NO: 32.

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

[0031] In some embodiments, NK-92® cells or cell lines are 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 domain, and FceRI gamma signaling domain amino acid sequences may comprise polypeptide or polynucleotide sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with 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 containing one or more internal ribosome entry sites (IRESs), enabling the initiation of translation from an internal region of 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 polypeptide 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 tumor cells. 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 selection or enables the survival of NK-92® cells expressing a cytokine such as IL-2. In one embodiment, the cytokine targets the endoplasmic reticulum.In one embodiment, CAR scFv may contain a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39.

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

[0033] In one embodiment, 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 embodiment, the 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® cells. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is DNA.

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

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

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

[0038] Compositions and kits containing modified NK-92® cells are also provided. Methods for producing the modified cells and methods for treating cancer using the cells are also provided.

[0039] In another embodiment, methods for treating cancer or reducing tumor size are described. In some embodiments, the method for treating cancer or reducing tumor size comprises administering a therapeutically effective dose of modified NK-92® cells described herein to a subject in need thereof, wherein the administration treats cancer or reduces the size of a tumor in the subject. In some embodiments, the method comprises administering a therapeutically effective dose of modified NK-92® cells to a subject, comprising nucleic acids encoding homing receptors, Fc receptors such as ABP or CAR, CD16 or CD16-158V, and / or cytokines such as erIL-2 or erIL-15. In some embodiments, the method comprises administering a therapeutically effective dose of modified NK-92® cells to a subject, comprising nucleic acids encoding secreted cytokines, Fc receptors such as ABP or CAR, CD16 or CD16-158V, and / or cytokines such as erIL-2 or erIL-15.

[0040] In some embodiments, the NK cells described herein are administered together with a TGF-β inhibitor to block TGF-β and help eliminate immunosuppression. In some embodiments, the NK cells described herein are administered together with other immunotherapeutic agents to help reduce or eliminate tumors. For example, TGF-β can be inhibited by intratumoral injection of inhibitory peptides combined 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 embodiment, 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 agents for treating a disease. In some embodiments, the modified NK-92® cells described herein are provided for use in the treatment of a disease. In some embodiments, the modified NK-92® cells include a homing receptor, an Fc receptor such as ABP or CAR, CD16 or CD16-158V that specifically binds to a target antigen, and / or a nucleic acid encoding a cytokine such as erIL-2 or erIL-15. In some embodiments, the modified NK-92® cells include a secreted cytokine, an Fc receptor such as ABP or CAR, CD16 or CD16-158V that specifically binds to a target antigen, and / or a nucleic acid encoding a cytokine such as erIL-2 or erIL-15. In some embodiments, the disease is cancer.

[0042] Details of one or more embodiments are described in the accompanying drawings and the following description. Other features, purposes, and advantages will become apparent from the description and drawings, as well as the claims. [Brief explanation of the drawing]

[0043] [Figure 1] This is a schematic diagram showing the plasmid pNKAT-CCR7-LP3, which contains the CCR7 receptor for insertion into the AAVS1 gene locus of NK-92(registered trademark) cells. [Figure 2] This is a schematic diagram showing the plasmid pCRENFAT-CCL21, which contains the NFAT-responsive CCL21 gene. [Figure 3]This graph shows the 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] This graph shows the cytotoxic activity of modified NK-92® cells expressing CCR7 against K562 cells. [Figure 5] This graph shows the cytotoxic activity of modified NK-92® cells expressing CCR7 against HL-60 cells. [Figure 6A] This graph shows the activation of the NFAT-luciferase reporter gene in NK-92 cells, as demonstrated by its binding to K562 and SUP-B15 cells (when CD19-CAR mRNA was electroporated into NK-92 cells). [Figure 6B] This graph shows the activation of the NFAT-luciferase reporter gene in NK-92 cells, as demonstrated by its binding to K562 and SUP-B15 cells (when CD19-CAR mRNA was electroporated into NK-92 cells). [Figure 7] This graph shows modified NK-92® cells expressing CCR7 (Mi-aNK) that migrated toward the chemokines CCL19 and CCL21. [Figure 8]A diagram illustrating an exemplary method for in vitro testing of modified NK-92® cells as described herein is shown. 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 the modified Boyden chamber Transwell assay shown. [Figure 9] This specification shows representative cytotoxicity assays using the 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 showed the highest percentage of target cell lysis, and this percentage increased when the K562 target cells expressed both CCL19 and CCL21. [Figure 10] This is a schematic diagram showing the plasmid pNKAT-CCR7-CD19CAR-CD16-ERIL2, which is called a "quadricistronic vector" and can be used to stably transfect cells at a single insertion site. [Figure 11] This is a schematic diagram showing the linearized plasmid from Figure 10. [Figure 12] This shows the cell surface expression of CCR7, CD16, and CD19 CAR 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 nucleic acid constructs 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 indicated time intervals after NK cell administration. Data are mean ± SEM. The - and + signs indicate the expression status of the CCR7 receptor (first sign) and CCL19 ligand (second sign). * indicates P<0.05 by multiple comparisons using one-way ANOVA followed by Tukey's test. The last panel shows time-course curves. [Figure 14] Direct comparison of non-CR and Mi-T-haNK cell infiltration into parental or CCL19-expressing tumors in single animals 24 hours after administration. Three out of four animals administered Mi-T-haNK cells showed higher infiltration into CCL19+ tumors, while three out of four animals administered 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] The survival curves of IV Raji-19.5 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 the log-rank (Mantel-Cox) test. ***, P=0.0002;****, P<0.0001. [Figure 16] This shows weight changes in an IV Raji-19.5 tumor model. The curves show weight changes (percentage change relative to day 0) in 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 administration days. Weight measurements performed on administration days were taken prior to administration. At all time points prior to day 20, the curves for the NK cell treatment group showed a statistically significant difference (P<0.05) compared to the vehicle control group, as determined by two-way ANOVA followed by Tukey's test for multiple comparisons. [Figure 17] This shows the SC Raji-19.5 tumor size at the time of randomization. Individual tumor sizes at randomization are also shown. Black squares enclose large tumors (over 200 mm³), and blue squares enclose small tumors (less than 200 mm³). Group mean ± SEM is also shown. [Figure 18]This shows tumor growth in a large tumor subpopulation of 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 using Tukey's test. Statistical significance was not achieved. (B) Individual curves. Red arrows indicate administration days. Tx: Treatment. [Figure 19] This shows tumor growth in a small tumor subpopulation of 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 using Tukey's test. No statistical significance was detected between the two groups at any time point. (B) Individual curves. Red arrows indicate administration days. Tx: Treatment. [Figure 20] This shows weight changes in the SC Raji-19.5 tumor model. The curves show weight change (percentage change relative to day 1) in 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 the administration day. Weight measurements performed on the administration day were taken prior to administration. At all time points prior to day 16, the curves for the NK cell treatment group reached a statistically significant difference compared to the vehicle control group, as determined by two-way mixed-effects analysis followed by Tukey's test for multiple comparisons. [Figure 21] This shows one embodiment of a quadricistronic TGFβ trap armored PD-L1 CAR structure. [Figure 22] This shows the expression analysis of PD-L1CAR and CD16 in PD-L1 (TGFβ trap) t-haNK clones. [Figure 23] This shows that the TGFβ trap is secreted into the culture supernatant of the TGFβ trap / PD-L1 t-haNK clone. [Figure 24] This demonstrates the cytotoxicity of the quadricistronic TGFβ trap construct against K562 target cells. [Figure 25] This demonstrates CAR death in PD-L1-expressing SUP-B15 target cells using a quadricistronic TGFβ trap construct. [Figure 26] This demonstrates CAR death in MDA-MB231 target cells using a quadricistronic TGFβ trap construct. [Figure 27] This shows the ADCC of a quadricistronic TGFβ trap construct for SUP-B15CD19-CD20+. [Figure 28] This study demonstrates that TGFβ / SMAD luciferase reporter HEK293 cells are induced by TGFβ. [Figure 29] In the HEK293T reporter assay, the secreted TGFβ trap sequestered TGFβ and inhibited luciferase expression. [Figure 30] This shows IL-12 secretion from NK-92® cell lines transduced with IL-12 virus. [Figure 31] This shows one embodiment of the Quadricistronic IL-12 / PD-L1 t-hanK structure. [Figure 32] This shows cytotoxic data for CCR7 CD19 t-haNK cells. [Figure 33] This shows IL-12 secretion from the IL-12 / PD-L1 t-haNK(trademark) cell line. [Modes for carrying out the invention]

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

[0045] In this specification and the appended claims, several terms are used as to be defined as having the following meanings:

[0046] The terms used herein are intended solely to describe specific embodiments and are not intended to limit the invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context explicitly indicates otherwise.

[0047] All numerical specifications, including ranges and variations, such as pH, temperature, time, concentration, quantity, and molecular weight, are typically encountered by those skilled in the art. Therefore, numerical values ​​may include variations of + or - increments of 0.1 or 1.0, as necessary, depending on the significant figures involved. It should be understood that all numerical notations may be preceded by the term “approximately,” although not necessarily explicitly stated. As used herein, the term “approximately” may also mean that the value can vary by ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0048] Although not always explicitly stated, it should be understood that the reagents described herein are merely examples, and that equivalents of such reagents may be known in the art.

[0049] "Optional" or "optional" means that the event or situation described thereafter may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not occur.

[0050] The term “contains” is intended to mean that a composition and method includes the elements described, but does not exclude other elements. “Essentially consisting of” as used to define a composition and method shall mean excluding any other elements that are essentially important to the combination. For example, a composition essentially consisting of elements as defined herein does not exclude other elements that do not substantially affect the basic and novel features of the claims. “Consists of” shall mean excluding other components and substantial steps of the described method in amounts greater than trace amounts. Embodiments defined by each of these transitional 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 causes a cell to migrate toward a target cell or tissue. For example, homing receptors expressed by leukocytes are used by leukocytes and lymphocytes to enter secondary lymphoid tissue 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, but are not limited to, chemokine receptors such as 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); and G protein-coupled receptors such as integrins, including α4β7 integrin, LPAM-1, and LFA-1. Homing receptors generally bind to recognition ligands on target tissues or cells. In some embodiments, homing receptors bind to adresins on the endothelium of venules, such as mucosal vascular adresin 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 can induce cytotoxicity upon contact with target cells.

[0053] As used herein, “natural killer (NK) cells” are cells of the immune system that kill target cells in the absence of specific antigen stimulation and without restriction by major histocompatibility complex (MHC) class. NK cells are distinguished by the presence of the CD56 surface marker and the CD3 surface marker. It is characterized by the absence of surface markers.

[0054] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), 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 may be used for autologous or allogeneic treatment by 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), the rights of which are owned by NantKwest® (hereinafter, "NK-92® cells"). The immortal NK cell line was originally obtained from patients with non-Hodgkin 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 exogenous genes). NK-92® cells and their exemplary and non-limiting modifications are described in U.S. Patent Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; and 9,150,636, and published as U.S. Patent Application No. 10 / 008,955, all of which are incorporated herein by reference in their entirety, 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 skilled 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), the rights of which are 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 its cell surface, the rights of which are described in Gong et al. (1994), the rights of which are 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 their 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, the rights of which are described in Gong et al. (1994), the rights of which are 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 modified to express CD16 on its cell surface and a chimeric antigen receptor, as described by Gong et al. (1994) and whose rights are owned by NantkWest (hereinafter referred to as "CAR-modified CD16+NK-92(registered trademark) cells" or "t-haNK cells"). In some embodiments, t-haNK cells express a high-affinity CD16 receptor on their cell surface.

[0057] The terms "chemokine-targeted t-haNK" and "Mi-T-haNK" refer to t-haNK cells that have been modified to express chemokine receptors on their 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. Generally, cytotoxic activity relates to the killing of target cells by any of the following biological, biochemical, or biophysical mechanisms. Cytolysis, more specifically, refers to the activity of an effector that dissolves the plasma membrane of a target cell, thereby destroying its physical integrity. This results in the death of the target cell. While we do not wish to be bound by theory, the cytotoxic effect of NK-92® cells is considered to be due to cytolysis.

[0059] The term "die" in reference to cells / cell populations is intended to include any type of operation that leads to the death of those cells / cell populations.

[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 defense 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 a cell's Fc receptor (FcR) stimulates phagocytic or cytotoxic activity of the cell via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fc-γ receptors (FCγR) bind to IgG class antibodies. FCγRIII-A (also known as 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 that fuses with an intracellular signaling domain. CARs can be expressed on T cells or NK cells and can enhance cytotoxicity. Generally, the extracellular antigen-binding domain is an scFv specific to the antigen found on the cell of interest. CAR-expressing NK-92® cells target cells that express a particular antigen on their cell surface, based on the specificity of the scFv domain. The scFv domain can be genetically engineered to recognize any antigen, including tumor-specific antigens. For example, the CD19CAR recognizes CD19, a cell surface marker expressed by some cancers.

[0062] As used herein, the term "tumor-specific antigen" refers to an antigen that is present on cancer or neoplastic cells but is undetectable on normal cells originating from the same tissue or lineage as the cancer cells. As used herein, tumor-specific antigen also refers to tumor-associated antigens, i.e., antigens that are expressed at higher levels on cancer cells compared to normal cells originating from the same tissue or lineage as the cancer cells.

[0063] The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably and refer to polymeric forms of any length of nucleotides, whether deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. 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 may include modified nucleotides such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure may be conjugated before or after the assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. This term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present invention that is a polynucleotide encompasses both a double-stranded form and each of two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

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

[0065] "Homologousity," "identity," or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Sequence similarity can be determined by comparing the positions in each sequence that can be aligned for 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 across a given comparison window. Sequences may 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 terms "identical" or "percent identical" refer to two or more nucleic acid or polypeptide sequences that are identical or have a specific percentage of amino acid residues or nucleotides, as measured by the BLAST or BLAST2.0 sequence comparison algorithm using the default parameters described below, or by manual alignment and visual inspection (i.e., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical in a particular region when compared and aligned to obtain the greatest match in a comparison window or designated region) (see, for example, the NCBI website). In such cases, such sequences are said to be substantially identical. This definition may also refer to or apply to complements of test sequences. The definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Preferred algorithms can account for gaps, etc., as described below. In some embodiments, identity exists over a region having a length of at least about 25 amino acids or nucleotides, or over a region having a length of 50 to 100 amino acids or nucleotides.

[0067] In sequence comparison, typically one sequence acts as a reference sequence, and the test sequence is compared to it. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer; sub-sequence coordinates are specified as needed; and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters.

[0068] The comparison window used herein includes references to any one segment of any number of adjacent positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, and after the two sequences have been optimally aligned, the sequences can be compared to reference sequences of the same number of adjacent positions. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, 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 the similarity search of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from 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 BLAST2.0 algorithms, 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 BLAST2.0, along with the parameters described herein, are used to determine the percent sequence identity of nucleic acids or proteins. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information, as is well known in the Art. This algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of selected length (W) in the query sequence that, when aligned with words of the same length in the database sequence, match or satisfy a portion of a positive threshold score T. T is called the neighbor word score threshold (Altschul et al., cited above). These first neighbor word hits serve as a seed to initiate a search for longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as they can increase the cumulative alignment score. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for matching residue pairs; always greater than 0) and N (penalty score for mismatched residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped if: the cumulative alignment score has decreased by a quantity X from the maximum achieved value; the cumulative score becomes zero or less due to the accumulation of residue alignments with one or more negative scores; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The expected value (E) represents the number of different alignments that have a score equal to or greater than what is expected to occur by chance in a database search.The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and a comparison of both strands by default. For amino acid sequences, the BLASTP program uses a word length of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), alignment (B) of 50, an expected value (E) of 10, M=5, and N=-4 by default.

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

[0071] When used in reference to cells, the term transformed refers to cells that have been transformed as described herein to carry exogenous or heterologous genetic material (e.g., recombinant nucleic acids). The term transformed may 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" has its broadest meaning and is intended to encompass introduction of nucleic acids into cells or organisms, for example, introduction by transformation methods (e.g., calcium chloride-mediated transformation, electroporation, particle impact), and introduction by other methods including transduction, conjugation, and conjugation. Optionally, constructs are used to introduce nucleic acids into cells or organisms.

[0073] When used in relation to cells, nucleic acids, polypeptides, vectors, etc., the terms modified and recombinant refer to cells, nucleic acids, polypeptides, vectors, etc., that have been modified by laboratory methods or are the result of such modifications, and do not occur naturally. For example, modified cells include cells produced or modified by laboratory methods, such as transformation methods for introducing nucleic acids into cells. Modified cells may contain nucleic acid sequences not found in the cell's natural (non-recombinant) form, or may contain modified nucleic acid sequences linked to, for example, non-natural promoters.

[0074] As used herein, the term "exogenous" refers to a substance, such as a nucleic acid (e.g., a nucleic acid containing a regulatory sequence and / or a gene) or polypeptide, that is artificially introduced into a cell or organism and / or does not occur naturally in the cell in which it resides. In other words, a substance, such as a nucleic acid or polypeptide, originates from outside the cell or organism into which it is introduced. Exogenous nucleic acids may have the same nucleotide sequence as nucleic acids naturally present in the cell. For example, NK-92® cells can be genetically engineered to contain nucleic acids having the NK-92® sequence, such as heparanase. By optional selection, the endogenous NK-92® heparanase sequence is operably linked to a gene that does not involve a regulatory sequence under natural conditions. While the NK-92® heparanase sequence may occur naturally in the host cell, the introduced nucleic acid is exogenous according to this disclosure. Exogenous nucleic acids may have a nucleotide sequence different from the nucleotide sequence of any nucleic acid naturally present in the cell. For example, exogenous nucleic acids may be heterologous nucleic acids, i.e., nucleic acids from a different species or organism. Therefore, exogenous nucleic acids may be identical to nucleic acids naturally found in the source organism, but may have a different nucleic acid sequence than the cell into which the exogenous nucleic acid is introduced. As used herein, the term endogenous refers to a nucleic acid sequence intrinsic to the cell. As used herein, the term heterogeneous refers to a nucleic acid sequence that is not intrinsic to the cell, i.e., originates from an organism other than the cell. The terms exogenous and endogenous or heterogeneous are not mutually exclusive. Therefore, nucleic acid sequences can be exogenous and endogenous. This means that a nucleic acid sequence can be introduced into a cell, but has a sequence that is identical or similar to a nucleic acid sequence naturally present in the cell. Similarly, nucleic acid sequences can be exogenous and heterogeneous. This means that a nucleic acid sequence can be introduced into a cell, but has a sequence that is not intrinsic 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, test cells, e.g., cells transformed with a nucleic acid sequence encoding the gene for the Fc receptor, can be compared to known normal (wild-type) cells (e.g., standard control cells). A standard control may also represent an average measurement or value collected from a population of cells (e.g., standard control cells) that do not express the Fc receptor, have no Fc receptor activity, or have it at a minimal level. 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 "expressed" 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 cell's genome. When referring to expression, the term "stable" means that the polynucleotide is integrated into the cell's genome, or that a positive selection marker (i.e., an exogenous gene expressed by cells that benefit under specific growth conditions) is used to maintain the 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 (ILs), particularly IL-2, IL-12, IL-15, IL-18, and IL-21. In preferred embodiments, 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 the antitumor response. Certain cytokines can inhibit the endogenous immune system's response to tumors, thus reducing the effectiveness of immunotherapy in cancer treatment. Therefore, the term also includes inhibitors of tumor growth-promoting cytokines, such as peptide inhibitors and / or ligands or receptors that bind to tumor growth-promoting cytokines, e.g., ligand traps.

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

[0080] As used herein, the term “targeting” is intended to include, but is not limited to, directing a protein or polypeptide to a suitable intracellular or extracellular destination when referring to protein expression. Targeting is typically achieved via a signal peptide or targeting peptide, which is a sequence 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 manipulated to have a signal peptide at their C-terminus. Signal peptides can direct polypeptides to extracellular regions, placement in the plasma membrane, Golgi, endosomes, endoplasmic reticulum, or other intracellular compartments. For example, a polypeptide having a specific amino acid sequence (e.g., KDEL) at its C-terminus may be retained in or returned to the ER lumen.

[0081] As used herein, the term “target” refers to the ability of NK-92® cells to recognize and kill tumor cells (i.e., target cells) when referring to tumor targeting. In this context, the term “targeted” refers, for example, to the ability of CARs expressed by NK-92® cells to recognize and bind to cell surface antigens expressed by tumors.

[0082] As used herein, the term “transfect” refers to the insertion of nucleic acids into cells. Transfection can be carried out using any means that allows nucleic acids to enter cells. DNA and / or mRNA can be transfected into cells. Preferably, the transfected cells express the gene product (i.e., protein) encoded by the nucleic acid.

[0083] Titles or subtitles 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 defined more specifically below.

[0084] Genetically engineered cells using a cytotoxic activated natural killer cell line (NK-92) as a basis for improving immunotherapy for cancer and tumors and / or increasing homing (migration) to a target of interest are provided herein. In some embodiments, NK-92® cells are genetically engineered to express homing receptors known to direct lymphocytes to lymph nodes when expressed. In some embodiments, NK-92® cells are genetically engineered to express secreted cytokines that modulate the tumor microenvironment, or inhibitors that block cytokines that modulate the tumor microenvironment.

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

[0086] In the proof-of-concept embodiment, the four genes used to generate a modified gene expression profile in a therapeutic cell line are CCR7, the CD19 chimeric antigen receptor, a high-affinity variant of CD16, and endoplasmic reticulum-bound IL-2. ER-bound IL-2 functions as a selector, in addition to stimulating the cytotoxicity of the NK-92®-based cell line into which it is incorporated. The IL-2-producing gene is located at the end of the quadracistronic vector, furthest from the promoter, making it the element most likely to be lost if the gene constructs a fragment (leading to negative selection and auto-excision from the pool, as cells require IL-2 for continued survival). However, if all elements are successfully incorporated into the genome, cells are selected by removing IL-2 from the culture medium, as only cells incorporating their own source of IL-2 will survive. Because the IL-2 element is furthest from the promoter, this is advantageous for the complete incorporation of the entire cassette of the four elements. This can be further verified by flow cytometry staining analysis of the other components (see Examples).

[0087] The constructs described herein offer the advantages of reducing development time when generating new therapeutic cell lines and minimizing stress and adverse effects on cells caused by multiple rounds of genomic manipulation and subsequent selection. Furthermore, by placing the selector (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 for cells to silence any given component of the construct without causing IL-2 starvation. Therefore, stable cell lines constructed in this manner should retain the 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 targets were addressed by the four components that make up this quadracistronic construct. IL-2 functions as a selective agent, is a known agonist of NK-92® cells with cytotoxic effects, and is an effective cancer treatment agent. The CCR7 element (CC chemokine receptor type 7) is a chemokine receptor that plays a role in causing immune cells expressing it to migrate in the direction of the chemokine gradient of 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 with SEQ ID NO: 1 (CCR7 sequence). In one embodiment, CCL21 as intended herein may include a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2 (CCL21 sequence). In one embodiment, CCL19 as intended herein may include a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16 (CCL19 sequence).

[0089] CD19 CARs (chimeric antigen receptors) are constructs used to enhance the cytotoxicity of cells when they encounter CD19 differentiation clusters on the surface of the cells they encounter. These differentiation clusters are commonly expressed on both normal and malignant B cells and have shown efficacy in direct immunotherapy trials for B-cell lymphoma. High-affinity CD16 receptors allow 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 like rituxumab and Herceptin. When immune cells armed with CD16 receptors encounter cells coated with one of these antibodies, they trigger ADCC (antibody-dependent cell-mediated cytotoxicity), attempting to destroy the cells. In practice, this makes it possible to use such armed cells in combination therapy regimens with monoclonal antibodies against cancer-generating antigens, etc. While each of these components has merit on its own, it has been proposed that combining this particular combination could 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, initiating CAR-mediated cytotoxicity or acting with monoclonal antibodies such as rituxumab to evade antigen escape. This proof-of-concept example is non-limiting, and it will be understood that NK-92® cells can be modified using the methods described herein to express other homing receptors and / or CARs targeting other antigens of interest in order to produce effective immunotherapy cell lines.

[0090] As described herein, modified NK-92® cells are generated with stable, long-term expression of the CCR7 lymph node homing receptor driven by the elongation factor 1a (EF1a) promoter after electroporation using a linearized gene construct containing a CCR7 expression cassette along with a removable selection cassette containing a selectable marker. One week after puromycin selection, monoclonal cell lines retaining high levels of CCR7 expression were established after serial dilution cloning. These CCR7-overexpressing NK cells exhibit a functional response to lymph node-associated chemokines CCL21 and CCL19 in migration / infiltration 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, the chemokines and homing receptors intended herein are SEQ ID NO: 44 (CCR7 aa acid sequence), or SEQ ID NO: 45 (CCL19 aa acid 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 It may contain a polypeptide sequence or polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the aa sequence.

[0092] The target involvement of susceptible cell lines has been shown to be recognized in NK-92® cells by activation of the NFAT transcription factor and its nuclear translocation. Target binding involving the FceRIg or CD3zeta pathway (including ADCC or CAR-mediated target recognition) is sufficient to induce NFAT activation in NK-92® cells. This was demonstrated by inserting a reporter cassette containing three arrest regions adjacent to the NFAT binding domain and a minimal promoter driving firefly luciferase. Luciferase expression was achieved by NFAT activation via 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 nonspecific cytotoxicity).

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

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

[0095] In one embodiment, the complete NFAT response cassette contemplated herein (polyA+ rest 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 with SEQ ID NO: 6.

[0096] In one embodiment, the complete sequence of the first insertion intended herein (the EF1a promoter, the CCR7 gene with polyA, and the LoxP-adjacent puromycin resistance gene driven by the ubiquitin promoter, all enclosed 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 with SEQ ID NO: 7.

[0097] In one embodiment, the complete sequence of the second insertion contemplated herein (including a NFAT-responsive cassette driving CCL21+Poly-A and a blastosidine resistance gene embedded with a CMV-driven FRT) 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 be modified by substituting the LoxP-adjacent puromycin resistance cassette from the first insertion, and the FRT sequence surrounding the blastosidine gene in this sequence, to allow for the later removal or substitution 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 male diagnosed with non-Hodgkin lymphoma (Gong, et al., Leukemia. 8:652-8 (1994)). NK-92® cells exhibit CD56 negativity in the absence of CD3, CD8, and CD16. bright It is characterized by the expression of CD2 and CD56. bright / CD16 negThe / low phenotype is typical of a minor subset of peripheral blood NK cells that possess immunomodulatory functions as cytokine-producing cells. Unlike normal NK cells, NK-92® lacks expression of most killer cell inhibitor receptors (KIRs) (Maki, et al., J Hematother Stem Cell Res. 10:369-83 (2001)). Only KIR2DL4, a KIR receptor with activating and inhibitory functions expressed in all NK cells, was detected on the surface of NK-92. KIR2DL4 is thought to mediate its inhibitory effect through binding to the HLA allele G (Suck, Cancer Immunol. Immunother. 65(4):485-92 (2015)). The primary pathway of cytotoxic death in 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 range 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 or 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 human melanoma models resulted in improved survival rates and suppression of tumor growth, including complete remission in several mouse tumors (Tam, et al., J Hematother. 8:281-90 (1999), Yan, et al., Clin Cancer Res. 4:2859-68 (1998)). Its safety profile has been confirmed in Phase I clinical trials. The characteristics of the NK-92® cell line are described in International Publication No. 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, which are incorporated herein by reference in their entirety.

[0100] By optional selection, 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 defense 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 a cell’s Fc receptor (FcR) stimulates phagocytic or cytotoxic activity of the cell via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified according to the type of antibody they recognize. For example, Fc-γ receptors (FCγRs) bind to IgG class antibodies. 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 can be found in entry P08637 of the SwissProt database.

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

[0102] By optional selection, modified NK-92® cells contain nucleic acid sequences that have 70%, 80%, 90%, or 95% identity with SEQ ID NO: 13. By optional selection, modified NK-92® cells contain nucleic acid sequences that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. By optional selection, modified NK-92® cells contain polypeptides that have 70%, 80%, 90%, or 95% identity with SEQ ID NO: 12 (having valine at position 176 of the full-length polypeptide). By optional selection, modified NK-92® cells contain polypeptides that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12.

[0103] The cytotoxicity of NK-92® cells depends on the presence of cytokines (e.g., interleukin-2 (IL-2)). Therefore, by optional selection, modified NK-92® cells are further modified to express at least one cytokine. By optional selection, at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. By optional selection, at least one cytokine is IL-2, IL-15, or a combination thereof. By optional selection, IL-2 and / or IL-15 are expressed with a signaling sequence that directs the cytokine to the endoplasmic reticulum. By directing IL-2 to the endoplasmic reticulum, IL-2 expression becomes possible at a level sufficient for autocrine activation without releasing a substantial amount of IL-2 extracellularly. See Konstantinidis et al, “Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92(registered trademark) cells,” Exp Hematol. 2005 Feb;33(2):159-64. A representative nucleic acid encoding IL-2 is shown in SEQ ID NO: 14, and a representative polypeptide of IL-2 is shown in SEQ ID NO: 15.

[0104] By optional selection, modified NK-92® cells contain a nucleic acid sequence encoding IL-2 with 70%, 80%, 90%, or 95% identity to SEQ ID NO: 14. By optional selection, modified NK-92® cells contain a nucleic acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14. By optional selection, modified NK-92® cells contain an IL-2 polypeptide with 70%, 80%, 90%, or 95% identity to SEQ ID NO: 15. By optional selection, modified NK-92® cells contain an IL-2 polypeptide with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15. The modified NK-92® cells provided can, advantageously, be maintained in the absence of IL-2 without secreting clinically adverse levels of IL-2.

[0105] In one embodiment, the subject of the present invention includes modified NK-92® cells capable of modulating the tumor microenvironment. The modified NK-92® cells preferably comprise 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, where the nucleic acid sequence is operably linked to a promoter. In one embodiment, the quadracistronic vectors intended herein are shown in Figures 21, 30, and 31, respectively. The IL-12 envisioned herein may include nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57 (p35 nt sequence) or SEQ ID NO: 59 (p40 nt sequence). The IL-12 envisioned herein may also include amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58 (p35 aa sequence, isoform 1 precursor) or SEQ ID NO: 60 (p40 aa sequence, precursor).

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

[0107] The TGF beta traps envisioned herein may comprise polynucleotide sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 63 (TGFBRII extracellular domain) or SEQ ID NO: 65 (TGFb trap sequence). The TGF beta traps envisioned herein may also comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with 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.T.er.2012, Vol 11(7), 1477-1487.

[0108] Furthermore, the nucleic acid constructs of the subject matter of the present invention may also include sequences encoding 2A peptides, such as T2A, P2A, E2A, or F2A peptides, in order to generate equimolar polypeptides encoded by the same mRNA. The 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. The 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 exemplary and non-limiting example, the plasmids disclosed herein may comprise polynucleotide sequences 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 reader), SEQ ID NO: 25 (CD19 CAR-negative signal peptide), SEQ ID NO: 26 (high affinity CD16), SEQ ID NO: 27 (IRES), SEQ ID NO: 28 (SC40 poly-A), SEQ ID NO: 29 (3' homology arm of AAVS1), and / or SEQ ID NO: 30 (homologous arm of AAVS1).

[0110] Chimeric antigen receptor By optional selection, modified NK-92® cells are further genetically engineered to express chimeric antigen receptors (CARs) on their cell surface. By optional selection, the CARs are specific to tumor-specific antigens. Tumor-specific antigens are described, without limitation, in U.S. Patent Application Publication No. 2013 / 0189268; International Publication No. 1999024566 A1; U.S. Patent No. 7098008; and International Publication No. 2000020460 A1, each of which is incorporated herein by reference in whole. 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. CARs can be genetically engineered as described, for example, in International Publication No. 2014039523; U.S. Patent Application Publication No. 20140242701; U.S. Patent Application Publication No. 20140274909; U.S. Patent Application Publication No. 20130280285; and International Publication No. 2014099671, etc., each of which is incorporated herein by reference in whole. By choice, the CAR is a CD19 CAR, a CD33 CAR, or a CSPG-4 CAR. In one exemplary and non-limiting example, CD19CAR_CD3a may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41.

[0111] Homing receptors Modified NK-92® cells comprising nucleic acids encoding homing receptors are provided herein. 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 the receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, or CXCL14. In some embodiments, the nucleic acid encoding CCR7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. By optional selection, the homing receptor is expressed on the cell surface of modified NK-92® cells. By optional selection, the modified NK-92® cells further contain a CAR. By optional selection, the CAR is CD19. By optional selection, the modified NK-92® cells further contain an Fc receptor. By optional selection, the Fc receptor is CD16. By optional selection, the modified NK-92® cells further contain cytokines 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 43.

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

[0113] In some embodiments, the provided expression vector is a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1; a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25 (CD19 CAR) or 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 The expression vector contains nucleic acids having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the aa sequence or sequence number 14. In some embodiments, the expression vector provided includes nucleic acids having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to sequence number 47; nucleic acids having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to sequence number 25 (CD19 CAR) or sequence number 12; and sequence number 13 (CD16 The recombinant nucleic acid comprises nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 158V) and / or nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15(erIL-2) or 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, the nucleic acid encoding erIL-15 having 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] The expression analysis of PD-L1 CAR and CD-16 in PD-L1 (TGFβ trap) t-haNK cells is shown in Figure 22. Furthermore, as shown in Figure 23, the TGFβ trap is secreted into the culture supernatant of TGFβ trap / PD-L1 t-haNK cells. Similarly, the right column of Figure 30 shows the secretion of IL-12 from the NK-92® cell line, which has been virally transduced with IL-12.

[0115] Method for producing modified NK-92(registered trademark) cells A method for producing modified NK-92® cells containing the nucleic acid molecules described herein is provided herein. The method comprises transforming NK-92® cells with an expression vector containing the nucleic acid described herein, operably linked to a promoter.

[0116] As used herein, the terms promoter, promoter element, and regulatory sequence refer to a polynucleotide operably ligated to a promoter that modulates the expression of a selected polynucleotide sequence and influences the expression of a selected polynucleotide sequence in a cell. In some embodiments, the promoter element is or includes the untranslated region (UTR) at the 5' position of the coding sequence. The 5'UTR is an integral part of protein expression in eukaryotes because it forms part of the mRNA transcript. Post-transcription, the 5'UTR can regulate protein expression at both the transcriptional and translational levels. Promoter controls transcription from vectors in mammalian host cells can be obtained from various sources, such as the genomes of viruses including polyomas, Simian virus 40 (SV40), adenoviruses, retroviruses, 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). Promoter having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 3, 9, 10, or 11 is provided herein.

[0117] As used herein, the term “selectable marker” refers to either a nucleotide sequence encoding a selectable product (polypeptide), such as a gene, or the gene product (such as a polypeptide) itself. The term “selectable marker” is used herein as is commonly understood in the art, and refers to a marker whose presence in a cell or organism provides a significant advantage or disadvantage to growth or survival under specific, defined culture conditions (selective conditions). As used herein, the term “selective agent” refers to a drug that introduces selective pressure into a cell or cell population, either favorably or unfavorably to a cell or cell population having a selectable marker. For example, a selective agent is an antibiotic, and a selectable marker is an antibiotic resistance gene. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin.

[0118] As used herein, nucleic acids refer to deoxyribonucleotides or ribonucleotides, as well as polymers and their complements. This term includes deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded forms. This term encompasses nucleic acids containing known nucleotide analogs or modified skeletal residues or linkages, which are synthetic, naturally occurring, and non-natural, possess similar binding properties to the reference nucleic acid, and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified herein, conservatively modified variants of nucleic acid sequences (e.g., degenerate codon substitutions) and complementary sequences may be used in place of specific nucleic acid sequences described herein. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced with a mixed base and / or a deoxyinosine residue (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] Nucleic acids are operably ligated when they are placed in a functional relationship with another nucleic acid sequence. For example, DNA encoding a pre-sequence or secreted leader is operably ligated to the polypeptide-encoding DNA if it is expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is operably ligated to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably ligated to a coding sequence if it is positioned to facilitate translation. Generally, operably ligated means that the ligated DNA sequences are close to each other, adjacent in the case of a secreted leader, and in the reading phase. However, enhancers do not need to be adjacent. For example, a nucleic acid sequence operably ligated to a second nucleic acid sequence is directly or indirectly covalently bound to such a second sequence, but any effective three-dimensional association is permitted. A single nucleic acid sequence can be operably ligated to multiple other sequences. For example, a single promoter can lead the transcription of multiple RNA species. Ligation can be achieved by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0120] Treatment method Methods for treating the cancer or tumors of interest are described herein. As used herein, the term “cancer” means all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, carcinomas, and sarcomas. Exemplary cancers include cancers of the brain, breast, cervix, colon, head and neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovarian, sarcoma, stomach, uterus, and medulloblastoma. Further examples include Hodgkin’s disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the pancreatic endocrine and exocrine parts, and prostate cancer.

[0121] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” may be used interchangeably and refer to the spread of cancer from one organ or another non-adjacent organ or part of the body, a proliferative disorder or disorder. Cancer originates in a site of origin, for example, the breast, which is called a primary tumor, for example, primary breast cancer. Cancer cells from the primary tumor or part of the site of origin acquire the ability to penetrate and invade the surrounding normal tissue in the local area, and / or penetrate the walls of the lymphatic or vascular system, circulating through the system to reach other parts and tissues in the body. A second clinically detectable tumor formed from cancer cells of the primary tumor is called a metastatic or secondary tumor. When cancer cells metastasize, it is assumed that the metastatic tumor and its cells are similar to those of the original tumor. For example, if lung cancer metastasizes to the breast, the secondary tumor in the breast site will consist of abnormal lung cells, not abnormal mammary gland cells. The secondary tumor in the breast is called metastatic lung cancer. Therefore, the expression metastatic cancer refers to a disease in which the subject has or has had a primary tumor in the past and has one or more secondary tumors. The expression "non-metastatic cancer" or "cancer that is not metastatic" refers to a 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 a disease in a subject who has a primary lung tumor or a history thereof and has one or more secondary tumors in a second or multiple location, such as the breast.

[0122] As used herein, “treating” a condition, disease, or disorder, or symptoms associated with a condition, disease, or disorder, or such “treatment” refers to an approach to obtain beneficial or desirable outcomes, including clinical outcomes. Beneficial or desirable clinical outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions; reduction of the severity of a condition, disorder, or disorder; stabilization of the condition, disorder, or disorder; prevention of the onset of a condition, disorder, or disorder; prevention of the spread of a condition, disorder, or disorder; delay or slowing of the progression of a condition, disorder, or disorder; delay or slowing of the onset of a condition, disorder, or disorder; improvement or relief of a condition, disorder, or disorder; and partial or overall remission. “Treatment” may also mean extending the subject’s survival beyond what would be expected without treatment. “Treatment” may also mean temporarily inhibiting or temporarily slowing the progression of a condition, disorder, or disorder, but in some cases, this may involve permanently halting the progression of a condition, disorder, or disorder. As used herein, the terms treatment, to treat, or to treat refer to one or more symptoms of a disease or condition characterized by protease expression, or a method of reducing the effects of symptoms of a disease or condition characterized by protease expression. Accordingly, in the disclosed methods, treatment may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptoms of a disease or condition. For example, a method for treating a disease is considered a treatment if, compared to a control, one or more symptoms of the disease in the subject are reduced by 10%. Accordingly, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between 10% and 100% compared to natural or control levels. It is understood that treatment does not necessarily refer to a cure or complete disappearance of a disease, condition, or symptoms of a disease or condition. Furthermore, as used herein, references to reduction, decrease, or inhibition include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control level, and such terms may include, but do not necessarily include, complete elimination.

[0123] The terms subject, patient, and individual are not intended to be limiting and are generally interchangeable. That is, an individual described as a patient may not necessarily have a specific disease and may simply be seeking medical advice. As used throughout, subject can be vertebrates, more specifically mammals (e.g., humans, horses, cattle, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, and guinea pigs), birds, reptiles, amphibians, fish, and any other animal. The terms are not intended to indicate a specific age or sex. Therefore, adult and neonatal subjects are intended to be subject regardless of whether they are male or female. Where used herein, patient, individual, and subject may be used interchangeably and are not intended to be limiting. That is, an individual described as a patient may not necessarily have a specific disease and may simply be seeking medical advice. The terms patient or subject include human and animal subjects.

[0124] As used herein, “administer” or “to administer” means providing, contacting, and / or delivering one or more compounds by any suitable route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intra-bursal, intrasternal, intrathecal, intralesional, or intracranial injection), percutaneous, topical, buccal, transrectal, transvaginal, transnasal, transocular, inhalation, and implantation. Optionally, NK-92® cells are administered parenterally. Optionally, NK-92® cells are administered intravenously. Optionally, NK-92® cells are administered peritumorally.

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

[0126] In some embodiments, the method further comprises administering a therapeutically effective dose of modified NK-92® cells described herein to a target, wherein the administration treats cancer or reduces the size of the target tumor. In some embodiments, the method comprises administering modified NK-92® cells to a target comprising nucleic acids encoding i) IL-12 or TGFb traps, ii) ABP or CAR that specifically binds to a target antigen, iii) Fc receptors such as CD16 or CD16-158V, and / or iv) cytokines such as erIL-2 or erIL-15. In some embodiments, the method comprises administering modified NK-92® cells to a target comprising nucleic acids encoding i) homing receptors, ii) ABP or CAR that specifically binds to a target antigen, iii) Fc receptors such as CD16 or CD16-158V, and / or iv) cytokines such as erIL-2 or erIL-15.

[0127] Figure 24 shows the cytotoxicity of TGFβ trap / PD-L1 against K562 target cells. Furthermore, SUP-B15 PD-L1+ CAR death in target cells is shown in Figure 25, and CAR death in MDA-MB231 target cells is shown in Figure 26. SUP-B15 CD19-CD20+Figure 27 shows the ADCC of TGFβ trap / PD-L1 against TGFβ. Figure 28 shows that TGFβ / SMAD luciferase reporter HEK293 cells are induced by TGFβ. As shown in Figure 29, in the HEK293T reporter assay, the secreted TGFβ trap sequestered TGFβ and inhibited luciferase expression. Reporter cells were treated with 1 ng / mL of TGFβ1 for 19 hours. Among other options, preferred CAR molecules may have an amino acid sequence that specifically binds to PD-L1 and 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 can be administered to subjects via various routes. For example, NK-92® cells can be administered to subjects by infusion over a period of time (e.g., intravenous infusion). Typically, for a single dose of NK-92® cells, the duration is 5 to 130 minutes. Optionally, the duration is 90 to 120 minutes. Optionally, the duration is 15 to 30 minutes.

[0129] NK-92 (registered trademark) cells, and optionally other anti-cancer agents, can be administered once to a patient having cancer, or multiple times, for example, throughout the treatment period, 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 weeks or more than a week, or once every arbitrary range including the endpoints between any two numbers. Thus, for example, NK-92 (registered trademark) cells can be administered once daily to a subject for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more. Optionally, NK-92 (registered trademark) cells are administered for 2 days in a once-daily cycle. Then, following this cycle, there is a period of 1 hour or more, 1 day or more, or 1 week or more without treatment with NK-92 (registered trademark) cells. As used herein, the term "cycle" refers to a treatment that is repeated on a regular schedule with a rest period (e.g., no treatment, or treatment with other agents) in between. For example, a 1-week treatment followed by a 2-week rest is one treatment cycle. Such treatment cycles can be repeated one or more times. Thus, NK-92 (registered trademark) 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 (registered trademark) cells can be administered to a subject in an absolute number of cells, for example, the subject can receive from about 1000 cells / injection up to a maximum of about 10 billion cells / injection, for example, about, at least about, or at most about 1×10 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 x 10 3 , 5×10 3 Individual (or similar) NK-92® cells, or any range including the endpoints between any two numbers, may be administered. Optionally, 1 × 10⁶ cells may be administered. 8 ~1 × 10 10 Individual cells are administered to the target. Optionally, cells are administered once or twice a week for one week or more. Optionally, cells are administered once or twice a week for one, two, three, four, five, six, seven, eight, nine, ten weeks or more.

[0131] Subjects are selected at random, with approximately 1000 cells / injection / m² being administered. 2 Up to approximately 10 billion cells / injection / m 2 Up to, for example, about, at least about, or at most about 1 × 10 per injection. 10 / m 2 , 1 x 10 9 / m 2 , 1 x 10 8 / m 2 , 1 x 10 7 / m 2 , 5×10 7 / m 2 , 1 x 10 6 / m 2 , 5×10 6 / m 2 , 1 x 10 5 / m 2 , 5×10 5 / m 2 , 1 x 10 4 / m 2 , 5×10 4 / m 2 , 1 x 10 3 / m 2 , 5×10 3 / m 2 NK-92® cells (such as) or any range including the endpoints between any two numbers may be administered. Optionally, 1m 2 1 x 10 3 ~1 × 10 10Individual NK-92(registered trademark) cells are administered to the target. Optionally, 1 million cells are administered. 2 2 x 10 9 Individual NK-92(registered trademark) cells are administered to the target.

[0132] By choice, NK-92® cells can be administered to such individuals in relative numbers, for example, from about 1,000 cells per kilogram of the individual to a maximum of about 10 billion cells, for example, about, at least about, or at most about 1 × 10¹⁶ cells per kilogram of the individual. 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 , 5×10 7 , 1 x 10 6 , 5×10 6 , 1 x 10 5 , 5×10 5 , 1 x 10 4 , 5×10 4 , 1 x 10 3 , 5×10 3 Individual (or similar) NK-92® cells, or any range including the endpoints between any two numbers, may be administered.

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

[0134] Optionally, NK-92® cells are administered in a composition comprising NK-92® cells and a culture medium, such as human serum or its equivalent. Optionally, the culture medium may contain human serum albumin. Optionally, the culture medium may contain human plasma. Optionally, the culture medium may contain approximately 1% to 15% human serum or its equivalent. Optionally, the culture medium may contain approximately 1% to 10% human serum or its equivalent. Optionally, the culture medium may contain approximately 1% to 5% human serum or its equivalent. Optionally, the culture medium may contain approximately 2.5% human serum or its equivalent. Optionally, the serum is human AB serum. Optionally, a serum substitute acceptable for use in human therapy may be used instead of human serum. Such serum substitutes are known in the art. Optionally, NK-92® cells are administered in a composition comprising NK-92® cells and an isotonic solution to support cell viability. Optionally, NK-92® cells are administered using a composition reconstituted from cryopreserved samples.

[0135] According to the methods provided herein, subjects are administered an effective dose of one or more of the drugs provided herein. The terms effective dose and effective dosage are used interchangeably. The term effective dose is defined as any amount necessary to produce a desired physiological response (e.g., reduction of inflammation). The effective dose and schedule for administering a drug can be determined empirically by those skilled in the art. With respect to administration, the range of doses is large enough to produce the desired effect, in which one or more symptoms of a disease or disorder are affected (e.g., reduced or delayed). The dose should not be so large as to cause substantial adverse side effects, such as undesirable cross-reactions or anaphylactic reactions. In general, the dose can be determined by those skilled in the art, depending on age, condition, sex, type of disease, severity of disease or disorder, route of administration, or whether other drugs are included in the regimen. In the event of any contraindications, individual physicians may adjust the dose. The dose can vary and may be administered once or more times daily over a day or several days. Guidance on appropriate dosages for a given class of medicinal products can be found in the literature. For example, with respect to a given parameter, an effective dose shows an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Effectiveness can also be expressed as an increase or decrease of "~ times". For example, a therapeutically effective dose may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more compared to the control. The exact dose and formulation depend on the purpose of the treatment and can be determined by those skilled 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] Pharmacovigilant compositions may include a variety of carriers and excipients. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. 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). A pharmaceutically acceptable carrier means a material that is not biologically or otherwise undesirable; that is, the material is administered to a subject without causing undesirable biological effects or interacting in an adverse manner with other components of the pharmaceutical composition containing it. When administered to a subject, the carrier is optionally selected to minimize the degradation of the active ingredient and to minimize adverse side effects in the subject. As used herein, the term pharmaceutically acceptable is synonymous with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions generally include the active ingredient for buffering and storage, and may include buffers and carriers for appropriate delivery depending on the route of administration.

[0137] The composition may contain acceptable auxiliary substances required for the appropriate physiological state, such as pH adjusters and buffers, as well as toxicity modifiers, such as sodium acetate, sodium hydrochloride, potassium hydrochloride, calcium hydrochloride, and sodium lactate. The concentrations of cellular and / or other active ingredients in these formulations may vary and are primarily selected based on liquid volume, viscosity, and body weight, etc., according to the needs of the chosen specific dosage form and target population.

[0138] Combination therapy Optionally, NK-92® cells are administered to subjects in combination with one or more other treatments for the cancer being treated. While not bound by theory, it is believed that the simultaneous treatment of subjects with NK-92® cells and other therapies for cancer allows NK-92® cells and alternative therapies to give such endogenous immune systems a chance to eliminate cancer that has previously suppressed its endogenous action. Optionally, two or more other treatments for the cancer being treated may include, for example, antibodies, bispecific engagers, radiation, chemotherapy, stem cell transplantation, or hormone therapy.

[0139] Optionally, antibodies may be administered to patients in combination with NK-92® cells. Optionally, NK-92® cells and antibodies may be administered to the subject together, for example, in the same formulation; separately, for example, in separate formulations, simultaneously; or separately, for example, on different dosing schedules or at different times of the day. When administered separately, antibodies may be administered by any suitable route, such as intravenous or oral administration.

[0140] Antibodies can be used to target cancer cells or cells expressing cancer-related markers, at will. Some antibodies are approved for use alone in the treatment of cancer.

[0141] The methods provided can be further combined with other oncological therapies such as radiotherapy, surgery, hormone therapy, and / or immunotherapy. Therefore, the methods provided may further include the administration of one or more additional therapeutic agents. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, resuscitation agents, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antitumor agents, allosteric inhibitors, anabolic steroids, antirheumatic agents, psychotherapeutic agents, nerve blockers, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antiprotozoal agents, antivirals, dopamine agonists, hematological agents, immunotherapeutics, muscarinic agents, protease inhibitors, vitamins, growth factors, and hormones. The selection of agents and dosages can be readily determined by those skilled in the art based on the given disease being treated. Optional additional therapeutic agents include octreotide acetate, interferon, pembrolizumab, glucopyranosyllipid A, carboplatin, etoposide, or any combination thereof.

[0142] Optionally, additional therapeutic agents are chemotherapeutic agents. A chemotherapy regimen may include the administration of one chemotherapeutic agent or a combination of chemotherapeutic agents to the target. 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] A combination of drugs or compositions may be administered in combination (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines), or sequentially (e.g., one drug is administered first, followed by the second drug). Therefore, the term "combination" is used to refer to the combined, simultaneous, or sequential administration of two or more drugs or compositions. The course of treatment is best determined individually, depending on the specific characteristics of the subject and the type of treatment chosen. Treatments such as those disclosed herein may be administered to a subject daily, twice daily, every other week, monthly, or in any applicable standard that is therapeutically effective. Treatments may be administered alone or in combination with other treatments disclosed herein or known in the art. Additional treatments may be administered concurrently with the first treatment, at different times, or on a completely different treatment schedule (e.g., the first treatment daily, and the additional treatment weekly).

[0144] kit Kits comprising modified NK-92® cells as described herein are provided herein. In some embodiments, the kit comprises modified NK-92® cells comprising one or more nucleic acid sequences operably linked to a promoter, encoding i) a homing receptor, ii) 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. Optionally, one or more proteins encoded by the nucleic acid sequence are expressed on the cell surface of the modified NK-92® cells. In some embodiments, the kit comprises modified NK-92® cells comprising nucleic acids encoding the receptor for CC chemokine receptor type 7 (CCR7), CXCR2, or 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. By optional selection, the homing receptor is expressed on the cell surface of modified NK-92® cells. By optional selection, the promoter contains one or more NFAT binding elements and a minimal promoter. By optional selection, the promoter has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. By optional selection, one or more proteins encoded by the nucleic acid sequence are expressed on the cell surface of modified NK-92® cells.

[0145] Optionally, modified NK-92® cells are provided in a composition containing pharmaceutically acceptable excipients. Optionally, the kit may include additional compounds, such as therapeutically active compounds or drugs, to be administered before, simultaneously with, or after administration of modified NK-92® cells. Optionally, the kit's instructions for use may include instructions for using the kit components in the treatment of cancer. The instructions may further include information on how to prepare antibodies and NK-92® cells (e.g., thawing and / or culturing lyophilized proteins). The instructions may further include guidance on dosage and frequency of administration.

[0146] Materials, compositions, and components that can be used in, in combination with, or in the preparation thereof, or are products thereof, are disclosed herein. Where these and other materials are disclosed herein and combinations, subsets, interactions, groups, etc., of these materials are described, it is understood that while specific descriptions of various individual and collective combinations and permutations of these compounds may not be explicitly stated, each is specifically intended and described herein. For example, where a method is disclosed and discussed and several modifications that can be made to some molecules including the method are discussed, each and all combinations and permutations of the method, as well as possible modifications, are specifically intended unless otherwise specifically indicated. Similarly, any subset or combination of these is also specifically intended and disclosed. This concept applies to all aspects of this disclosure, including steps in methods using the compositions of the disclosure, but is not limited thereto. Thus, where various additional steps can be carried out, it is understood that each of these additional steps can be carried out by any particular method step or combination of method steps of the disclosed method, and that each of such combinations or subsets of combinations should be considered specifically intended and disclosed.

[0147] Publications cited herein and materials from which they are cited are incorporated herein by reference in their entirety. [1] Modified NK-92 cells containing nucleic acids encoding homing receptors operably linked to a promoter. [2] The modified NK-92 cells according to [1], 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 cells according to [2], wherein the chemokine receptor is selected from the receptors CCR7, CXCR2, or CXCL14, and the cell adhesion molecule is selected from L-selectin (CD62L), α4β7 integrin, LPAM-1, and LFA-1. [4] The modified NK-92 cells according to [3], wherein the nucleic acid encoding CCR7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO: 1. [5] The modified NK-92 cell according to any one of the above [1] to [4], wherein the modified NK-92® cell further comprises a nucleic acid encoding an antigen-binding protein operably linked to a promoter. [6] The modified NK-92 cells according to [5], wherein the antigen-binding protein specifically binds to a tumor-associated antigen. [7] Modified NK-92 cells as described in [6], 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 cells described in [5], wherein the antigen-binding protein comprises a chimeric antigen receptor (CAR). [9] The modified NK-92 cells according to [8], wherein the CAR has an amino acid sequence that is specifically bound to CD19 or 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 cells according to any one of the above [1] to [9], further comprising nucleic acids encoding cytokines operably linked to a promoter.

[11] The modified NK-92 cells according to

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

[12] The modified NK-92 cell according to

[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 with SEQ ID NO: 14.

[13] The modified NK-92 cell according to

[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 with SEQ ID NO: 67.

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

[13] , wherein the modified NK-92® cell further comprises a nucleic acid encoding an FC receptor operably linked to a promoter.

[15] The modified NK-92 cells according to

[14] , wherein the Fc receptor is a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CD16 or high affinity CD16 (sequence number 12), or sequence number 13.

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

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

[17] Modified NK-92 cells comprising one or more nucleic acids encoding i) a homing receptor, ii) 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 sequences are operably linked to a promoter.

[18] The modified NK-92 cells according to

[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 cells according to

[17] , wherein the Fc receptor is a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CD16 or high affinity CD16 (sequence number 12), or sequence number 13.

[20] Modified NK-92® cells according to any one of the above [1] to

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

[21] A composition comprising modified NK-92 cells as described in any one of the preceding paragraphs [1] to

[20] and a pharmaceutically acceptable excipient.

[22] A kit comprising NK-92 cells and instructions for use as described in any one of the preceding paragraphs [1] to

[21] .

[23] A method of treating a target cancer or tumor, comprising administering a therapeutically effective amount of modified NK-92 cells as described in any one of [1] to

[20] above or the composition as described in

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

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

[20] or the composition as described in

[21] , thereby reducing cancer metastasis of the target.

[25] 1m 2 1 x 10 3 ~1 × 10 10 The method according to

[23] or

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

[26] The method according to any one of the

[23] to

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

[27] The method according to any one of the above

[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, containing an NFAT transcription factor response element.

[29] The inducible promoter according to

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

[30] The inducible promoter described in

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

[31] The inducible promoter according to

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

[32] A method for generating a modified NK92 cell line, including targeted homologous recombination at a specific gene locus.

[33] The method according to

[32] , wherein the gene 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 with SEQ ID NO: 7.

[35] Modified NK-92 cells containing nucleic acids encoding secreted cytokines that modulate the tumor microenvironment and are operably linked to a promoter.

[36] The modified NK-92 cells 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 the claims

[35] to

[36] , wherein the modified NK-92 cell further comprises a nucleic acid encoding an antigen-binding protein operably linked to a promoter.

[38] Modified NK-92 cells according to any one of the above

[35] to

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

[39] Modified NK-92 cells as described in

[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 cells according to

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

[41] The modified NK-92 cells according to

[40] , wherein the CAR has an amino acid sequence that is specifically bound to PD-L1 or 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 the

[35] to

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

[43] The modified NK-92 cells described in

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

[44] The modified NK-92 cell according to

[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 with SEQ ID NO: 14.

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

[35] to

[44] , further comprising nucleic acids encoding an FC receptor operably linked to a promoter.

[46] The modified NK-92 cells according to

[45] , wherein the Fc receptor is a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with CD16 or high affinity CD16 (sequence number 12), or sequence number 13.

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

[35] to

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

[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 cells according to any one of

[35] to

[48] above and a pharmaceutically acceptable excipient.

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

[35] to

[49] above and instructions for use.

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

[35] to

[48] above or the composition according to

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

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

[35] to

[48] above or the composition according to

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

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

[51] or

[52] , wherein the NK-92 cells are administered to the subject at a dose of 1×10 to 1×10 cells per m.

[54] The method according to any one of

[51] to

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

[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, comprising administering a first composition of modified NK-92 cells as described in any one of the preceding paragraphs

[35] to

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

[57] The method according to

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

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

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

[0150] To confirm that CCR7 expression does not affect NK-92® cells, the expression of markers in NK-92® cells was measured. The results are shown in Figure 3. All data in Figure 3 were generated using the Intellicyt iQue Screener Plus. Cells were incubated at 4°C for 30 minutes with either an APC-conjugated antibody against the described phenotypic marker or an appropriate isotype as a negative control. Cells were then rinsed with PBS + 1% BSA, pelletized, and resuspended in 30 μL of PBS + 1% BSA. Readings were then gated to remove cell debris from the readings, as shown in the upper left quadrant, and the percentage of cells exceeding the fluorescence threshold was then displayed as two separate heatmaps, as shown in the upper right quadrant, with the percentages exceeding the “positive” threshold and the “very positive” threshold in the lower left and lower right quadrants, respectively. Figure 3 shows that driving CCR7 expression does not significantly affect the key phenotypic markers associated with the cell line. Specifically, CCR7 expression does not appear to affect the 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 a 96-well V-bottom plate, leaving 100k effectors in the highest concentration well, and undergoing seven further 2x dilutions across eight rows of the plate. Stained target cells (K562 (Figure 4), HL-60 (Figure 5)) were then seeded at 10k / well in all wells containing effectors, along with a control well containing only targets to measure background death. The plates were then briefly spun down and incubated at 37°C, 5% CO2 for 4 hours. The plates were then spun down again, the supernatant was aspirated and removed, and the cells were resuspended in PBS containing propidium iodide to measure cell death. The cells were then passed through an Intellicyt iQue screener plus, and the percentage of target cells (distinguished from effectors by their staining) that were also positive for PI staining were measured. Next, the percentage of dead cells was compared to the number of naturally dead cells in the control well, 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 upregulatory clone compared to the parental cell line versus K562 cells, and Figure 5 shows comparable cytotoxicity in the CCR7 upregulatory clone compared to the parental cell line versus HL-60 cells.

[0152] In vitro studies consisted of Boyden chamber assays and migration blocking using Matrigel layers. Modified cells expressing CCR7 showed migration to CCL21 and CCL19 (alternative CCR7 ligands) in these assays. Cells were placed in upper wells and separated from the lower chamber by a thin layer of Matrigel (ECM-like substrate) coated with 8 μM pores. Cells (25 kJ / well) were placed in the upper chamber in reduced serum medium (supplemented with 1% human serum and 500 U / mL IL-2), and the same reduced serum medium was used in the lower chamber, either alone or with the target chemokine added. In this case, CCL21 was used at 15 ng / mL, CCL19 at 15 ng / mL, and SDF-1a at 20 ng / mL. Each test was performed three times on either NK-92® cells or modified NK-92® cells expressing CCR7. Next, the plate was placed in an incubator overnight for an 18-hour invasion assay, after which the upper chamber was removed and the entire mixture was mixed. 150 μL (out of a total volume of 750 μL) was sampled from the lower wells and read using a MacsQuant FACS analyzer. Live cells in the lower chamber were counted, and the cell count was compared to the wells without chemokines to generate an invasion index. These figures were averaged, and statistical correlations were calculated using a two-sided t-test. Since the lower wells were sampled without cells detaching from the lower membrane, cells still adhering to the lower part of the ECM are not represented in these figures, which may explain the difference between CCL19 and CCL21. The results are shown in Figure 7. Specifically, Figure 7 shows a statistically significant increase in the invasion of modified NK-92® cells expressing CCR7 against CCL19, which is the 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 into the ECM and subsequent detachment, which is consistent with CCL19 gradient migration. CCL21 induces migration but not detachment from the matrix, requiring an additional step to demonstrate the potential for statistically significant invasion.

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

[0154] Example 3. Modified NK cell line expressing CCR7 and CCL21. The pCRENFAT-CCL21 plasmid is incorporated into NK-92® cells containing CCR7 via recombinase-mediated cassette exchange using a LoxP site embedded in the pNKAT-CCR7-LP3 construct. Following electroporation of the circular plasmid (pCRENFAT-CCL21), Cre recombinase is transiently expressed, which mediates the exchange of the old select cassette with a new LoxP-adjacent cassette. Selection with blastosidine is used to facilitate the incorporation of the new cassette, and monoclonal cell lines are subcloned from the population obtained 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 induce NFAT activation (K562 or other cell lines) in the lower wells of a Boyden chamber, while stained modified NK-92® cells are placed in the upper chamber. The system is considered functional if migration is demonstrated to be induced by co-culture with a sensitive cell line.

[0156] Example 4. In vitro cytotoxic assay using a modified NK cell line expressing CCR7. Figure 9 shows that NK-92® cells modified to express CCR7 maintain cytotoxicity against target cells after migration in the 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. Figure 12 shows that modified NK-92® cells transfected with nucleic acid constructs encoding CCR7, CD16, and CD19 CARs express each protein at high levels on the cell surface.

[0158] Example 6. In vivo distribution of CCR7-expressing chemokine-responsive NK cells in NSG mice with CCL19-positive subcutaneous K562 tumors. This study demonstrates chemokine-responsive aNK cells that home to target tissues expressing chemokines after intravenous administration. Since mouse and human chemokines do not cross-react, the inventors developed a localized ligand-expressing tumor model as a surrogate model.

[0159] Experimental methods (Table 1): a. Animals: i. Animal type: NSG mouse (JAX), female, 7-8 weeks old ii. Number of animals: 30 animals (28 animals that received NK cell injections [24 + 4 extra]; 2 additional mice that did not receive NK treatment were used as negative controls for flow cytometry) b. Tumor model: i. Cell line: K562, parent and subline expressing CCL19, K-19 ii. Route of vaccination: Subcutaneously; parent K562 in the left flank, K-19 in the right flank. iii. Inoculum: 1E6 cells in 100 μL serum-free medium / Matrigel (v / v 1:1) iv. Tumor load at the start of treatment: Average 107 mm on K-19 3 Parent K562 average 135mm 3 v. Randomization: Animals were randomized primarily based on the volume of the K-19 tumor. 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 immediately before in vivo administration, according to the manufacturer's instructions. 2. Cultured CFSE-labeled cells were harvested at each time point 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: The collected tumors were isolated into single-cell suspensions according to the in-house protocol (attached) and subjected to flow cytometry counting of CFSE-positive NK cells. e. Formulas and statistical analysis: i. Tumor volume = length × width 2 / 2 (Length and width are the longest and shortest diameters of the tumor, respectively) ii. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons using Tukey's test with GraphPad Prism version 7.0. P<0.05 is considered statistically significant.

[0160] [Table 1]

[0161] result: a. Safety: iii. Animals administered with both types of NK cells showed a mild to moderate acute reaction immediately after cell injection (G1-G2, mild to severe depression, lethargy, sluggishness, or unresponsiveness). iv.Two animals (out of 14) in the R7-19.1 group were found to have died within 24 hours of injection, while no deaths occurred in the CD19 t-haNK group. After 24 hours, the animals in the CD19 t-haNK group were able to recover, but the animals in the R7-19.1 group remained mildly depressed and appeared sluggish in response to stimuli (G1). They became more responsive at 48 hours (G0), but still appeared to have coat stiffness and tachypnea. b. NK cell homing: vi. The number of tumor-infiltrating NK cells at each time point is shown in Table 2 and in Figure 13 as a graph. 1. The four CCR7 receptor-CCL19 ligand combinations are as follows: a. CD19 t-haNK(registered trademark), K562 tumor: receptor-less - ligand-less [--]; b. CD19 t-haNK(registered trademark), K-19 tumor: receptor absent - ligand present [-+]; c.R7-19.1 cells, K562 tumor: receptor present - ligand absent [+-]; 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 homing of non-CR CD19 t-haNK cells to CCL19- or CCL19+ tumors ([--] and [-+], respectively; P<0.05). However, a direct comparison of R7-19.1 cell homing to CCL19-negative and CCL19-positive tumors in the same animals was not possible because many cells could not be recovered from two of the four K562 tumors in group B. viii. After 24 hours, there was no statistically significant difference in NK cell homing among any of the four CCR7 receptor-CCL19 ligand combinations. However, when comparing the homing of each NK cell line with CCL19+ and CCL19- tumors within the same animal, three out of four animals treated with R7-19.1 cells showed improved infiltration into CCL19+ tumors, while the majority of animals treated with CD19 t-haNK® showed similar levels of NK infiltration into both tumors, regardless of CCL19 expression (Figure 14). ix. After 48 hours, the total number of tumor-infiltrating NK cells decreased in all combinations, and there was no difference among the groups.

[0162] [Table 2]

[0163] This example demonstrates that the coexistence of the CCR7 receptor and CCL19 ligand resulted in more efficient NK cell infiltration at 3 hours post-administration. At 24 hours post-administration, in animals administered with non-CR aNK cells, NK cells showed similar levels of tumor homing in 3 out of 4 animals, regardless of CCL19 expression status. In contrast, in animals administered with R7-19.1 cells, NK cells were able to home to CCL19-positive tumors more efficiently in 3 out of 4 animals compared to parental non-ligand-expressing controls. The number of tumor-infiltrating NK cells decreased at 48 hours, regardless of receptor or ligand expression. R7-19.1 cells showed a particular tendency to home to CCL19 tumors at an earlier time (less than 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 NSG mice with intravenous CCL19-positive RAJI tumors. CCR7 is a chemokine receptor that induces cell migration toward gradients of chemokines CCL19 and CCL21, and is typically expressed in lymph nodes and other lymphoid organs, which are the primary disease sites of B-cell lymphoma. Based on the t-haNK platform, we generated chemokine-responsive NK-92® cells (i.e., R7-19.1 cells) expressing CD19-CAR, CCR7, CD16.158V, and ERIL-2. In addition to CCR7 expression, these cells possess cancer-targeted chimeric antigen receptors (CARs) for the CD19 cancer antigen, CD16 variants, 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] This study evaluated the antitumor effect of repeated intravenous (IV) administration of R7-19.1 cells in an IV xenograft model of Raji-19.5, which are Raji human Burkitt lymphoma cells genetically engineered to express CCL19 in NSG mice. Non-CCL7-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] Both NK cell lines showed a significant treatment effect in extending the survival of IV Raji-19.5 tumor-bearing animals compared to vehicle controls, while treatment with R7-19.1 cells yielded a significantly greater survival benefit than treatment with CD19 t-haNK cells. While clear treatment-related responses were observed with both NK cell lines, these responses are presumed to be mouse-specific issues associated with the administration of relatively high doses of human-derived cells.

[0167] Study Principles and Objectives: 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 effect of repeated IV administration of R7-19.1 cells was evaluated in an IV xenograft model of Raji-19.5 (a CCL19-expressing substrain of Raji) in NSG mice. Note that the original study protocol included additional animal groups (groups A-C) not included in this report. These were 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 substances. The test substances were R7-19.1 cells and CD19 t-haNK cells (non-CCR7-expressing control NK cells; clone 6). The R7-19.1 cells were cultured in a 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), the CD19 t-haNK cells were cultured in a 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 scid Il2rg tm1Wjl / SzJ (NSG) female mice, 10 - 11 weeks old at the start of the study (after quarantine and acclimation) and weighing 20 - 27 grams at randomization, were used. The number of animals used in the study was 30, and the supplier was The Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US). The animals were identified by ear tags, cage numbers, and tail mark numbers.

[0170] Raji-19.5 tumor model (cancer cell line) Cell culture medium: Raji-19.5 cancer cells were grown in ATCC-formulated RPMI-1640 modified 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 resuspended in serum-free medium at a concentration of 5×10 5 cells / mL and stored on ice prior to inoculation into animals. The cells used in the in vivo study had a viability of 97%.

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

[0173] Experimental Procedure Body weight: Animals were weighed before registration (after quarantine / acclimatization), before randomization, on each administration day (but before administration), on each post-administration day, and before euthanasia. Animals showing a weight loss of more than 20% compared to baseline (day 0) were euthanized in accordance with the institution's IACUC policy, and subsequently necropsy was performed at the discretion of the principal investigator.

[0174] Clinical observation: Animal mortality / morbidity rates were observed daily (G0-G4; see Table 2 in the study protocol in Appendix 1). Dying 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 quasi-randomized into three study groups of 10 mice each, based on animal body weight.

[0176] Test substance administration: Over four consecutive weeks, twice a week (days 3, 6, 10, 13, 17, 20, 24, and 27), R7-19.1 and control CD19 t-haNK cells that had grown in the logarithmic phase were collected by centrifugation and placed in serum-free growth medium in 5 × 10⁶ units. 7 At a concentration of cells / mL, with an injection volume of 200 μL, 1 × 10⁶ cells per mouse. 7 The cells were formulated for intravenous administration at the prescribed dose. All cell processing and formulation procedures were performed at room temperature. Cell viability was over 80% for all dose preparations. As shown in Table 3, group D was administered a vehicle control, while groups E and F were administered CD19 t-haNK cells and R7-19.1 cells, respectively.

[0177] Endpoints: Animals were euthanized when they met the criteria for paralysis, near death, or other endpoints defined by the institution's IACUC. The experiment was terminated on day 30 when the last surviving animal died from disease. Euthanasia was performed by CO2 inhalation followed by cervical dislocation. At the discretion of the principal investigator, some of the euthanized animals were autopsied to identify visible tumor nodules in the internal organs. Complete records of mortality and death events, as well as autopsy findings, are found in Appendix 5.

[0178] [Table 3]

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

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

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

[0182] result Effectiveness: The main measurement of effectiveness was the survival rate of the animals. Death events were counted if the animals were euthanized due to morbidity, paralysis, or weight loss exceeding 20%. Dead animals were not seen. As shown in Figure 15 and Table 4, treatment with both R7-19.1 and control CD19 t-haNK cells could significantly extend the survival of Raji-19.5 IV tumor-bearing animals compared to the vehicle control (P < 0.0001 for R7-19.1; P = 0.0002 for CD19 t-haNK, by log-rank test). The median survival periods increased by 6.5 days and 2.5 days respectively, which corresponded to increases of 30% and 12% respectively compared to the vehicle control. More importantly, the expression of the CCR7 chemokine-responsive receptor in R7-19.1 cells further increased the median survival period by 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 administration. In most cases, the animals were able to recover from the cell injection and showed weight recovery, resulting in variable weight change curves between doses. However, the first administration seemed 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 injection and are not specific to R7-19.1 cells. The weight recovery suggests that the weight loss was temporary and reversible. [[ID=1,6]]

[0185] Notably, towards the end of the study, the animals showed a rapid decrease in body weight, likely due to disease progression (Figure 16). Autopsies revealed tumor nodules in the liver, ovaries, and sometimes the spleen in almost all animals examined. One exception was the first mouse euthanized with CD19 t-haNK cells, which reached a weight loss of over 20% the day after the sixth dose. No visible tumor nodules were found during autopsy of this animal. Therefore, the exact cause of the weight loss justifying euthanasia could not be identified.

[0186] conclusion Newly prepared R7-19.1 cells 1 × 10 7 Intravenous administration of IV Raji-19.5 at cell / dose levels twice weekly for 4 weeks demonstrated a significant and statistically substantial antitumor effect in an IV Raji-19.5 xenograft model. This treatment resulted in a median survival increase of 6.5 days (30%) compared to the vehicle control group and 4 days (17%) compared to the CD19 t-haNK treatment group.

[0187] Clear treatment-related reactions were observed, but these were transient, and the surviving animals showed signs of recovery. These reactions are presumed to be mouse-specific issues associated with the administration of relatively high doses of human-derived NK cells, and are therefore unlikely to be translated into human.

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

[0189] Example 8. Comparative efficacy evaluation of CCR7-expressing R7-19.1 cells in NSG mice with subcutaneous CCL19-positive RAJI tumors. CCR7 is a chemokine receptor that induces cell migration toward gradients of chemokines CCL19 and CCL21, and is typically expressed in lymph nodes and other lymphoid organs, which are the primary disease sites of B-cell lymphoma. Based on the t-haNK platform, we generated chemokine-responsive NK-92® cells (i.e., R7-19.1 cells) expressing aCD19-CAR, CCR7, CD16.158V, and ERIL-2. In addition to CCR7 expression, these cells possess cancer-targeted chimeric antigen receptors (CARs) for the CD19 cancer antigen, CD16 variants, 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] This study evaluated the antitumor effect of repeated intravenous (IV) administration of R7-19.1 cells in a SC xenograft model of Raji-19.5, which are Raji human Burkitt lymphoma cells genetically engineered to express CCL19 in NSG mice. Non-CCL7-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 demonstrated a clear effect in suppressing tumor growth, with R7-19.1 treatment showing stronger inhibition than control CD19 t-haNK cells. Clear treatment-related responses were observed in both NK cell lines, but these responses are presumed to be mouse-specific issues associated with the administration of relatively high doses of human-derived cells.

[0192] Study Principles and Objectives: 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 effect of repeated IV administration of R7-19.1 cells was evaluated in IV and SC xenograft models of Raji-19.5 (a CCL19-expressing substrain of Raji) in NSG mice.

[0193] research materials Test substances: R7-19.1 cells (clone) and CD19 t-haNK cells (non-CCR7 expressing control NK cells; clone 6) were used as test substances, and growth medium was used as the 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] Study system: At the start of the study (after quarantine and acclimatization), 10-11 week old, with a body weight of 19-28 grams of NOD.Cg-Prkdc. scid Il2rg tm1Wjl Thirty female / SzJ(NSG) mice were used in the study. They were supplied by The Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US). The 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-based RPMI-1640 modified medium supplemented with 10% fetal bovine serum.

[0198] Cell harvesting: Logarithmic phase Raji-19.5 cells (16th generation) were harvested by centrifugation according to NantKwest's SOP_Suspension Cancer Cell Collection for In Vivo Studies. The cells were then washed, resuspended in serum-free medium, and mixed with an equal volume of Matrigel to form 2.5 × 10⁶ cells. 6 The cells were brought to a final concentration of cells / mL. Before inoculation into animals, the cells were stored on ice. The cells used in the in vivo study had a 97% viability rate.

[0199] Inoculation: 2.5 × 10⁶ doses were administered to 30 animals in a volume of 100 μL. 5 One cell was injected into the right flank of the abdomen. The skin was trimmed before injection.

[0200] Experimental Procedure Tumor volume measurement: After SC tumor transplantation, animals were examined at least twice a week for tumor engraftment. When the tumor was measurable, tumor volume (TV) was measured twice a week using a digital caliper 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 volume exceeding (T) or with ulcerative tumors were euthanized in accordance with the facility's IACUC policy and subsequently autopsied. Tumor growth inhibition (TGI) was calculated as follows: TGI = (T C -T t ) / ΔT C ×100% (in the formula, T C and T t ΔT is the mean tumor volume of the control and treatment groups at a specific time point; C (This refers to the change in mean tumor volume in the control group.)

[0201] Body weight: Animals were weighed before registration (after quarantine / acclimatization), before randomization, on each administration day (but before administration), on each post-administration day, and before euthanasia. Animals showing a weight loss of more than 20% compared to baseline (day 1) were euthanized in accordance with the facility's IACUC policy and subsequently necropped.

[0202] Clinical observation: Animal mortality / morbidity rates were observed daily (G0-G4; see Table 5). Dying or paralyzed animals were euthanized and subsequently necropsied.

[0203] Randomization: Mean tumor volume of 190 mm 3When this was reached, 30 mice were pseudo-randomized into three study groups with 10 mice per group to achieve similar tumor volumes between the groups. This was defined as day 1. Notably, due to the variation in tumor size by randomization, each group contained two subpopulations: 4 - 5 animals had large (>200 mm 3 ) tumors and 5 - 6 had small (<200 mm 3 ) tumors (see Figure 17).

[0204] Test substance administration: For 4 consecutive weeks, twice a week (on days 1, 4, 9, 12, and 15), R7-19.1 and control CD19 t-haNK cells that had proliferated in the logarithmic phase were recovered by centrifugation and formulated for intravenous administration at a dose of 1×10 7 cells / mouse at a concentration of 5×10 7 cells / mL and an injection volume of 200 μL. All cell processing and formulation procedures were carried out at room temperature. The cell viability was over 80% for all dose preparations. As shown in Table 5, group A was administered the vehicle control, and groups B and C were administered 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 facility's IACUC. Euthanasia was performed by CO2 inhalation followed by cervical dislocation. Based on the judgment of the study investigator, a portion of the euthanized animals was necropsied to identify visible tumor nodules in the internal organs.

[0206]

Table 5

[0207] Data analysis Calculation of tumor volume: Tumor volume = length × 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 These are the mean tumor volume of the control group and the treatment group at a specific time point, respectively, and T C (This refers to the change in mean tumor volume in the control group.)

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

[0210] result Efficacy: The primary measure of this study is tumor growth inhibition. In SC tumors with different initial tumor volumes, the vascular system and CCL19 gradient may develop differently, which could affect the distribution of the test substance 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 Figure 18, in a subpopulation of animals with large early tumors, treatment with NK cell lines was effective for both the vehicle control (tumor volume of 2000 mm²) and the vehicle control. 3 Compared to the NK cell-treated group (where 3 out of 4 animals had to be euthanized on day 9 due to exceeding a certain threshold or the presence of ulcerative tumors), the R7-19.1 group showed clear inhibition of tumor growth. In contrast, the majority of animals in the NK cell-treated group survived until day 12 or 15. More importantly, on day 15, the R7-19.1 group showed a superficial enhancement of tumor growth inhibition compared to the non-CCR7-expressing counterpart, achieving a 35% TGI. This difference was not statistically significant, likely due to the small cohort size (2 N in the CD19 t-haNK group and 4 N in the R7-19.1 group).

[0212] However, in animals that had 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 is thought to be due to the following three factors: 1) The vascular system of small tumors was underdeveloped, resulting in low exposure to / reach of the test substance, which may have led to failure of both NK cell therapies. 2) Chemotaxis via CCL19 requires a chemokine gradient, not just a response to the mere presence of chemokines. Such a gradient for CCL19 was not sufficiently probable in small tumors, which may have resulted in a lack of distinguishability between R7-19.1 cells and CD19 t-haNK controls. 3) There were some "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 administration. In most cases, animals were able to recover from cell injection and showed weight recovery, resulting in a fluctuating weight change curve between doses. However, the first administration appeared to produce the most acute and severe reaction, associated with the longest recovery time in both clinical symptoms and weight change. Such reactions are not uncommon in animals receiving intravenous NK infusion and are not specific to R7-19.1 cells. Weight recovery suggests that the weight loss was temporary and reversible.

[0214] conclusion Newly prepared chemokine-responsive R7-19.1 cells 1 × 10 7At cell / dose levels, IV administration twice weekly for 4 weeks resulted in clear inhibition of tumor growth in animals with large SCRaji-19.5 tumors; however, such an effect did not reach statistical significance, likely due to the small cohort size. This treatment regimen did not show therapeutic effect in animals with small initial tumor volumes. This may be due to the underdeveloped tumor vascular system and / or insufficient establishment of the chemokine gradient in small tumors. Clear treatment-related responses were observed; however, these responses were transient, and the animals showed signs of recovery. These responses are presumed to be mouse-specific issues associated with the administration of relatively high doses of human-derived NK cells and are therefore unlikely to be translated into human.

[0215] Example 9. Production of aTGFβ / PD-L1 CAR-modified NK-92(registered trademark) cells A TGFβ trap, composed of a single-stranded dimer of the extracellular domain of TGFβRII, was cloned into a quadricistronic plasmid vector also containing PD-L1 CAR, CD16, and the erIL-2 transgene (Figure 21). Modified NK-92 cells were generated by electroporating aNK cells with the quadricistronic plasmid. Modified NK-92® cells were selected using IL-2 depletion medium because IL-2-dependent non-transformed aNK cells 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] Limit dilution cloning Aliquots of polyclonal aTGFβ / PD-L1 t-haNK™ pooled cultures were diluted to a density of 3 cells / ml in growth medium without the addition of IL-2. This cell suspension was dispensed into 96-well plates at a volume of 200 μl per well, which corresponds to an average of 0.6 cells per well. The plates were incubated at 37°C for 10 days, after which cell growth was visually checked. Clones were harvested and transferred to larger containers for expansion culture 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 that does not contain 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 that did not contain 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 / antifungal cocktail. K562 cells were passaged every 2–5 days, or whenever the culture turned 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 μM beta-mercaptoethanol, and an antibiotic / antifungal cocktail. Otherwise, cells were passaged as K562 cells as described above.

[0222] Antibody staining for flow cytometry analysis: Cells were collected 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 appropriate conjugate antibodies (or isotype controls) in the dark at 4°C for 20 minutes, and then washed twice with FACS buffer. To detect CAR proteins, cells were incubated with biotinylated anti-F(ab')2 fragment antibody, followed by incubation with streptavidin-APC antibody. Samples were analyzed using a MACSQuant flow cytometer.

[0223] Cytotoxicity: Suspension-growing cell lines were resuspended by pipetting of 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 antibiotics / antifungal agents. Effector and target cells were mixed in 96-well plates in 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, and 0.15:1) and co-cultured for 4 hours 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 using a MACSquant flow cytometry instrument.

[0224] ADCC: Suspension-grown cell lines were resuspended by pipetting of the cell culture. Cell viability was determined by automated counting (trypan blue exclusion method). Target cells were labeled with PKH67-GL dye, and both target cells and effector cells were diluted to the required cell concentration in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotics / antifungal agents. Target cells were pre-incubated at room temperature for 30 minutes with monoclonal antibodies trastuzumab, rituximab, or without antibody. Antibody-labeled target cells (and unantibody controls) were then mixed with effector cells 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, and 0.15:1) and co-cultured for 4 hours in a 37°C incubator under a 5% CO2 atmosphere. Next, PI was added for fluorescent labeling of dead cells, and the assay was analyzed using a MACSquant flow cytometry instrument.

[0225] Quantification of TGFβ traps secreted from aTGFβ / PD-L1 t-haNK cells The sample supernatant for analysis was prepared by a first centrifugation step of 500×g for 5 minutes to remove cells, followed by a second centrifugation step of 2000×g for 5 minutes to remove cell debris. The sample supernatant was frozen at -80°C until analysis. The cell pellet from the 500×g centrifugation step was resuspended, the triplicate was pooled, and the cell density was recorded. The concentration of TGFβ traps in the sample supernatant was measured using the Human TGFβRII ELISA Detection Kit according to the manufacturer's instructions and compared to the provided standard. The TGFβ trap concentration was normalized to cell number and expressed as pg / ml / 10 6 Represented as cells. Figure 23 shows that all aTGFβ PD-L1 t-haNK clones are trapped in large amounts of TGFβ (approximately 6-13 ng / ml / 10 6 This indicates that the cells secreted something.

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

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

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

[0229] Figure 27 shows SUP-B15 combined with an anti-CD20 rituximab monoclonal antibody or an anti-Her2-neutrastuzumab monoclonal antibody. CD19KO / CD20+ This study demonstrates the ADCC activity of aTGFβ / PD-L1 t-haNK™ cells against cells (CD19-, CD20+, Her2-neu-, NK resistant). In a 4-hour cytotoxicity assay, aTGFβ / PD-L1 t-haNK cells showed resistance to SUP-B15 when combined with the anti-CD20 antibody rituximab. CD19KO / CD20+ We were able to effectively target and kill the target SUP-B15. Both haNK(registered trademark) and aTGFβ / PD-L1 t-haNK(trademark) clones, when combined with the anti-Her2 / neu control antibody trastuzumab, effectively targeted and killed the target SUP-B15. CD19KO / CD20+ The cells could not be killed.

[0230] Example 10. TGFβ traps secreted from modified NK-92(registered trademark) cells inhibit TGFβ activity. HEK293 cells genetically engineered with a TGFβ-responsive element (SMAD-binding promoter) that directly expresses a luciferase reporter gene show a dose-dependent increase in luciferase activity when treated with TGFβ (Figure 28).

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

[0232] Example 11. Production of IL-12 by modified NK-92(registered trademark) cells. NK-92 cells were transduced with a lentiviral construct (contract) encoding a functional IL-12 p70 dimer as a single-stranded polypeptide, either p35-p40 or p40-p35 oriented (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] Production of IL-12 / PD-L1 CAR-modified NK-92(registered trademark) cells The single-stranded dimer of IL-12 (scIL-12 p70) was cloned into a quadricistronic plasmid vector containing the PD-L1 CAR, CD16, and erIL-2 transgene. Modified NK-92 cells were generated by electroporating aNK cells with the quadricistronic plasmid (Figure 31). Modified NK-92® cells were selected using IL-2 depletion medium because IL-2-dependent non-transformed aNK cells could not survive in IL-2 depletion medium. Figure 33 shows that IL-12 / PD-L1 CAR modified NK-92® cells were able to secrete significant amounts of the scIL-12p70 cytokine.

[0234] The examples and embodiments described herein are for illustrative purposes only, and it will be understood that various modifications or changes in that regard should be suggested to those skilled in the art and should be included in the spirit and scope of this application and in the appended claims. All publications, sequence accession numbers, patents and patent applications cited herein are incorporated herein 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 concept of the invention as described herein. Therefore, the subject matter of the invention should not be limited beyond the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible way that is consistent with the context. In particular, the terms “includes” and “contains” should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the referenced element, component, or step exists, is utilized, or can be combined with other elements, components, or steps not explicitly referenced. Where a claim in 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, and not A and N, or B and N, etc.

Claims

1. These are recombinant modified natural killer (NK)-92 cells that have been stably transfected with nucleic acids. The nucleic acid encodes an anti-differentiation cluster 123 (CD123) chimeric antigen receptor (CAR), an Fc receptor, and a cytokine; The anti-CD123 CAR comprises an intracellular signaling domain derived from FcεRIγ; The recombinant modified cells express the anti-CD123 CAR and the Fc receptor on the cell surface of the recombinant cells. Recombinant modified NK-92 cells.

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

12.

3. Recombinant modified NK-92 cells according to claim 2, wherein the amino acid sequence of sequence number 12 has the F158V mutation.

4. Recombinant modified NK-92 cells according to claim 1, wherein the cytokine has IL-12 activity.

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

61.

6. The recombinant modified NK-92 cell according to claim 4, wherein the cytokine is a heterodimer 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 according to claim 1, wherein the anti-CD123 CAR comprises a CD8 hinge region and a CD28 transmembrane domain bound to an intracellular signaling domain from FcεRIγ.

8. The recombinant modified NK-92 cell according to claim 1, wherein the 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.

9. The recombinant modified NK-92 cell according to claim 8, wherein the anti-CD123 CAR comprises, in a single polypeptide chain, an anti-CD123 scFv moiety, 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 derived from FcεRIγ having the amino acid sequence of SEQ ID NO:

31.

10. Recombinant modified NK-92 cells that are stably transfected with recombinant nucleic acids and express anti-CD123 chimeric antigen receptor (CAR), Fc receptor, and cytokines from the recombinant nucleic acids, The anti-CD123 CAR contains an intracellular signaling domain derived from FcεRIγ, The anti-CD123 CAR and the Fc receptor are expressed on the cell surface of the recombinant modified NK-92. Recombinant modified NK-92 cells.

11. The recombinant modified NK-92 cell according to claim 10, wherein the Fc receptor has the amino acid sequence of SEQ ID NO: 12 and may have the F158V mutation.

12. Recombinant modified NK-92 cells according to claim 10, wherein the cytokine is single-stranded IL-12 having the amino acid sequence of SEQ ID NO:

61.

13. Recombinant modified NK-92 cells according to claim 10, wherein the cytokine is a heterodimer 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.

14. The recombinant modified NK-92 cell according to claim 10, wherein the 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.

15. The recombinant modified NK-92 cell according to claim 10, wherein the anti-CD123 CAR comprises, in a single polypeptide chain, an anti-CD123 scFv moiety, 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 derived from FcεRIγ having the amino acid sequence of SEQ ID NO: 31.

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