Genetically modified tetra-cis-trans subsystems comprising homing receptors or cytokines and chimeric antigen receptors for immunotherapy

Abstract

Provided herein are modified NK-92 cells comprising one or more nucleic acids encoding i) a homing receptor, 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 wherein the nucleic acid sequence is operably linked to a promoter. Further provided herein are modified NK-92 cells comprising one or more nucleic acids encoding i) an IL-12 and / 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 wherein the nucleic acid sequence is operably linked to a promoter. Also provided are compositions and kits comprising the modified NK-92 cells, as well as methods of using the modified cells to treat cancer.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of August 1, 2019, application number 201980051071.5, and invention name "Genetically modified tetracistronic system comprising homing receptors or cytokines and chimeric antigen receptors for immunotherapy".

[0002] This application claims priority to our co-pending U.S. Provisional Applications Serial No. 62 / 713,264 filed on August 1, 2018, Serial No. 62 / 713,278 filed on August 1, 2018, Serial No. 62 / 713,310 filed on August 1, 2018, and Serial No. 62 / 713,323 filed on August 1, 2018. Each of these applications is incorporated herein by reference in its entirety. Technical Field

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

[0004] The Background Description includes information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is an admission made that any publication specifically or implicitly referenced is prior art.

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

[0006] Cancer immunotherapy based on adoptively transferred tumor-specific cytotoxic lymphocytes holds promise for treating patients with malignancies. Despite early success in certain cancers, treatment of tumors remains a challenge, 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, pp. 2473-2480, 2012. In addition to modified T cells, NK cell-based immunotherapy is being explored. Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Natural killer (NK) cells, which typically account for approximately 10%-15% of circulating lymphocytes, bind and kill target cells that are not specific for an antigen and have no prior immune sensitization, including virus-infected cells and many malignant cells. Herberman et al., Science 214:24 (1981). NK-92® is a cytolytic cancer cell line that was found in the blood of subjects with non-Hodgkin's lymphoma and then immortalized in vitro. NK-92® cells are derived from NK cells but lack the major inhibitory receptors displayed by normal NK cells while retaining most of the activating receptors. However, NK-92® cells do not attack normal cells and do not cause unacceptable immune rejection in humans.

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

[0008] Thus, there remains a need for improvements in NK cells and NK cell-based therapies, particularly in the context of NK cell homing to and modulation of the tumor microenvironment. Summary of the invention

[0009] Provided herein are modified NK-92® cells comprising nucleic acids encoding a variety of functional elements. Functional elements are typically proteins or polypeptides that provide specific functions that improve the effectiveness of cells as cell lines for immunotherapy. On the one hand, NK-92® cells comprise a nucleic acid construct encoding four functional elements. In certain embodiments, the nucleic acid construct comprises a sequence encoding four functional elements operably connected to a promoter (referred to as a "tetracistronic construct").

[0010] In some embodiments, the first element encoded by the nucleic acid construct is a cytokine that provides selection for NK-92® cells expressing the 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 the endoplasmic reticulum IL-2 ("erIL-2"). In another embodiment, IL-15 is expressed with a signal sequence that directs IL-15 to the 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-γ receptor (FCγR). In some embodiments, the Fc-γ receptor is FCγRIII-A (also known as CD16), which is a low affinity Fc receptor that binds to IgG antibodies and activates ADCC. In some embodiments, the CD16 receptor comprises a phenylalanine (F)-valine (V) substitution at amino acid position 158 (F158V) of the mature form of the polypeptide (SEQ ID NO: 12) (corresponding to position 176 of the full-length form of the polypeptide containing the signal sequence). In one embodiment, the Fc receptor comprises the nucleic acid sequence of SEQ ID NO: 13 or the amino acid sequence of SEQ ID NO: 12.

[0012] In some embodiments, the first element and the second element 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, the third element encoded by the nucleic acid construct is a homing receptor. In some embodiments, the homing receptor is a cytokine receptor, a G protein coupled receptor, a chemokine receptor, a cell adhesion molecule, a selectin or an integrin. In some embodiments, the homing receptor is operably connected to a promoter that allows nucleic acid transcription. The modified NK-92® cells can migrate to the source of chemokines, which are ligands of receptors. Different from normal blood-derived NK cells, the modified NK-92® cells can develop into cell lines associated with human clinical trials, thereby providing obvious advantages for immunotherapy. Examples of homing receptors include G protein-coupled receptors, such as chemokine receptors, including but not limited to CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC or CXCL14 receptors; cytokine receptors; cell adhesion molecules, such as selectins, including L-selectin (CD62L); integrins, such as α4β7 integrin, LPAM-1 and LFA-1. In some embodiments, the homing receptor is a cell adhesion molecule, such as LFA-1. In some embodiments, the homing receptor is a selectin, such as L-selectin (CD62L). In some embodiments, the homing receptor is an integrin, such as α4β7 integrin, LPAM-1 or VLA-4. In some embodiments, the homing receptor is a CC or CXC chemokine receptor. Thus, in some embodiments, the third element encoded by the nucleic acid is a homing receptor described herein.

[0014] In some embodiments, the third element encoded by the nucleic acid construct is a secretory cytokine, whereby the cytokine enhances or improves the function of NK-92® cells as an immunotherapeutic agent. The cytokine can also regulate the tumor microenvironment. In some embodiments, the secretory cytokine that regulates the tumor microenvironment is IL-12 or IFN-α. Therefore, in some embodiments, the third element encoded by the nucleic acid construct is a cytokine, such as IL-12 or IFN-α.

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

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

[0017] It is known that TGF-β expression within a tumor inhibits the anti-tumor 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-chain dimer of the extracellular domain of the TGFβRII molecule, and most preferably comprises a single-chain dimer of the TGF-β receptor II extracellular domain.

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

[0019] In some embodiments, the fourth element encoded by the nucleic acid construct is an antigen binding protein ("ABP"). In some embodiments, the antigen binding protein specifically binds to a tumor-associated antigen. In some embodiments, the ABP comprises a fragment of an antibody, such as an scFv. In some embodiments, the antigen binding protein comprises a chimeric antigen receptor (CAR) or is a part of a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes an ABP or CAR that specifically binds to the following substances: 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 aspect, the modified NK-92® cells comprise a nucleic acid encoding IL-12. In another aspect, the modified NK-92® cells comprise a nucleic acid encoding a TGF-β trap. In some embodiments, the TGF-β trap comprises a single chain dimer of the extracellular domain of the TGFβRII molecule.

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

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

[0023] In some embodiments, the modified NK-92® cells comprise a nucleic acid encoding an antigen binding protein ("ABP"). In some embodiments, the antigen binding protein specifically binds to a tumor-associated antigen. In some embodiments, the ABP comprises a fragment of an antibody, such as an scFv. In some embodiments, the antigen binding protein comprises a chimeric antigen receptor (CAR) or is a portion of a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes an ABP or CAR that specifically binds to the following substances: 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 aspect, the modified NK-92® cells comprise a nucleic acid encoding a secreted cytokine that modulates the tumor microenvironment. In some embodiments, the cytokine that modulates the tumor microenvironment is a chemokine, such as XCL1, CCL5, CCL21, or CCL16. In some embodiments, the modified NK-92® cells comprise a nucleic acid encoding a Toll-like receptor (TLR) agonist. In some embodiments, the modified NK-92® cells comprise a nucleic acid encoding IL-12 or IFN-α. In some embodiments, the modified NK-92® cells comprise a nucleic acid encoding a TGF-β inhibitor (e.g., a peptide that inhibits TGF-β). In some embodiments, the modified NK-92® cells comprise a nucleic acid encoding a TGF-β trap. In some embodiments, the TGF-β trap comprises an extracellular domain of a TGFβRII molecule, or a single-chain dimer of an extracellular domain of a TGFβRII molecule.

[0025] On the one hand, the modified NK-92® cells comprise one or more, or multiple nucleic acid molecules encoding homing receptors, ABPs or CARs, Fc receptors and / or cytokines, which provide selection for NK-92® cells expressing the cytokine or allow the survival of NK-92® cells expressing the cytokine. Therefore, in some embodiments, the modified NK-92® cells comprise nucleic acid molecules encoding chemokine receptors, CARs, CD16, and erIL-2. In some embodiments, the modified NK-92® cells comprise nucleic acid molecules encoding CCR7 or CXCR2, CARs, CD16, and erIL-2. In some embodiments, the modified NK-92® cells comprise nucleic acid molecules encoding IL-12 or TGF-β traps, CARs, CD16, and erIL-2.

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

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

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

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

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

[0031] In some embodiments, 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 amino acid sequence of the CD8 hinge region, CD28 transmembrane and FcεRIγ signaling domain comprises a polypeptide or polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the nucleic acid construct further comprises a promoter that promotes transcription of the nucleic acid sequence. In some embodiments, the promoter is an inducible promoter. In some embodiments, the nucleic acid construct is a polycistronic vector comprising one or more internal ribosome entry sites (IRES) to enable translation initiation from the internal region of mRNA transcribed from the nucleic acid sequence. In some embodiments, the nucleic acid construct comprises a sequence encoding a 2A peptide, such as a T2A, P2A, E2A, or F2A peptide, to produce an equimolar level of polypeptide encoded by the same mRNA. In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding an antigen binding protein (ABP). In some embodiments, the ABP is a scFv or a codon-optimized scFv. In some embodiments, the ABP specifically binds to an antigen expressed by a tumor cell. In some embodiments, the ABP is part of a chimeric antigen receptor (CAR). In some embodiments, the construct comprises a nucleic acid encoding a cytokine, which provides selection of NK-92® cells expressing the cytokine (such as IL-2) or allows the NK-92® cells to survive. In one embodiment, the cytokine targets the endoplasmic reticulum. In one embodiment, the CAR scFv may comprise a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 39.

[0032] In some embodiments, the construct comprises Fig.10 In some embodiments, the NK-92® cell or cell line is genetically modified to express CD16 on the cell surface. In one embodiment, the NK-92® cell or cell line is genetically modified to express high affinity CD16 (F158V) on the cell surface.

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

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

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

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

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

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

[0039] In another aspect, methods for treating cancer or reducing tumor size are described. In some embodiments, methods for treating cancer or reducing tumor size include administering a therapeutically effective amount of modified NK-92® cells described herein to a subject in need thereof, wherein the administration treats cancer in the subject or reduces tumor size in the subject. In some embodiments, these methods include administering to the subject a therapeutically effective amount of modified NK-92® cells, the modified NK-92® cells comprising nucleic acids encoding homing receptors, ABPs or CARs that specifically bind to a target antigen, Fc receptors (such as CD16 or CD16-158V), and / or cytokines (such as erIL-2 or erIL-15). In some embodiments, these methods include administering to the subject a therapeutically effective amount of modified NK-92® cells, the modified NK-92® cells comprising nucleic acids encoding secreted cytokines, ABPs or CARs that specifically bind to a target antigen, Fc receptors (such as CD16 or CD16-158V), and / or cytokines (such as erIL-2 or erIL-15).

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

[0041] In another aspect, the use of the compositions described herein for treating a disease is provided. In some embodiments, the modified NK-92® cells described herein are provided for use as a drug for treating a disease. In some embodiments, the modified NK-92® cells described herein are provided for treating a disease. In some embodiments, the modified NK-92® cells comprise nucleic acids encoding homing receptors, ABPs or CARs that specifically bind to a target antigen, Fc receptors (such as CD16 or CD16-158V), and / or cytokines (such as erIL-2 or erIL-15). In some embodiments, the modified NK-92® cells comprise nucleic acids encoding secreted cytokines, ABPs or CARs that specifically bind to a target antigen, Fc receptors (such as CD16 or CD16-158V), and / or cytokines (such as erIL-2 or erIL-15). In some embodiments, the disease is cancer.

[0042] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a schematic diagram showing the plasmid pNKAT-CCR7-LP3 containing the CCR7 receptor at the AAVS1 locus for insertion into NK-92® cells.

[0044] Figure 2 It is a schematic diagram showing the plasmid pCRENFAT-CCL21 containing the NFAT-responsive CCL21 gene.

[0045] Figure 3 is a graph showing 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

[0046] Figure 4 is a graph showing the cytotoxic activity of modified NK-92® cells expressing CCR7 against K562 cells.

[0047] Figure 5is a graph showing the cytotoxic activity of modified NK-92® cells expressing CCR7 against HL-60 cells.

[0048] Fig. 6A and Figure 6B is a graph showing activation of the NFAT-luciferase reporter gene in NK-92 cells confirmed when NK-92 cells were electroporated with mRNA of CD19-CAR in combination with K562 and SUP-B15 cells.

[0049] Figure 7 is a graph showing the migration of modified NK-92® cells expressing CCR7 (Mi-aNK) toward the chemokines CCL19 and CCL21.

[0050] Figure 8 A diagram showing an exemplary method for in vitro testing of modified NK-92® cells described herein is shown. Activated NK-92® cells (aNK) are modified to express a chemokine receptor (e.g., CCR7), and target cells are modified to express a chemokine that binds to the receptor (e.g., CCL19 or CCL21). As shown, the modified NK-92® cells are tested in a modified Boyden chamber Transwell assay.

[0051] Fig. 9 A representative cytotoxicity assay using modified NK-92® cells described herein is shown. Figure 8 The cytotoxicity of modified NK-92® cells against K562 target cells expressing and secreting one or two chemokine ligands was investigated. The ML4 clone showed the highest percentage of target cell lysis, and this percentage increased when the K562 target cells simultaneously expressed CCL19 and CCL21.

[0052] Fig.10 is a schematic diagram showing the plasmid pNKAT-CCR7-CD19CAR-CD16-ERIL2 (referred to as a "tetracistronic vector"), which can be used to stably transfect cells at a single insertion site.

[0053] Fig.11 Is displayed from Fig.10 Schematic diagram of the linearized plasmid.

[0054] Fig.12Cell surface expression of CCR7, CD16 and CD19 CAR by NK-92® cells is shown. "aNK" is a wild-type NK-92® cell line. "ML4" is an aNK cell line transfected with a nucleic acid construct encoding CCR7 operably linked to a promoter (i.e., Mi-aNK). "P2" is an aNK cell line transfected with a nucleic acid construct encoding CCR7, CD16, ER-IL2 and CD19 CAR (i.e., Mi-T-haNK).

[0055] Fig.13 . Homing of non-CR and Mi-T-haNK cells to parental or CCL19-expressing tumors at the indicated hours after NK cell administration. Data are mean ± SEM. - and + signs indicate the expression status of CCR7 receptor (first symbol) and CCL19 ligand (second symbol). *, P < 0.05 as determined by one-way ANOVA followed by Tukey's test for multiple comparisons. The last figure presents the time course curves.

[0056] Fig.14 . Head-to-head comparison of infiltration of non-CR and Mi-T-haNK cells into parental or CCL19 expressing tumors from individual animals 24 hours after dosing. Three of the four animals that received Mi-T-haNK cells showed higher infiltration into CCL19+ tumors, while three of the four animals that received non-CR CD19 t-haNK cells showed similar levels of infiltration into K562 and K-19 tumors. One "outlier" animal in each group is indicated by a dotted line.

[0057] Fig.15 Survival curves of IV Raji-19.5 tumor-bearing animals are illustrated. Survival curves of Raji-19.5 IV tumor-bearing NSG mice treated with vehicle, CD19 t-haNK cells, or R7-19.1 cells. Statistical analysis was performed by log-rank (Mantel-Cox) test. ***, P = 0.0002; ****, P < 0.0001.

[0058] Fig.16Body weight changes in the IV Raji-19.5 tumor model are illustrated. Body weight change curves (% change from body weight on day 0) of IV Raji-19.5 tumor-bearing animals treated with vehicle, CD19 t-haNK cells, or R7-19.1 cells. Data are mean ± SEM. Red arrows indicate dosing days. Weight measurements made on the day of dosing were performed prior to dosing. For all time points until day 20, the curves for the NK cell-treated group were statistically significantly different (P < 0.05) from those for the vehicle control group by 2-way ANOVA followed by Tukey's test for multiple comparisons.

[0059] Fig.17 The sizes of SC Raji-19.5 tumors at the time of randomization are illustrated. The sizes of individual tumors at the time of randomization are shown. Black boxes enclose tumors > 200 mm 3 The blue box encloses large tumors < 200 mm 3 Group mean ± SEM is also shown.

[0060] Fig.18 Tumor growth of a subpopulation of large tumors in SC Raji-19.5 tumor-bearing mice is illustrated. (A) Group analysis. Data are mean ± SEM. Statistical analysis was performed using 2-way mixed-effects analysis followed by multiple comparisons by Tukey's test. Statistical significance was not reached. (B) Individual curves. Red arrows indicate dosing days. Tx: treatment.

[0061] Fig.19 Tumor growth of a small tumor subpopulation in SC Raji-19.5 tumor-bearing mice is illustrated. (A) Group analysis. Data are mean ± SEM. Statistical analysis was performed using a two-way mixed effects analysis followed by multiple comparisons by Tukey's test. No statistical significance was detected between any 2 groups at any time point. (B) Individual curves. Red arrows indicate dosing days. Tx: treatment.

[0062] Fig. 20The weight changes in the SC Raji-19.5 tumor model are illustrated. The weight change curves of SC Raji-19.5 tumor-bearing animals treated with vehicle, CD19 t-haNK cells or R7-19.1 cells (% change in body weight compared to day 1). Data are mean ± SEM. Red arrows indicate dosing days. Body weight measurements made on the day of dosing were performed before dose administration. For all time points before day 16, the curves of the NK cell-treated group were statistically significantly different from those of the vehicle control group by two-factor mixed effects analysis followed by multiple comparisons by Tukey's test.

[0063] Fig.21 An example of a PD-L1 CAR construct equipped with a tetracistronic TGFβ-trap is illustrated.

[0064] Fig. 22 Expression analysis of PD-L1 CAR and CD16 in PD-L1 (TGFβ-trap) t-haNK clones is illustrated.

[0065] Fig.23 It is demonstrated that TGFβ trap is secreted into the culture supernatant of TGFβ trap / PD-L1 t-haNK clones.

[0066] Fig.24 The cytotoxicity of the tetracistronic TGFβ-trap construct towards K562 target cells is demonstrated.

[0067] Fig.25 CAR killing of SUP-B15 target cells expressing PD-L1 by a tetracistronic TGFβ-trap construct is demonstrated.

[0068] Fig.26 CAR killing of MDA-MB 231 target cells by a tetracistronic TGFβ-trap construct is demonstrated.

[0069] Fig. 27 Description of the tetracistronic TGFβ-trap construct for SUP-B15 CD19-CD20+ ADCC.

[0070] Fig.28 TGFβ / SMAD luciferase reporter HEK293 cells induced by TGFβ are shown.

[0071] Fig.29 Secreted TGFβ-trap sequestered TGFβ and suppressed luciferase expression in a HEK293T reporter assay are illustrated.

[0072] Fig.30 IL-12 secretion from IL-12 virus-transduced NK-92® cell line is demonstrated.

[0073] Fig.31 An example of a tetracistronic IL-12 / PD-L1 t-haNK construct is described.

[0074] Fig.32 Cytotoxicity data for CCR7CD19 t-haNK cells are illustrated.

[0075] Fig.33 IL-12 secretion by the IL-12 / PD-L1 t-haNK™ cell line is demonstrated. definition

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the field of immunology and immunotherapy.

[0077] Throughout this specification and the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0078] The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the present invention.As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0079] All numerical designations (e.g., pH, temperature, time, concentration, amount, and molecular weight, including ranges) include variations commonly encountered by those of ordinary skill in the art. Thus, as appropriate, numerical values ​​may include (+) or (-) variations in increments of 0.1 or 1.0, depending on the relevant significant digits. It should be understood that, although not always explicitly stated, all numerical designations may be preceded by the term "about." As used herein, the term "about" may also mean that the value may vary by ± 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0080] It is also understood, although not always explicitly stated, that the reagents described herein are exemplary only and that equivalents thereof are known in the art.

[0081] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0082] The term "comprising" is intended to mean that compositions and methods include the listed elements, but not excluding other elements. When used to define compositions and methods, "consisting essentially of shall mean excluding other elements of any significance to the combination. For example, a composition consisting essentially of the elements defined herein will not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of shall mean excluding more than trace amounts of other ingredients and substantial method steps listed. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0083] The term "homing receptor" refers to a receptor that activates a cellular pathway that directly or indirectly causes the cell to migrate to a target cell or target tissue. For example, homing receptors expressed by leukocytes are used by leukocytes and lymphocytes to enter secondary lymphoid tissues via high endothelial venules. Homing receptors can also be used by cells to migrate to a source of a chemical gradient such as a chemokine gradient. Examples of homing receptors include G protein-coupled receptors, such as chemokine receptors, including but not limited to CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, and DARC; cytokine receptors; cell adhesion molecules, such as selectins, including L-selectin (CD62L); integrins, such as α4β7 integrin, LPAM-1, and LFA-1. A homing receptor typically binds to a cognate ligand on a target tissue or target cell. In some embodiments, a homing receptor binds to an addressin on the venular endothelium, such as mucosal vascular addressin protein cell adhesion molecule 1 (MAdCAM-1).

[0084] As used herein, "immunotherapy" refers to the use of modified or unmodified NK-92® cells, naturally occurring or modified NK cells, or T cells, alone or in combination, and capable of inducing cytotoxicity upon contact with target cells.

[0085] As used herein, "natural killer (NK) cells" are cells of the immune system that kill target cells in the absence of specific antigenic stimulation and without restriction based on major histocompatibility complex (MHC) class. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.

[0086] The term "endogenous NK cells" is used to refer to NK cells that originate from a donor (or patient), as distinct from the NK-92® cell line. Endogenous NK cells are typically a heterologous cell population in which NK cells have been enriched. Endogenous NK cells can be used for autologous or allogeneic treatment of a patient.

[0087] The term "NK-92" refers to natural killer cells derived from a highly potent unique cell line described by Gong et al. (1994), the rights to which are owned by NantKwest® (hereinafter referred to as "NK-92® cells"). The immortalized NK cell line was originally obtained from a patient with non-Hodgkin's lymphoma. Unless otherwise indicated, the term "NK-92®" refers to the original NK-92® cell line as well as NK-92® cell lines that have been modified (e.g., by the introduction of foreign genes). NK-92® cells and exemplary and non-limiting modifications thereof are described in U.S. Pat. Nos. 7,618,817, 8,034,332, 8,313,943, 9,181,322, 9,150,636, and published U.S. 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 of ordinary skill in the art, and such cells are available from NantKwest, Inc. to those of ordinary skill in the art.

[0088] The term "aNK" refers to unmodified natural killer cells derived from the highly efficient unique cell line described by Gong et al. (1994) (the rights of which are owned by Nant West) (hereinafter referred to as "aNK® cells"). The term "haNK" refers to natural killer cells derived from the highly efficient unique cell line described by Gong et al. (1994) (the rights of which are owned by Nant West) that have been modified to express CD16 on the cell surface (hereinafter referred to as "CD16+ NK-92® cells" or "haNK® cells"). In some embodiments, CD16+ NK-92® cells contain a high affinity CD16 receptor on the cell surface. The term "taNK" refers to natural killer cells derived from the highly efficient unique cell line described by Gong et al. (1994) (the rights of which are owned by Nant West) that have been modified to express a chimeric antigen receptor (hereinafter referred to as "CAR-modified NK-92® cells" or "taNK® cells"). The term "t-haNK" refers to natural killer cells derived from a highly efficient unique cell line described by Gong et al. (1994) (whose rights are owned by Nant West), modified to express CD 16 on the cell surface and to express a chimeric antigen receptor (hereinafter referred to as "CAR-modified CD16+ NK-92® cells" or "t-haNK cells"). In some embodiments, t-haNK cells express a high affinity CD16 receptor on the cell surface.

[0089] The terms "chemokine-targeted t-haNK" and "Mi-T-haNK" refer to t-haNK cells that have been modified to express chemokine receptors on the cell surface.

[0090] 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. Typically, cytotoxic activity involves killing target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of effectors that lyse the plasma membrane of target cells, thereby destroying their physical integrity. This results in the killing of target cells. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK-92® cells is due to cytolysis.

[0091] The term "killing" of a cell / cell population is intended to include any type of manipulation that will result in the death of that cell / cell population.

[0092] The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to a portion of an antibody called the Fc region. Binding of the Fc region of an antibody to the cell's Fc receptor (FcR) stimulates the cell's phagocytic or cytotoxic activity through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified according to the type of antibody they recognize. For example, Fc-gamma receptors (FCγRs) 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 commonly found on NK cells. NK-92® cells do not express FCγRIII-A. Fc-ε receptors (FcεRs) bind to the Fc region of IgE antibodies.

[0093] As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen binding domain fused to an intracellular signaling domain. CARs can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen binding domain is a scFv that is specific for an antigen found on the target cell. Based on the specificity of the scFv domain, NK-92® cells expressing CARs target cells that express certain antigens on the cell surface. The scFv domain can be engineered to recognize any antigen, including tumor-specific antigens. For example, the CD19 CAR recognizes CD19, a cell surface marker expressed by certain cancers.

[0094] As used herein, the term "tumor-specific antigen" refers to an antigen present on cancer cells or neoplastic cells but not detectable on normal cells derived from the same tissue or lineage as the cancer cells. As used herein, tumor-specific antigens also refer to tumor-associated antigens, i.e., antigens expressed at higher levels on cancer cells than on normal cells derived from the same tissue or lineage as the cancer cells.

[0095] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can 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 can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, nucleotide structural modifications can be performed before or after polynucleotide assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the invention includes both a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0096] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and when the polynucleotide is RNA, uracil (U) replaces thymine. Thus, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule.

[0097] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Sequence similarity can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The percentage of sequence similarity between sequences is a function of the number of matching positions or homologous positions that the sequences have in common within a given comparison window. A sequence may be at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence described herein.

[0098] In the case of two or more nucleic acid sequences or peptide sequences, the term identical or percentage identity refers to as measured using BLAST or BLAST 2.0 sequence comparison algorithms with the following default parameters, or by artificial comparison and visual inspection measurement method (see, for example, NCBI website, etc.), two or more sequences or subsequences are identical, or have a specified percentage of identical amino acid residues or nucleotides (that is, when comparing and comparing maximum correspondence on a comparison window or a specified area, there is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity on a specified area). Such sequences are then called to be substantially identical. This definition also refers to or can be applied to the complementary sequence of a test sequence. This definition also includes sequences with deletions and / or additions, and those sequences with substitutions. As described below, preferred algorithms can consider room, etc. In some embodiments, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0099] For sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer; if necessary, subsequence coordinates are specified; 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 percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0100] As used herein, a comparison window includes reference to a segment having any number of consecutive positions selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, and a sequence can be compared to a reference sequence having the same number of consecutive positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be accomplished, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology [Current Protocols in Molecular Biology] (Ausubel et al., ed., 1995 Supplement)).

[0101] The preferred example of the algorithm suitable for determining the percentage of sequence identity and sequence similarity is BLAST and BLAST 2.0 algorithm, which are respectively described in Altschul et al., Nuc. Acids Res. [Nucleic Acids Research] 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. [Molecular Biology] 215:403-410 (1990). Using BLAST and BLAST 2.0, the percentage of sequence identity of nucleic acid or protein is determined with the parameters described herein. The software for performing BLAST analysis can be publicly obtained through the National Center for Biotechnology Information (National Center for Biotechnology Information), as known in the art. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of a selected length (W) in the query sequence, which match or satisfy some positive threshold score T when compared with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for starting searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score can be calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction terminates when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative scoring residue alignments; 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 expectation (E) represents the number of different alignments whose scores are equal to or higher than the expected score that would occur by chance in a database search. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) with alignments (B) of 50, expectation (E) of 10, M = 5, and N = -4.

[0102] The term transformation as used herein refers to the process of introducing an exogenous or heterologous nucleic acid molecule (e.g., a vector or a recombinant nucleic acid molecule) into a recipient cell. An exogenous or heterologous nucleic acid molecule may or may not be integrated into (i.e., covalently linked to) the chromosomal DNA that constitutes the host cell genome. For example, an exogenous or heterologous polynucleotide may be maintained on an additional element such as a plasmid. Alternatively or additionally, an exogenous or heterologous polynucleotide may be integrated into a chromosome so that it is inherited by daughter cells through chromosome 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; biolistic delivery; retroviral infection; lipofection; and direct microinjection of DNA into cells.

[0103] As used with respect to cells, the term "transformed" refers to a cell that has undergone transformation as described herein such that the cell carries exogenous or heterologous genetic material (e.g., recombinant nucleic acid). The term "transformed" may also or alternatively be used to refer to a cell, cell type, tissue, organism, etc. that contains exogenous or heterologous genetic material.

[0104] As used herein with respect to introducing a nucleic acid into a cell or organism, the term "introduced" is intended to have the broadest meaning, and is intended to encompass, for example, introduction by transformation methods (e.g., calcium chloride-mediated transformation, electroporation, particle bombardment), and also encompasses introduction by other methods including transduction, conjugation, and mating. Optionally, the nucleic acid is introduced into a cell or organism using a construct.

[0105] The terms "modified" and "recombinant" when used with respect to cells, nucleic acids, polypeptides, vectors, etc., indicate that the cells, nucleic acids, polypeptides, vectors, etc. have been modified by or are the result of laboratory methods, and are not naturally occurring. Thus, for example, modified cells include cells produced or modified by laboratory methods, such as by transformation methods for introducing nucleic acids into cells. Modified cells may include nucleic acid sequences that are not found in the native (non-recombinant) form of the cell, or may include nucleic acid sequences that have been altered, such as a nucleic acid sequence linked to a non-native promoter.

[0106] As used herein, the term "exogenous" refers to a substance, such as a nucleic acid (e.g., a nucleic acid including a regulatory sequence and / or a gene) or a polypeptide, that is artificially introduced into a cell or organism and / or is not naturally present in the cell in which it is located. In other words, a substance such as a nucleic acid or a polypeptide originates from outside the cell or organism into which it is introduced. An exogenous nucleic acid may have a nucleotide sequence identical to a nucleic acid naturally present in a cell. For example, an NK-92® cell may be engineered to include a nucleic acid having an NK-92® sequence, such as heparanase. Optionally, an endogenous NK-92® heparanase sequence is operably linked to a gene having a regulatory sequence that is not involved under natural conditions. Although the NK-92® heparanase sequence may naturally exist in a host cell, according to the present disclosure, the introduced nucleic acid is exogenous. An exogenous nucleic acid may have a nucleotide sequence different from any nucleic acid naturally present in a cell. For example, an exogenous nucleic acid may be a heterologous nucleic acid, i.e., a nucleic acid from a different species or organism. Therefore, an exogenous nucleic acid may have a nucleic acid sequence identical to a nucleic acid naturally found in a source organism, but different from a cell into which the exogenous nucleic acid is introduced. As used herein, the term "endogenous" refers to a nucleic acid sequence that is natural to a cell. As used herein, the term "heterologous" refers to a nucleic acid sequence that is not natural to a cell, i.e., is from an organism different from the cell. The terms "exogenous" and "endogenous" or "heterologous" are not mutually exclusive. Therefore, a nucleic acid sequence can be exogenous and endogenous, meaning that the 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, a nucleic acid sequence can be exogenous and heterologous, meaning that the nucleic acid sequence can be introduced into a cell, but has a sequence that is non-natural to the cell, such as a sequence from a different organism.

[0107] As used herein, a control or standard control refers to a sample, measurement or value used as a reference (usually a known reference) for comparison with a test sample, measurement or value. For example, a test cell, such as a cell transformed with a nucleic acid sequence encoding an Fc receptor gene, can be compared with a known normal (wild type) cell (e.g., a standard control cell). A standard control can also represent an average measurement or average value collected from a population of cells that do not express an Fc receptor or do not have Fc receptor activity or have a minimal level of Fc receptor activity (e.g., a standard control cell). The skilled person will recognize that a standard control can be designed to assess many parameters (e.g., RNA levels, polypeptide levels, specific cell types, etc.).

[0108] The term "expression" refers to the production of a gene product (e.g., a protein). The term "transient" when referring to expression means that the polynucleotide is not incorporated into the genome of the cell. The term "stable" when referring to expression means that the polynucleotide is incorporated into the genome of the cell, or that a positive selection marker (i.e., an exogenous gene expressed by the cell that has a benefit under certain growth conditions) is used to maintain expression of the transgene.

[0109] 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 a preferred embodiment, the cytokine is IL-2.

[0110] The term "cytokines that modulate the tumor microenvironment" refers to molecules expressed by NK-92® cells and that act to increase anti-tumor responses. Certain cytokines can suppress the response of the endogenous immune system to tumors and thus reduce the effectiveness of immunotherapy in treating cancer. Therefore, the term also includes inhibitors of cytokines that promote tumor growth, such as peptide inhibitors and / or ligands or receptors that bind to cytokines that promote tumor growth (e.g., ligand traps).

[0111] As used herein, the term "vector" refers to a non-chromosomal nucleic acid containing an intact replicon so that when placed in a permissive cell, for example, by a transformation process, the vector can be replicated. A vector can replicate in one cell type (such as bacteria), but has limited or no ability to replicate in another cell (such as a mammalian cell). A vector can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles, comprising DNA condensed with cationic polymers, such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine, contained in liposomes in some cases; and the use of ternary complexes comprising 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.

[0112] As used herein, the term "targeted" is intended to include, but is not limited to, directing a protein or polypeptide to a suitable destination within or outside a cell when it comes to protein expression. Targeting is typically achieved by a signal peptide or targeting peptide, which is a segment of amino acid residues in a polypeptide chain. These signal peptides can be located anywhere within the polypeptide sequence, but are typically located at the N-terminus. The polypeptide can also be engineered to have a signal peptide at the C-terminus. The signal peptide can direct the polypeptide to be extracellularly cleaved and localized to the plasma membrane, Golgi apparatus, endosomes, endoplasmic reticulum, and other cellular compartments. For example, a polypeptide having a specific amino acid sequence at its C-terminus (e.g., KDEL) is retained in the ER lumen or transported back to the ER lumen.

[0113] As used herein, the term "targeting" refers to the ability of NK-92® cells to recognize and kill tumor cells (i.e., target cells) when referring to the targeting of tumors. The term "targeted" in this context refers to the ability of, for example, CAR expressed by NK-92® cells to recognize and bind to cell surface antigens expressed by tumors.

[0114] As used herein, the term "transfection" refers to the insertion of a nucleic acid into a cell. Transfection can be performed using any means that allow nucleic acid to enter a cell. DNA and / or mRNA can be transfected into a cell. Preferably, the transfected cell expresses a gene product (i.e., a protein) encoded by the nucleic acid.

[0115] For the convenience of the reader, titles or subtitles may be used in the specification, but this is not intended to affect the scope of the present invention. In addition, some terms used in this specification are defined in more detail below. DETAILED DESCRIPTION

[0116] Provided herein are engineered cells that use a cytotoxic activated natural killer cell line (NK-92) as a basis to improve immunotherapy against cancer and tumors and / or increase homing (migration) to a target of interest. In some embodiments, NK-92® cells are engineered to express homing receptors that are known to direct lymphocytes to lymph nodes when expressed. In some embodiments, NK-92® cells are engineered to express secreted cytokines that modulate the tumor microenvironment or inhibitors that block cytokines that modulate the tumor microenvironment.

[0117] The present disclosure provides the benefits of using tetracistronic vectors to insert multiple genes driven by a single highly active promoter to generate stable immunotherapeutic cell lines for clinical immunotherapy. Tetracistronic vectors string together four genes under the control of a single promoter using one or more approaches, including P2A peptides and IRES elements, and are linked to the expression of a final element - in this case, a selection agent whose expression is required to maintain cell survival and / or expansion.

[0118] In the proof-of-concept example, the four genes used to generate modified gene expression profiles in therapeutic cell lines are: CCR7, CD19 chimeric antigen receptor, high affinity variant of CD16, and endoplasmic reticulum-bound IL-2. In addition to its role in stimulating the cytotoxic capacity of the NK-92®-based cell line into which it is integrated, ER-bound IL-2 is also used as a selection agent. The gene producing IL-2 is placed at the end of the tetracistronic vector, farthest from the promoter, so the most likely element to be lost should be the gene construct fragment (leading to negative selection and self-excision from the library, because IL-2 is required for the continued survival of cells). However, if all elements are successfully integrated into the genome, cells will be selected by removing IL-2 from the culture medium, because only those cells that have integrated their own IL-2 source can survive. Since the IL-2 element is farthest from the promoter, this is conducive to the complete integration of the entire box of the four elements-can be further verified by flow cytometric staining analysis of other components (see Examples).

[0119] The constructs described herein offer the advantage of reducing development time for generating new therapeutic cell lines, as well as reducing stress and adverse effects on cells from multiple rounds of genome manipulation and subsequent selection. Additionally, by placing the selection agent (in this case ER-IL-2) at the end of the construct, it is expected that it will be difficult for cells to silence any given component of the construct without resulting in IL-2 starvation due to the nature of RNA transcription and processing. Therefore, stable cell lines constructed in this manner should maintain expression of all components already contained in the vector as long as they continue to produce their own IL-2.

[0120] To demonstrate proof of concept, four specific objectives were addressed by the 4 components that are part of this tetracistronic construct. IL-2 acts as a selector and is a known agonist of the cytotoxic effects of NK-92® cells, making these cells effective cancer therapeutics. The CCR7 element (CC chemokine receptor type 7) is a chemokine receptor responsible for migrating immune cells expressing it in the direction of a chemokine gradient of ligands CCL19 and CCL21 that are usually expressed in lymph nodes. In one embodiment, the CCR7 element contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 (CCR7 sequence). In one embodiment, CCL21 contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 (CCL21 sequence). In one embodiment, CCL19 contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 16 (CCL19 sequence).

[0121] The CD19 CAR (chimeric antigen receptor) is a construct designed to increase the cytotoxicity of cells when they encounter a CD19 differentiation population on the surface of the cells they encounter (a differentiation population that is commonly expressed on normal and malignant B cells), and has shown efficacy in targeted immunotherapy trials against B-cell lymphomas. The high affinity CD16 receptor allows the modified NK-92® cells to recognize and respond to cells that have been recognized by IgG antibodies, such as those used as monoclonal antibodies in cancer treatment regimens, such as Rituxumab and Herceptin. When an immune cell armed with the CD16 receptor encounters a cell coated in one of these antibodies, it triggers ADCC (antibody-dependent cellular cytotoxicity) and attempts to destroy the cell. In effect, this allows cells that have been so armed to be used in combination treatment regimens with monoclonal antibodies against cancer neoantigens, etc. While each of these components has its own utility, it is proposed that this particular combination, in combination, will result in an effective therapy for B cell lymphomas that are able to migrate to common sites of tumor growth (lymph nodes), where they recognize the B cell antigen CD19 and initiate CAR-mediated cytotoxicity, or work in conjunction with a monoclonal antibody such as rituximab to avoid the possibility of antigen escape. It should be understood that this proof-of-concept example is non-limiting, and that NK-92® cells can be modified using the methods described herein to express other homing receptors and / or CARs targeting other target antigens in order to generate effective immunotherapeutic cell lines.

[0122] As described herein, modified NK-92® cells have been produced, and after electroporation with a linearized gene construct containing a CCR7 expression cassette together with a removable selection cassette with a selection marker, the CCR7 lymph node homing receptor driven by the elongation factor 1a (EF1a) promoter has stable long-term expression. After one week of puromycin selection, followed by serial dilution cloning, a monoclonal cell line that maintains high levels of CCR7 expression was established. These NK cells overexpressing CCR7 have functional responses to lymph node-associated chemokines CCL21 and CCL19 in migration / invasion assays. In one embodiment, the EF1a promoter contemplated herein may include 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).

[0123] In some exemplary embodiments, the chemokines and homing receptors contemplated herein may comprise a nucleotide sequence corresponding to SEQ ID NO: 44 (CCR7 amino acid sequence), or SEQ ID NO: 45 (CCL19 amino acid sequence), or SEQ ID NO: 46 (CCL21 amino acid sequence), or SEQ ID NO: 47 (CXCR2 nucleotide sequence), or SEQ ID NO: 48 (CXCR2 amino acid sequence), or SEQ ID NO: 49 (CXCL14 nucleotide sequence), or SEQ ID NO: 50 (CXCL14 amino acid sequence), or SEQ ID NO: 51 (CD62L nucleotide sequence), or SEQ ID NO: 52 (CD62L amino acid sequence), or SEQ ID NO: 53 (IL-8 nucleotide sequence), or SEQ ID NO: 54 (IL-8 amino acid sequence), or SEQ ID NO: 55 (CXCL1 nucleotide sequence), or SEQ ID NO: 56 (CXCL1 amino acid sequence) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0124] Target engagement in NK-92® cells that recognize susceptible cell lines was confirmed by activation of the NFAT transcription factor and its nuclear translocation. Target engagement involving the FceRIg or CD3ζ pathways, including ADCC or CAR-mediated target recognition, was sufficient to induce NFAT activation in NK-92® cells. This was demonstrated by insertion of a reporter gene cassette containing 3 terminator regions flanking the NFAT binding domain and a minimal promoter driving firefly luciferase. Activation of NFAT through the CD3ζ pathway was achieved by electroporation of CD19 CAR mRNA into this reporter cell line, followed by co-culture with SUP-B15 (CD19+, but resistant to nonspecific cytotoxicity), resulting in luciferase expression.

[0125] In one embodiment, the NFAT response element sequence (binding site for NFAT activation) contemplated herein may comprise a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0126] In one embodiment, the minimal promoter contemplated herein (downstream of the three NFAT response elements) may comprise a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5.

[0127] In one embodiment, the complete NFAT response cassette contemplated herein (poly A + pause site, followed by 3 NFAT response elements, followed by a minimal promoter) may comprise a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6.

[0128] In one embodiment, the complete sequence contemplated herein for initial insertion (EF1a promoter, CCR7 gene with poly A, and LoxP-flanked puromycin resistance gene driven by the ubiquitin promoter, all wrapped in homology arms targeting the AAVS1 locus) may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7.

[0129] In one embodiment, the complete sequence contemplated herein for the second insertion (containing a NFAT response cassette driving CCL21+poly A and a FRT embedded blasticidin resistance gene driven by CMV) may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8. The SEQ ID NO: 8 sequence may replace the LoxP-flanked puromycin resistance cassette from the first insertion, i.e., the FRT sequence surrounding the blasticidin gene in this sequence, to allow for subsequent removal or replacement of the cassette using similar Flp-FRT recombination.

[0130] 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's lymphoma (Gong et al., Leukemia. 8:652-8 (1994)). NK-92® cells are characterized by the expression of CD56 in the absence of CD3, CD8, and CD16. 亮 and CD2.CD56 亮 / CD16 阴性The / low phenotype is typical of a small part of NK cells in peripheral blood, which has an immunomodulatory function as a cytokine producer. Unlike normal NK cells, NK-92® lacks the expression of most killer cell inhibitor receptors (KIR) (Maki et al., J Hematother Stem Cell Res. [Hematopoietic Stem Cell Research Magazine] 10:369-83 (2001)). Only KIR2DL4 was detected on the surface of NK-92, which is a KIR receptor with activation function and inhibitory potential expressed by all NK cells. KIR2DL4 is believed to mediate inhibition by binding to HLA allele G (Suck, Cancer Immunol. Immunother. [Cancer Immunology Immunotherapy] 65 (4): 485-92 (2015)). The major pathway of cytotoxic killing by NK-92® cells is through the perforin / esterase pathway; NK-92® express high levels of perforin and granzyme B (Maki et al., J Hematother Stem Cell Res. 10:369-83 (2001)).

[0131] NK-92® cells have a very broad cytotoxic spectrum and are active against cell lines derived from hematological malignancies and solid tumors (Klingemann, Blood, 87(11):4913-4 (1996); Swift, Haematologica. 97(7):1020-8 (2012); Yan et al., Clin Cancer Res. 4:2859-68 (1998)). Safety evaluations in severe combined immunodeficient (SCID) mice did not show effects associated with NK-92® treatment, 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 to a mouse model of human melanoma resulted in improved survival and inhibition of tumor growth, including complete remission in some mouse tumors (Tam et al., J Hematother. [Journal of Blood Therapy] 8:281-90 (1999), Yan et al., Clin Cancer Res. [Clinical Cancer Research] 4:2859-68 (1998)). Phase I clinical trials have demonstrated its safety. Characterization of the NK-92® cell line is disclosed in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044, both of which are incorporated herein by reference in their entirety.

[0132] Optionally, the 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 protective function of immune cells by binding to a portion of an antibody called the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates the phagocytic or cytotoxic activity of the cell through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified by the type of antibody they recognize. For example, Fc-γ receptors (FCγRs) 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 commonly 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 the SwissProt database under entry P08637.

[0133] In some embodiments, the CD16 receptor comprises a phenylalanine (F)-valine (V) substitution at amino acid position 158 (F158V) in the IgG binding domain of the mature CD16 receptor (corresponding to Val at position 176 of the full-length protein), which affects the antibody-dependent cellular cytotoxicity (ADCC) function of NK cells. The CD16 158V variant binds to human IgG1 and IgG3 with higher affinity than the 158F variant.

[0134] Optionally, the modified NK-92® cells comprise a nucleic acid sequence that is 70%, 80%, 90% or 95% identical to SEQ ID NO: 13. Optionally, the modified NK-92® cells comprise a nucleic acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. Optionally, the modified NK-92® cells comprise a polypeptide that is 70%, 80%, 90% or 95% identical to SEQ ID NO: 12 (with valine at position 176 of the full-length polypeptide). Optionally, the modified NK-92® cells comprise a polypeptide that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12.

[0135] The cytotoxicity of NK-92® cells depends on the presence of cytokines, such as interleukin 2 (IL-2). Thus, optionally, the modified NK-92® cells are further modified to express at least one cytokine. Optionally, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. Optionally, the at least one cytokine is IL-2, IL-15, or a combination thereof. Optionally, IL-2 and / or IL-15 are expressed in the presence of a signal sequence that directs the cytokine to the endoplasmic reticulum. Directing IL-2 to the endoplasmic reticulum allows IL-2 to be expressed at levels sufficient for autocrine activation without releasing large amounts of IL-2 outside the cell. See Konstantinidis et al. "Targeting IL-2 to the endoplasmicreticulum confines autocrine growth stimulation to NK-92® 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.

[0136] Optionally, the modified NK-92® cells comprise a nucleic acid sequence encoding IL-2 that is 70%, 80%, 90% or 95% identical to SEQ ID NO: 14. Optionally, the modified NK-92® cells comprise a nucleic acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14. Optionally, the modified NK-92® cells comprise an IL-2 polypeptide that is 70%, 80%, 90% or 95% identical to SEQ ID NO: 15. Optionally, the modified NK-92® cells comprise an IL-2 polypeptide that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15. The provided modified NK-92® cells are advantageously capable of being maintained in the absence of IL-2 without secreting an amount of IL-2 that causes clinical side effects.

[0137] In one aspect, the subject matter of the present invention includes modified NK-92® cells that are capable of modulating the tumor microenvironment. The modified NK-92® cells preferably comprise a tetracistronic vector comprising one or more nucleic acids encoding: i) IL-12 or TGF-β trap, ii) an antigen binding protein (ABP) or a chimeric antigen receptor (CAR) that specifically binds to a target antigen, iii) an Fc receptor, such as CD16 or CD16-158V, and / or iv) a cytokine, such as erIL-2 or erIL-15, wherein the nucleic acid sequence is operably linked to a promoter. In one embodiment, the modified NK-92® cells preferably comprise a tetracistronic vector comprising one or more nucleic acids encoding: i) IL-12 or TGF-β trap, ii) an antigen binding protein (ABP) or a 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. Fig.21 , Fig.30 and Fig.31 Tetracistronic vectors contemplated herein are described in . The IL-12 contemplated herein may comprise a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 57 (p35 nucleotide sequence) or SEQ ID NO: 59 (p40 nucleotide sequence). The IL-12 contemplated herein may also comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 58 (p35 amino acid sequence, isoform 1 precursor) or SEQ ID NO: 60 (p40 amino acid sequence, precursor).

[0138] In an exemplary embodiment, the IL-12 single-chain p40_p35 sequence in the IL-2 / PD-L1 tetracistronic vector may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 61, or may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 62.

[0139] The TGF-β trap contemplated herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 63 (TGFBRII extracellular domain) or SEQ ID NO: 65 (TGFb trap sequence). The TGF-β trap contemplated herein may also comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 64 (TGFBRII extracellular domain) or SEQ ID NO: 66 (TGFb trap sequence). Other suitable TGF-β traps include those described in Mol. Canc. THer. [Molecular Cancer Therapy] 2012, Vol. 11 (7), 1477-1487.

[0140] In addition, the nucleic acid constructs of the subject matter of the present invention may also comprise sequences encoding 2A peptides, such as T2A, P2A, E2A or F2A peptides, so as to produce equimolar levels of polypeptides encoded by the same mRNA. The E2A peptide 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: 17. The T2A peptide 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: 18.

[0141] In an exemplary, non-limiting example, the plasmid disclosed herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19 (5' homology arm of AAVS1), SEQ ID NO: 20 (EF1a promoter), SEQ ID NO: 21 (T7 promoter), SEQ ID NO: 22 (CCR7 cDNA), SEQ ID NO: 23 (P2A element), SEQ ID NO: 24 (IgHC leader sequence), SEQ ID NO: 25 (CD19 CAR minus signal peptide), SEQ ID NO: 26 (high affinity CD16), SEQ ID NO: 27 (IRES), SEQ ID NO: 28 (SC40 poly A), SEQ ID NO: 29 (3' homology arm of AAVS1) and / or SEQ ID NO: 30 (homology arm of AAVS1).

[0142] Chimeric Antigen Receptor

[0143] Optionally, the modified NK-92® cells are further engineered to express a chimeric antigen receptor (CAR) on the cell surface. Optionally, the CAR is specific for a tumor-specific antigen. As non-limiting examples, tumor-specific antigens are described in US 2013 / 0189268, WO 1999024566 A1, US 7098008, and WO 2000020460 A1, each of which is incorporated herein by reference in its entirety. Tumor specific antigens include, but are not limited to, NKG2D, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, CD19, CD33, B7-H4, CD20, and 41BB. CARs can be designed as described in, for example, patent publications WO 2014039523, US20140242701, US 20140274909, US 20130280285, and WO 2014099671, each of which is incorporated herein by reference in its entirety. Optionally, the CAR is CD19 CAR, CD33 CAR or CSPG-4 CAR. In an exemplary non-limiting example, CD19CAR_CD3a can include and SEQ ID NO: 41 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity of the polynucleotide sequence.

[0144] Homing receptors

[0145] Provided herein are modified NK-92® cells comprising nucleic acids encoding homing receptors. 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 selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC or CXCL14 receptors. In some embodiments, the nucleic acid encoding CCR7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. Optionally, the homing receptor is expressed on the cell surface of the modified NK-92® cells. Optionally, the modified NK-92® cells further include CAR. Optionally, CAR is CD19. Optionally, the modified NK-92® cells further comprise an Fc receptor. Optionally, the Fc receptor is CD16. Optionally, the modified NK-92® cells further comprise a cytokine, such as IL-2. 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 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 SEQ ID NO: 43.

[0146] Expression vector

[0147] Provided herein are expression vectors comprising nucleic acids operably linked to promoters. The nucleic acids encoding different elements of the vector can each be operably linked to the same or different promoters. Exemplary promoters include, but are not limited to, CMV promoters, ubiquitin promoters, PGK promoters, and EF1 promoters. Optionally, provided herein are 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. Optionally, the nucleic acid is operably linked to the promoter. Optionally, the promoter is selected from the group consisting of promoters having SEQ ID NO:3, 9, 10 or 11 or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3, 9, 10 or 11. Optionally, the nucleic acid is operably linked to the promoter. Optionally, the promoter comprises SEQ ID NO: 4 and / or SEQ ID NO: 5. Optionally, the promoter comprises SEQ ID NO: 6, or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6.

[0148] In some embodiments, an expression vector is provided comprising SEQ ID NO: 1, or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1; SEQ ID NO: 25 (CD19 CAR) or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 (CD16 158V), or a nucleic acid or polypeptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 (erIL-2 nucleotide sequence); and / or SEQ ID NO: 15 (erIL-2 amino acid sequence), or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14. In some embodiments, an expression vector is provided comprising SEQ ID NO: 47 (CXCR2), or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 47; SEQ ID NO: 25 (CD19 CAR), or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12; and SEQ ID NO: 13 (CD16158V), and / or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14; and / or SEQ ID NO: 15 (erIL-2), or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14. Suitable expression vectors are known in the art and can be used. In another aspect, the recombinant nucleic acid comprises a segment encoding erIL-15, and the nucleic acid encoding erIL-15 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 67. Optionally, the expression vector is a plasmid.

[0149] exist Fig. 22 The expression analysis of PD-L1 CAR and CD-16 in PD-L1 (TGFβ-trap) t-haNK cells is described in. Moreover, Fig.23 As shown, TGFβ-trap was secreted into the culture supernatant of TGFβ-trap / PD-L1 t-haNK cells. Fig.30 The right column shows IL-12 secretion by IL-12 virus-transduced NK-92® cell line.

[0150] Methods for preparing modified NK-92® cells

[0151] Provided herein are methods for preparing modified NK-92® cells comprising nucleic acid molecules described herein. These methods include transforming NK-92® cells with an expression vector comprising a nucleic acid described herein operably linked to a promoter.

[0152] As used herein, the terms promoter, promoter element and regulatory sequence refer to a polynucleotide that regulates the expression of a selected polynucleotide sequence operably linked to the promoter and achieves the expression of the selected polynucleotide sequence in the cell. In some embodiments, the promoter element is or comprises an untranslated region (UTR) at the 5' position of the coding sequence. The 5'UTR forms part of the mRNA transcript and is therefore an integral part of protein expression in eukaryotic organisms. After transcription, the 5'UTR can regulate protein expression at the transcriptional and translational levels. Promoters for controlling transcription from vectors in mammalian host cells can be obtained from various sources, for example, viruses such as polyoma viruses, simian virus 40 (SV40), adenoviruses, retroviruses, hepatitis B viruses, and cytomegalovirus genomes (e.g., SEQ ID NO: 11), or from heterologous mammalian promoters, such as β-actin promoters, eukaryotic translation elongation factor 1 α1 (EF1α) promoters (e.g., SEQ ID NO: 3), phosphoglycerate kinase (PGK) promoters (e.g., SEQ ID NO: 10) and ubiquitin promoters (e.g., SEQ ID NO: 9). The promoters provided herein are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, 9, 10 or 11.

[0153] As used herein, the phrase selection marker refers to a nucleotide sequence (e.g., gene) encoding a product (polypeptide) that allows selection, or refers to a gene product (e.g., polypeptide) itself. The term selection marker is used herein as generally understood in the art, and refers to a marker that, under certain limited culture conditions (selective conditions), its presence in a cell or organism gives the cell or organism a significant growth or survival advantage or disadvantage. As used herein, the phrase selection agent refers to an agent that introduces selection pressure to a cell or cell colony to favor or resist the cell or cell colony with a selection marker. For example, the selection agent is an antibiotic and the selection marker is an antibiotic resistance gene. Examples of suitable selection markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin and puromycin.

[0154] Nucleic acid as used herein refers to deoxyribonucleotides or ribonucleotides and polymers and complements thereof. The term includes deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form. The term covers nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs). Unless otherwise indicated, conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences of nucleic acid sequences may be used to replace the specific nucleic acid sequences described herein. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0155] When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is operably connected. For example, if the DNA encoding the presequence or secretory leader sequence is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably connected to the DNA encoding the polypeptide; if a promoter or enhancer affects the transcription of the sequence, the promoter or enhancer is operably connected to the coding sequence; or if the ribosome binding site is positioned to facilitate translation, it is operably connected to the coding sequence. Generally, operably connected means that the DNA sequences connected are close to each other, and in the case of a secretory leader sequence, they are continuous and in the reading phase. However, enhancers do not have to be continuous. For example, a nucleic acid sequence operably connected to a second nucleic acid sequence is directly or indirectly covalently connected to such a second sequence, but any effective three-dimensional association is acceptable. A single nucleic acid sequence can be operably connected to multiple other sequences. For example, a single promoter can guide the transcription of multiple RNA species. The connection can be completed by connecting at a convenient restriction site. If there is no such site, a synthetic oligonucleotide adapter or joint is used according to conventional practice.

[0156] Treatment

[0157] The method for treating cancer or tumor in a subject is described herein. As used herein, the term "cancer" refers to all types of cancers, neoplasms or malignant tumors found in mammals, including leukemia, carcinoma and sarcoma. Exemplary cancers include brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer and medulloblastoma. Other examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, primary macroglobulinemia, primary brain tumor, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine and exocrine pancreatic neoplasms and prostate cancer.

[0158] As used herein, the terms "metastasis", "metastatic" and "metastatic cancer" can be used interchangeably and refer to a proliferative disease or condition such as cancer spreading from one organ or another non-adjacent organ or body part. Cancer occurs at the site of origin, such as breast cancer, which is called a primary tumor, such as primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and infiltrate surrounding normal tissues in the local area, and / or penetrate the wall of the lymphatic system or the wall of the vascular system, and circulate to other parts and tissues in the body through the system. The second clinically detectable tumor formed by the cancer cells of the primary tumor is called a metastatic tumor or secondary tumor. When cancer cells metastasize, it is speculated that the metastatic tumor and its cells are similar to the original tumor. Therefore, if lung cancer metastasizes to the breast, the secondary tumor at the breast site is composed of abnormal lung cells rather than abnormal breast cells. Secondary tumors in the breast are called metastatic lung cancer. Therefore, the phrase metastatic cancer refers to a disease in which a subject suffers from or has suffered from a primary tumor and has one or more secondary tumors. The phrase non-metastatic cancer or a subject with non-metastatic cancer refers to a disease in which a 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 with a primary lung tumor or a history of a primary lung tumor and one or more secondary tumors in a secondary location or locations (e.g., in the breast).

[0159] As used herein, "treating" a condition, disease or disorder and symptoms associated with a condition, disease or disorder, or "treatment" of a condition, disease or disorder and symptoms associated with a condition, disease or disorder refers to a method of obtaining a beneficial or desired result (including a clinical result). Beneficial or desired clinical results may include, but are not limited to, alleviating or improving one or more symptoms or conditions, reducing the extent of a condition, disorder or disease, stabilizing the state of a condition, disorder or disease, preventing the development of a condition, disorder or disease, preventing the spread of a condition, disorder or disease, delaying or slowing the progression of a condition, disorder or disease, delaying or slowing the onset of a condition, disorder or disease, improving or alleviating a condition, disorder or disease state, and partial or complete relief. "Treatment" may also mean extending the survival of a subject beyond the expected survival period when no treatment is performed. "Treatment" may also mean temporarily inhibiting the progression of a condition, disorder or disease, temporarily slowing the progression of a condition, disorder or disease, but in some cases, it involves permanently stopping the progression of a condition, disorder or disease. As used herein, the term treatment refers to the method of reducing the effect of one or more symptoms of a disease or condition characterized by protease expression or the symptom of a disease or condition characterized by protease expression. Therefore, in the disclosed method, treatment can refer to the severity of a determined disease, condition or the symptom of the disease or condition being reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. For example, if compared with a control, one or more symptoms of a disease in a subject are alleviated by 10%, it is considered that the method for treating a disease is treatment. Therefore, compared with a natural or control level, the mitigation amount can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between 10% and 100%. It should be understood that treatment does not necessarily refer to the cure or complete ablation of a disease, condition or the symptom of the disease or condition. Furthermore, as used herein, references to reducing, alleviating or inhibiting include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater compared to control levels, and such terms may include but do not necessarily include complete elimination.

[0160] The terms subject, patient, individual, etc. are not intended to be limiting and are generally interchangeable. That is, an individual described as a patient does not necessarily suffer from a given disease, but may simply be seeking medical advice. As used throughout, a subject may be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), a bird, a reptile, an amphibian, a fish, and any other animal. The term does not specify a specific age or gender. Therefore, it is intended to cover adult and newborn subjects, whether male or female. As used herein, patient, individual, and subject may be used interchangeably, and these terms are not intended to be limiting. That is, an individual described as a patient does not necessarily suffer from a given disease, but may simply be seeking medical advice. The term patient or subject includes human and veterinary subjects.

[0161] As used herein, "administering" refers to providing, contacting and / or delivering a compound or compounds by any appropriate route to achieve a desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ocular, inhalation and implantation. Optionally, NK-92® cells are administered parenterally. Optionally, NK-92® cells are administered intravenously. Optionally, NK-92® cells are administered peritumorally.

[0162] Thus, provided herein are methods of reducing cancer metastasis in a subject, the methods comprising administering to the subject a therapeutically effective amount of the modified NK-92® cells described herein, thereby reducing cancer metastasis in the subject. Also provided are methods of treating cancer in a subject, the methods comprising the steps of selecting a subject having cancer and administering to the subject a therapeutically effective amount of the modified NK-92® cells described herein, wherein the administration treats the cancer in the subject. Optionally, the methods further comprise administering to the subject an additional therapeutic agent.

[0163] In some embodiments, the methods further comprise administering to a subject a therapeutically effective amount of the modified NK-92® cells described herein, wherein the administration treats cancer in the subject or reduces tumor size in the subject. In some embodiments, the methods comprise administering to a subject modified NK-92® cells comprising nucleic acids encoding i) IL-12 or TGFb trap, 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 methods comprise administering to a subject modified NK-92® cells 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.

[0164] exist Fig.24 The cytotoxicity of TGFβ trap / PD-L1 on K562 target cells was demonstrated in Fig.25 The SUP-B15 PD-L1+ CAR killing of target cells, while Fig.26 CAR killing of MDA-MB 231 target cells is shown in FIG. Fig. 27 The effect of TGFβ trap / PD-L1 on SUP-B15 CD19-CD20+ ADCC. Fig.28 This indicates that TGFβ / SMAD luciferase reporter HEK293 cells are induced by TGFβ. Fig.29 Secreted TGFβ-trap sequestered TGFβ and inhibited luciferase expression in a HEK293T reporter assay are shown in . Reporter cells were treated with 1 ng / mL TGFβ1 for 19 hours. In other options, the preferred CAR molecule specifically binds to PD-L1 and may have an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69 (which may be encoded by a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 68.

[0165] NK-92® cells can be administered to a subject by a variety of routes. For example, NK-92® cells can be administered to a subject by infusion (e.g., intravenous infusion) over a period of time. Typically, for a single dose of NK-92® cells, the period of time is 5 to 130 minutes. Optionally, the period of time is between 90 and 120 minutes. Optionally, the period of time is between 15 and 30 minutes.

[0166] NK-92® cells and optionally other anticancer agents can be administered to a patient with cancer once during therapy or they can be administered multiple times, for example once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more, or any range (including endpoints) between any two of these numbers. Thus, for example, NK-92® cells can be administered to a subject once a day for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more. Optionally, NK-92® cells are administered once a day for a period of two days. After the cycle, NK-92® cells are not treated for one or more hours, one day or days, or one week or weeks. As used herein, the term "cycle" refers to a treatment repeated on a regular schedule with a rest period (e.g., no treatment or treatment with other agents) in between. For example, a week of treatment followed by a two-week break is a treatment cycle. Such treatment cycles can be repeated one or more times. Thus, NK-92® cells can be administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cycles.

[0167] NK-92® cells can be administered to a subject in absolute numbers of cells, for example, about 1000 cells per injection up to about 10 billion cells per injection, such as about, at least about, or at most about 1×10 cells per injection. 10 , 1 × 10 9 , 1 × 10 8 , 1 × 10 7 , 5 × 10 7 , 1 × 10 6 , 5 × 10 6 , 1 × 10 5 , 5 × 10 5 , 1 × 10 4 , 5 × 10 4 , 1 × 103 , 5 × 10 3 NK-92® cells, or any range between any two of these numbers (including the endpoints). Optionally, 1 × 10 8 Up to 1 × 10 10 The cells are administered to a subject. Optionally, the cells are administered once or more per week for one or more weeks. Optionally, the cells are administered once or twice per week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

[0168] Optionally, about 1000 cells / injection / m 2 Up to approximately 10 billion cells / injection / m 2 Administer to a subject, such as about, at least about, or at most about 1×10 10 pcs / m 2 , 1 × 10 9 pcs / m 2 , 1 × 10 8 pcs / m 2 , 1 × 10 7 pcs / m 2 , 5 × 10 7 pcs / m 2 , 1 × 10 6 pcs / m 2 , 5 × 10 6 pcs / m 2 , 1 × 10 5 pcs / m 2 , 5 × 10 5 pcs / m 2 , 1 × 10 4 pcs / m 2 , 5 × 10 4 pcs / m 2 , 1 × 10 3 pcs / m 2 , 5 × 10 3 pcs / m 2 (and so on) NK-92® cells, or any range (including endpoints) between any two of these numbers. Optionally, the subject is administered 2 Administer 1 × 10 3 Up to 1 × 10 10 NK-92® cells. Optionally, the subject is administered 2 Administer 2 × 10 9 NK-92® cells.

[0169] Optionally, NK-92® cells may be administered to such an individual in a relative number of cells, for example, from about 1000 cells / kg of the individual to up to about 10 billion cells / kg of the individual, such as 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 × 10 4 , 5 × 10 4 , 1 × 10 3 , 5 × 10 3 (and so on) NK-92® cells / kg individual, or any range between any two of these numbers (including the endpoints).

[0170] Optionally, the total dose can be calculated based on body surface area m 2 Calculations are performed, including every m 2 Approx. 1 × 10 11 pcs, 1 × 10 10 pcs, 1 × 10 9 pcs, 1 × 10 8个 , 1 × 10 7 or any range between any two of these numbers (including endpoints). Optionally, about 1 billion to about 3 billion NK-92® cells are administered to the patient. Optionally, the amount of NK-92® cells injected per dose can be calculated based on body surface area m2, including per m 2 1 × 10 11 pcs, 1 × 10 10 pcs, 1 × 10 9 pcs, 1× 10 8 pcs, 1 × 10 7 pcs, 1 × 10 6 pcs, 1 × 10 5 pcs, 1 × 10 4 pcs, 1 × 10 3 indivual.

[0171] Optionally, the NK-92® cells are administered in the form of a composition comprising NK-92® cells and a culture medium, such as human serum or its equivalent. Optionally, the culture medium comprises human serum albumin. Optionally, the culture medium comprises human plasma. Optionally, the culture medium comprises about 1% to about 15% human serum or a human serum equivalent. Optionally, the culture medium comprises about 1% to about 10% human serum or a human serum equivalent. Optionally, the culture medium comprises about 1% to about 5% human serum or a human serum equivalent. Optionally, the culture medium comprises about 2.5% human serum or a human serum equivalent. Optionally, the serum is human AB serum. Optionally, a serum substitute acceptable for human therapeutic agents is used instead of human serum. Such serum substitutes may be known in the art. Optionally, the NK-92® cells are administered in the form of a composition comprising NK-92® cells and an isotonic liquid solution that supports cell viability. Optionally, the NK-92® cells are administered in the form of a composition reconstituted from a cryopreserved sample.

[0172] According to the methods provided herein, an effective amount of one or more agents provided herein is administered to a subject. The terms effective amount and effective dose can be used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiological response (e.g., to reduce inflammation). Those skilled in the art can empirically determine the effective amount and schedule of administering the agent. The dosage range administered is those dosage ranges that are large enough to produce the desired effect, in which one or more symptoms of the disease or condition are affected (e.g., alleviated or delayed). The dosage should not be so large as to cause serious adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Generally, the dosage will vary with age, condition, sex, type of disease, degree of disease or condition, route of administration, or whether other drugs are included in the regimen, and can be determined by those skilled in the art. If any contraindications occur, the dosage can be adjusted by an individual physician. The dosage can vary and can be administered once or multiple times a day for one or several days. For a given class of drug products, guidance for appropriate dosages can be found in the literature. For example, for a given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100%. Efficacy can also be expressed as an increase or decrease of "how many times". For example, a therapeutically effective amount can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times or more relative to a control. The exact dosage and formulation will depend on the purpose of the treatment, and will be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms [drug dosage form] (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding [drug compounding field, science and technology] (1999); Remington: The Science and Practice of Pharmacy [Remington: Pharmaceutical Science and Practice], 22nd Edition, Gennaro edited (2012), and Pickar, Dosage Calculations [dose calculation] (1999)).

[0173] Pharmaceutically acceptable compositions may include a variety of carriers and excipients. Various aqueous carriers may be used, for example, buffered saline, etc. These solutions are sterile and generally free of unwanted substances. Suitable carriers and formulations thereof are described in Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012). Pharmaceutically acceptable carriers are meant to be materials that are not biologically or otherwise unsuitable, i.e., the material is administered to a subject without causing unsuitable biological effects or interacting with other components contained in the pharmaceutical composition in a harmful manner. If administered to a subject, a carrier is optionally selected to minimize degradation of the active ingredient and minimize adverse side effects of the subject. As used herein, the term pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions generally include agents for buffering and preservation in storage, and may include buffers and carriers for appropriate delivery depending on the route of administration.

[0174] These compositions may contain acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of cells in these formulations and / or other agents may vary and will be selected primarily based on fluid volumes, viscosities, body weight, etc., depending on the particular mode of administration chosen and the needs of the subject.

[0175] Combination therapy

[0176] Optionally, NK-92® cells are administered to the subject along with one or more other treatments for the cancer being treated. Without being bound by theory, it is believed that co-treatment of the subject with NK-92® cells and another cancer therapy will allow the NK-92® cells and the alternative therapy to provide the endogenous immune system with an opportunity to clear cancers that have so far overwhelmed such endogenous effects. Optionally, the two or more other treatments for the cancer being treated include, for example, antibodies, bispecific adapters, radiation, chemotherapeutic agents, stem cell transplants, or hormone therapy.

[0177] Optionally, the antibody is administered to the patient together with the NK-92® cells. Optionally, the NK-92® cells and the antibody are administered to the subject together, for example, in the same formulation; for example, in separate formulations, administered to the subject separately at the same time; or can be administered separately, for example, at different dosing schedules or at different times of the day. When administered alone, the antibody can be administered by any suitable route, such as intravenous or oral administration.

[0178] Optionally, the antibodies can be used to target cancer cells or cells expressing cancer-associated markers.Many antibodies have been approved for use alone in the treatment of cancer.

[0179] The provided method can further be combined with other tumor therapies such as radiotherapy, surgery, hormone therapy and / or immunotherapy. Therefore, the provided method can further include applying one or more additional therapeutic agents to the subject. Suitable additional therapeutic agents include but are not limited to analgesics, anesthetics, stimulants, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antitumor agents, allosteric inhibitors, anabolic steroids, antirheumatic agents, psychotherapeutics, nerve blockers, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinics, antimycobacterial agents, antiprotozoal agents, antivirals, dopaminergics, hematological agents, immunological agents, muscarinics, protease inhibitors, vitamins, growth factors and hormones. Based on the given disease treated, those skilled in the art can easily determine the selection of medicaments and dosages. Optionally, the additional therapeutic agent is octreotide acetate, interferon, pembrolizumab, glucopyranosyl lipid A, carboplatin, etoposide, or any combination thereof.

[0180] Optionally, the additional therapeutic agent is a chemotherapeutic agent. The chemotherapy treatment regimen may include administering to the subject a chemotherapeutic agent or a combination of chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, taxanes, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based compounds, retinoids, vinca alkaloids and derivatives thereof. Optionally, the chemotherapeutic agent is carboplatin.

[0181] The combination of agents or compositions can be administered concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines), or sequentially (e.g., one agent is administered first, followed by a second agent). Thus, the term combination is used to refer to the concomitant, simultaneous, or sequential administration of two or more agents or compositions. The course of treatment is preferably determined on an individual basis according to the particular characteristics of the subject and the type of treatment selected. The treatment, such as those disclosed herein, can be administered to the subject once a day, twice a day, once every two weeks, once a month, or on any applicable basis for which the treatment is effective. The treatment can be administered alone or in combination with any other treatment disclosed herein or known in the art. The additional treatment can be administered simultaneously with the first therapy, at a different time, or on a completely different treatment schedule (e.g., the first therapy can be administered once a day, while the additional treatment is administered once a week).

[0182] Reagent test kit

[0183] Provided herein are kits comprising modified NK-92® cells as described herein. In some embodiments, the kit comprises modified NK-92® cells comprising nucleic acids encoding the following substances operably linked to a promoter: i) homing receptors, ii) ABPs or CARs specifically binding to a target antigen, iii) Fc receptors (such as CD16 or CD16-158V), and / or iv) cytokines (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 CC chemokine receptor type 7 (CCR7), CXCR2, or CXCL14 receptors operably linked to a promoter. Optionally, the nucleic acid encoding CCR7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. Optionally, the homing receptor is expressed on the cell surface of the modified NK-92® cells. Optionally, the promoter comprises one or more NFAT binding elements and a minimal promoter. Optionally, the promoter has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6. Optionally, one or more proteins encoded by the nucleic acid sequence are expressed on the cell surface of the modified NK-92® cells.

[0184] Optionally, the modified NK-92® cells are provided in a composition comprising a pharmaceutically acceptable excipient. Optionally, the kit may contain other compounds, such as therapeutically active compounds or drugs to be administered before, simultaneously with, or after administration of the modified NK-92® cells. Optionally, the instructions for use of the kit will include guidance for using the kit components in cancer treatment. The instructions may further contain information on how to prepare the antibodies (e.g., dilution or reconstitution in the case of freeze-dried proteins) and NK-92® cells (e.g., thawing and / or culturing). The instructions may further include guidance on dosage and frequency of administration.

[0185] The following materials, compositions and components are disclosed, which can be used for the products of the disclosed methods and compositions, can be used in combination with these products, can be used to prepare these products, or disclose these products. These and other materials are disclosed herein, and it should be understood that when the combinations, subsets, interactions, groupings, etc. of these materials are disclosed, although the specific references to each individual and overall combination and arrangement of these compounds may not be explicitly disclosed, each is specifically considered and described herein. For example, unless explicitly stated to the contrary, if a method is disclosed and discussed, and many modifications that can be made to many molecules (including the method) are discussed, each and every combination and arrangement of the method and possible modifications are specifically considered. Similarly, any subset or combination of these is also specifically considered and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to steps in the method using the disclosed composition. Therefore, if multiple additional steps can be performed, it should be understood that each of these additional steps can be performed with any specific method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is specifically considered and should be considered disclosed.

[0186] Publications cited herein and the materials for which they are cited are hereby incorporated by reference in their entirety.

[0187] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein and are not intended to limit the scope of the claims. Examples

[0188] Example 1. Modified NK cell line expressing the cytokine CCR7 that modulates the tumor microenvironment.

[0189] The NEON transfection system (ThermoFisher Scientific, Waltham, MA) was used to transfect the cells with the linearized pNKAT-CCR7-LP3 plasmid ( Figure 1) NK-92® cells were electroporated to prepare modified NK-92® cells. After 1 week of puromycin selection, the resulting polyclonal populations were tested for CCR7 expression and monoclonal cell lines were derived by serial dilution in growth medium supplemented with 5% human serum and IL-2. The modified NK-92® cells contain the EF1α promoter, the CCR7 gene with a poly A tail, and a LoxP-flanked puromycin resistance gene driven by the ubiquitin promoter, all wrapped in homology arms targeting the AAVS1 locus (SEQ ID NO:7).

[0190] To verify that the expression of CCR7 did not affect NK-92® cells, the expression of NK-92® cell markers was measured. Figure 3 shown. Figure 3 All data in were generated using Intellicyt iQue screener plus. Cells were incubated with APC-conjugated antibodies against the phenotypic markers or the appropriate isotype used as a negative control at 4°C for 30 minutes. The cells were then rinsed in PBS +1% BSA, pelleted, and resuspended in 30 uL PBS +1% BSA. The readout was then gated as shown in the upper left quadrant to eliminate cell debris from the reading, and the percentage of cells above the fluorescence threshold shown in the upper right quadrant was then displayed as two separate heat maps, showing the percentage above the "positive" threshold and the "strongly positive" threshold in the lower left and lower right quadrants, respectively. Figure 3 We show that driving CCR7 expression does not meaningfully affect the major phenotypic markers associated with our cell lines. Specifically, CCR7 expression does not appear to affect CD54, NKp30, or NKG2D expression.

[0191] To determine 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, with 100,000 effector cells remaining in the well with the highest concentration and seven additional 2-fold dilutions in eight rows of the plate. The stained target cells (K562 ( Figure 4 )、HL-60( Figure 5)) were seeded at 10,000 / well into all wells containing effector cells, as well as into control wells whose goal was simply to measure background death. The plate was then centrifuged briefly and incubated for 4 hours at 37°C and 5% CO2. The plate was then centrifuged, the supernatant aspirated, and the cells resuspended in PBS containing propidium iodide to measure cell death. The cells were then run through the Intellicyt iQue screenerplus, and the proportion of target cells that also stained positive for PI (distinguished from effectors by their staining) was measured. The percentage of dead cells was then compared to the number of naturally dying cells in the control wells, and the percentage of cells specifically killed by effector cells was calculated. The results are shown in Figure 4 and Figure 5 shown. Figure 4 Clones showing upregulation of CCR7 were equally cytotoxic to K562 cells compared to the parental cell line, Figure 5 Clones showing upregulation of CCR7 were equally cytotoxic to HL-60 cells compared to the parental cell line.

[0192] In vitro testing consisted of using the Boyden chamber assay and a Matrigel layer to block migration. In these assays, modified cells expressing CCR7 were shown to migrate toward CCL21 and CCL19, an alternative CCR7 ligand. Cells were placed in the upper wells and separated from the lower chamber by a thin layer of Matrigel (ECM-like matrix) coated on 8uM pores. Cells (25,000 / well) were placed in low serum medium (supplemented with 1% human serum and 500U / mL IL-2) in the upper chamber and the same low serum medium was used in the lower chamber alone or with the chemokine of interest. 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 in triplicate for NK-92® cells or modified NK-92® cells expressing CCR7. The plates were then placed in an incubator overnight for an 18-hour invasion assay, after which the upper chamber was removed and 150 µL was sampled from the lower well after thorough mixing (total volume 750 µL) and read on a MacsQuant FACS analyzer. Viable cells in the lower chamber were counted and the cell number was then compared to wells without chemokine and an invasiveness index number was generated. These numbers were averaged and statistical correlations were calculated using a two-tailed t-test. Since the lower wells were sampled and no cells detached from the lower membrane, those cells that remained attached to the lower ECM would not be represented in these numbers, potentially creating a difference between CCL19 and CCL21. The results are shown in Figure 2. Figure 7 Specifically, Figure 7A statistically significant increase in the invasiveness of modified NK-92® cells expressing CCR7 toward CCL19, a CCR7 chemokine, was shown. The lack of a statistically significant response to CCL21 may be due to the nature of the assay performed. The assay measures invasion and subsequent detachment from the ECM, a behavior consistent with migration with a CCL19 gradient. CCL21, while inducing migration, does not induce detachment from the matrix, requiring additional steps to demonstrate statistically significant invasive potential.

[0193] Example 2. Generation of NFAT-responsive constructs for controlled expression of CCL21.

[0194] To identify NFAT response elements, a cell line stably expressing a NFAT-based luciferase expression cassette (NR2.2) was generated by electroporating a linearized construct into NK-92® cells containing a termination region, followed by three NFAT response elements (SEQ ID NO: 4), and a minimal promoter (SEQ ID NO: 5) so that the minimal promoter would drive the production of firefly luciferase in the presence of activated NFAT. A subset of these cells was then also electroporated with mRNA containing an anti-CD19 CAR (an antigen present on Sup-B15 cells that are otherwise resistant to NK-92® cell killing). These cells are denoted ENR2.2 in the left panel. The cells were then plated in triplicate in the absence or presence of target cells and incubated for 2.5 hours to 24 hours. At the end of the incubation period, step 1 reagent from the Promega DualGlo system was added to the wells to activate luciferase (by providing its substrate luciferin). The results were then read on a SpectraMax i3x microplate reader and expressed as mean values ​​with standard deviations calculated in Microsoft Excel. Fig. 6A and Figure 6B NFAT activation was demonstrated in the presence of target binding to K562 in a time-dependent manner and to Sup-B15 only when electroporated with CD19-CAR mRNA.

[0195] Example 3. Modified NK cell line expressing CCR7 and CCL21.

[0196] The pCRENFAT-CCL21 plasmid was incorporated into NK-92® cells containing CCR7 using the LoxP sites embedded in the pNKAT-CCR7-LP3 construct in a recombinase-mediated cassette exchange. After electroporation of the circular plasmid (pCRENFAT-CCL21), Cre recombinase was transiently expressed, mediating the exchange of the new LoxP-flanked cassette with the old selection cassette. Selection in blasticidin was used to favor the incorporation of the new cassette, and monoclonal cell lines were subcloned from the resulting population in the same manner as previously described in Example 1 to obtain modified NK-92® cells expressing CCR7 and CCL21.

[0197] To evaluate modified NK-92® cells expressing CCR7 and CCL21, co-culture unstained modified NK-92® cells with cells known to induce NFAT activation (K562 or other cell lines) in the lower well of the Boyden chamber and place stained modified NK-92® cells in the upper chamber. If migration is demonstrated to be induced by co-culture with sensitive cell lines, then the system is working.

[0198] Example 4: In vitro cytotoxicity assay using a modified NK cell line expressing CCR7

[0199] Fig. 9 It was shown that NK-92® cells modified to express CCR7, as described in Example 1, remained cytotoxic to target cells after migration in a modified Boyden chamber transwell assay.

[0200] Example 5: Cell surface expression of CCR7, CD16 and CD19 CAR in NK-92® cells transfected with nucleic acid constructs

[0201] Fig.12 It was shown that modified NK-92® cells transfected with nucleic acid constructs encoding CCR7, CD16 and CD19 CARs expressed high levels of the corresponding proteins on the cell surface.

[0202] Example 6: Biodistribution of CCR7-expressing chemokine-responsive NK cells in NSG mice bearing CCL19-positive subcutaneous K562 tumors

[0203] This study demonstrated that chemokine-responsive aNK cells home to chemokine-expressing target tissues following intravenous administration.Because mouse and human chemokines do not cross-react, the inventors developed a tumor model expressing local ligands as an alternative model.

[0204] Experimental methods (Table 1):

[0205] a. Animals:

[0206] i. Animal type: NSG mice (JAX), female, 7-8 weeks old

[0207] ii. Number of animals: 30 (28 animals received NK cell injection [24 + 4 extra]; 2 additional mice did not receive NK treatment and served as negative controls for flow cytometry)

[0208] b. Tumor Model:

[0209] i. Cell lines: K562, parental and CCL19-expressing subline, K-19

[0210] ii. Inoculation route: subcutaneous; parent K562 was inoculated in the left flank, K-19 was inoculated in the right flank

[0211] iii. Inoculum: 1E6 cells in 100 µL serum-free medium / Matrigel (v / v 1:1)

[0212] iv. Tumor burden at the start of treatment: K-19 average 107 mm 3 ; Parent K562 average 135 mm 3

[0213] v. Random grouping: main Animals were randomized into groups based on the volume of K-19 tumors.

[0214] c. Test sample:

[0215] i.NK cells:

[0216] 1. CD19 t-haNK (non-CR) (NantKwest Torrey Pines)

[0217] 2. Tetracistronic Mi-aNK R7-19.1 (NantKwest Woburn)

[0218] ii. Fluorescent labeling:

[0219] 1. According to the manufacturer's manual, both types of NK cells were labeled with CFSE immediately prior to in vivo administration.

[0220] 2. For each time point, cultured CFSE-labeled cells were harvested to serve as a positive control for flow cytometry.

[0221] iii. Administration method: intravenous

[0222] iv. Dosage:

[0223] 1.1E7 cells / mouse

[0224] v.Dosing frequency: Single dose

[0225] d. Tumor Collection:

[0226] i. Time point: 3, 24 and 48 hours after administration (± 2 hours)

[0227] ii. N = 4 mice / group / time point

[0228] iii. Tumor processing: Harvested tumors were dissociated into single-cell suspensions according to in-house protocols (Supplementary), and CFSE-positive NK cells were counted by flow cytometry.

[0229] e. Formula and statistical analysis:

[0230] i. Tumor volume = length × width 2 / 2 (length and width are the longest and shortest diameters of the tumor, respectively)

[0231] ii. Statistical analysis was performed by one-way ANOVA followed by multiple comparisons by Tukey's test using GraphPad Prism version 7.0. P < 0.05 was considered statistically significant.

[0232] Table 1. Experimental settings

[0233]

[0234] result:

[0235] a. Security:

[0236] iii. Animals receiving both types of NK cells showed mild to moderate acute reactions immediately after cell infusion (G1-G2, mild to marked depression, lethargy, slow or no response).

[0237] iv. Within 24 hours after injection, 2 animals (out of 14) in the R7-19.1 group were found dead, while there were no deaths in the CD19 t-haNK group.

[0238] After 24 h, animals in the CD19 t-haNK group were able to recover, while animals in the R7-19.1 group continued to appear mildly depressed and less responsive to stimulation (G1). They became more responsive at 48 h (G0), but still appeared to have rough fur and rapid breathing.

[0239] b.NK cell homing:

[0240] vi. The number of tumor-infiltrating NK cells at each time point is listed in Table 2 and Fig.13 A diagram is given in .

[0241] 1. The four CCR7 receptor-CCL19 ligand combinations are:

[0242] a.CD19 t-haNK®, K562 tumor: no receptor - no ligand [- -];

[0243] b.CD19 t-haNK®, K-19 tumor: no receptor - ligand present [- +];

[0244] c. R7-19.1 cells, K562 tumor: receptor present - ligand absent [+ -]; and

[0245] d. R7-19.1 cells, K-19 tumors: receptor present - ligand present [+ +]

[0246] vii. At 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, because we were unable to recover many cells from two of the four K562 tumors in Group B, direct comparison of homing of R7-19.1 cells to CCL19-negative versus positive tumors in the same animal was not possible.

[0247] viii. At 24 hours, there were no statistically significant differences in NK cell homing for any of the four CCR7 receptor-CCL19 ligand combinations. However, when comparing homing of each NK cell line to CCL19+ and - tumors in the same animal, 3 of 4 animals receiving R7-19.1 cells showed improved infiltration into CCL19+ tumors, while the majority of animals receiving CD19 t-haNK® showed similar levels of NK infiltration into both tumors, regardless of their CCL19 expression ( Fig.14 ).

[0248] ix. At 48 hours, the total number of tumor-infiltrating NK cells was reduced in all combinations and did not differ between any of the groups.

[0249] Table 2. Homing of non-CR vs. R7-19.1 to parental or CCL19 expressing tumors at indicated time points. Results are mean ± SEM. N = 4 unless otherwise stated.

[0250]

[0251] *, N = 2.

[0252] This example demonstrates that at 3 hours post-dose, the co-presence of the CCR7 receptor and the CCL19 ligand results in more efficient NK cell infiltration. At 24 hours post-dose, in animals receiving non-CR aNK cells, NK cells showed similar levels of tumor homing in 3 of 4 animals regardless of CCL19 expression status. In contrast, in animals receiving R7-19.1 cells, NK cells were able to home to CCL19-positive tumors more efficiently in 3 of 4 animals compared to the parental control that did not express the ligand. The number of tumor-infiltrating NK cells decreased at 48 hours, regardless of receptor or ligand expression. In particular, there was a clear trend for R7-19.1 cells to home more to CCL19 tumors in the early hours (< 48 hours post-dose), suggesting that if the cells were used in a therapeutic setting, exposure would be higher and cytotoxicity could be more potent.

[0253] Example 7: Comparative Efficacy Evaluation of R7-19.1 Cells Expressing CCR7 in NSG Mice Bearing Intravenous CCL19-Positive RAJI Tumors

[0254] CCR7 is a chemokine receptor that induces cell migration toward gradients of chemokines CCL19 and CCL21, which are typically expressed in lymph nodes and other lymphoid organs, which are the primary sites of disease manifestation in B-cell lymphomas. We have created chemokine-responsive NK-92® cells (i.e., R7-19.1 cells) expressing CD19-CAR, CCR7, CD16.158V, and ERIL-2 based on the t-haNK platform. In addition to CCR7 expression, these cells also have cancer-targeting chimeric antigen receptors (CARs) against the CD19 cancer antigen, CD16 variants, and ER-IL-2. Previous distribution studies have demonstrated that R7-19.1 cells preferentially home to subcutaneous (SC) tumors expressing CCL19 compared to their parental counterparts.

[0255] In this study, the antitumor effects of repeated intravenous administration of R7-19.1 cells were evaluated in an intravenous (IV) xenograft model of Raji-19.5 in NSG mice, which are Raji human Burkitt lymphoma cells engineered to express CCL19. CD19 t-haNK cells (NK-92® [anti-CD19-CAR, CD16.158V, ERIL-2]) that do not express CCR7 were used as a control NK cell line. A vehicle control group was also included.

[0256] Although both NK cell lines demonstrated significant therapeutic efficacy in prolonging survival of IV Raji-19.5 tumor-bearing animals compared to vehicle controls, treatment with R7-19.1 cells gave a significantly greater survival benefit than CD19 t-haNK cells. Although significant treatment-related responses were observed in both NK cell lines, it is speculated that these responses are mouse-specific issues related to the use of relatively high doses of human-derived cells.

[0257] Study Rationale 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 effects of repeated IV administration of R7-19.1 cells were evaluated in an IV xenograft model of Raji-19.5 (a CCL19-expressing subline of Raji) in NSG mice. Note that the original study protocol contained additional animal groups (AC groups) that were not included in this report. They are not relevant to the efficacy determination of R7-19.1 cells in this tumor model (see Table 3 for a simplified experimental design).

[0258] Research Materials:

[0259] One or more test articles. The test articles were R7-19.1 cells and CD19 t-haNK cells (control NK cells that do not express CCR7; clone 6). R7-19.1 cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68. For CD19 t-haNK cells (control NK cells that do not express CCR7; clone 6), CD19 t-haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68. Serum-free growth medium was used as a vehicle control.

[0260] Test System: Test Animals: NOD.Cg-Prkdc that were 10-11 weeks old at the start of the study (after quarantine and acclimatization) and weighed between 20-27 g at the time of randomization were used. scid Iq tm1Wjl / SzJ (NSG) female mice. 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). Animals were identified by ear tags, cage numbers, and tail numbers.

[0261] Raji-19.5 tumor model (cancer cell line)

[0262] Cell culture medium: Raji-19.5 cancer cells were grown in ATCC modified RPMI-1640 medium supplemented with 10% fetal bovine serum.

[0263] Cell harvesting: Raji-19.5 cells (passage 16) in the exponential phase were collected by centrifugation according to Nantes West SOP_Suspension Cancer Cell Collection for In Vivo Studies. The cells were then washed and cultured at 5 × 10 5 Cells were resuspended in serum-free medium at a concentration of 10 cells / mL and stored on ice prior to inoculation in animals. Cells used in in vivo studies had a viability of 97%.

[0264] Inoculation: 30 mice were inoculated via the lateral tail vein with 1 × 10 5 This was defined as day 0.

[0265] Experimental Procedure

[0266] Body Weight: Animals were weighed prior to enrollment (after quarantine / acclimatization), prior to randomization, on the day of each dosing (but prior to dosing), the day after each dosing, and prior to euthanasia. Animals that exhibited a weight loss of > 20% compared to baseline (Day 0) body weight were euthanized per institutional IACUC policy and necropsy was performed at the discretion of the study director.

[0267] Clinical Observations: Animals were observed daily for mortality / morbidity (G0 to G4; see Table 2 in the study protocol in Appendix 1). Moribund and paralyzed animals were euthanized and necropsy was performed at the discretion of the study supervisor.

[0268] Randomization: On day 3 (3 days after tumor cell inoculation), 30 tumor-bearing mice were pseudo-randomly divided into 3 research groups according to animal body weight, with 10 mice in each group.

[0269] Test article administration: R7-19.1 and control CD19 t-haNK cells growing in the exponential phase were harvested by centrifugation twice a week for 4 consecutive weeks (on days 3, 6, 10, 13, 17, 20, 24, and 27) and cultured at 5 × 10 7 The cells were prepared at a concentration of 1 × 10 cells / mL in serum-free growth medium and injected at a volume of 200 μL per mouse. 7The dose of 10 cells was administered IV. All cell handling and formulation procedures were performed at room temperature. The cell viability of all dose preparations was above 80%. As shown in Table 3, while group D received vehicle control, groups E and F received CD19 t-haNK and R7-19.1 cells, respectively.

[0270] Endpoints: Animals were euthanized when they became paralyzed, moribund, or met any other endpoint criteria defined by the Institutional IACUC. The experiment ended on Day 30 when the last surviving animal succumbed to disease. Euthanasia was performed via CO2 inhalation followed by cervical dislocation. At the discretion of the Study Director, necropsy was performed on some euthanized animals to identify visible tumor nodules on internal organs. A complete record of mortality and death events and necropsy findings can be found in Appendix 5.

[0271] Table 3: Study design (abbreviated)

[0272]

[0273] BIW: twice weekly; IACUC: Institutional Animal Care and Use Committee; IV: intravenous.

[0274] Data Analysis:

[0275] Body weight curves: Body weight curves were analyzed by two-way ANOVA (or by mixed-effects analysis when there were missing values; see revision 2 of Appendix 1 ) followed by Tukey's test for multiple comparisons.

[0276] Survival curves: Survival curves were analyzed by log-rank (Mantel-Cox) test.

[0277] Statistical analysis: All statistical analyses were performed using GraphPad Prism version 8. P < 0.05 was considered statistically significant.

[0278] result

[0279] Efficacy: The primary readout for efficacy was animal survival. Mortalities were counted when animals were euthanized due to morbidity, paralysis, or weight loss > 20%. No animal deaths were observed. Fig.15As shown in Table 4, treatment with both R7-19.1 and control CD19 t-haNK cells significantly prolonged the survival of Raji-19.5 IV tumor-bearing animals compared with vehicle control (P < 0.0001 for R7-19.1 and P = 0.0002 for CD19 t-haNK by log-rank test), with median survival increased by 6.5 and 2.5 days, respectively. This corresponds to an increase of 30% and 12%, respectively, relative to vehicle control. More importantly, expression of the CCR7 chemokine responsive receptor in R7-19.1 cells increased median survival by an additional 4 days (17% increase) compared with CD19 t-haNK cells (P < 0.0001).

[0280] Table 4: Summary of median survival of Raji-19.5 IV tumor-bearing NSG mice treated with vehicle, CD19 t-haNK cells, or R7-19.1 cells.

[0281]

[0282] NA: Not applicable.

[0283] Security

[0284] like Fig.16 As shown, animals treated with NK cells consistently showed 5%-10% weight loss after each treatment administration. In most cases, animals were able to recover from cell injections and showed weight recovery, causing oscillations in the weight change curve between doses. However, the initial dose seemed to cause the most severe reaction and was associated with the longest recovery time for clinical symptoms and weight changes. Such reactions are not uncommon in animals receiving IV NK infusions and are certainly not specific to R7-19.1 cells. The recovery of body weight indicates that weight loss is temporary and reversible.

[0285] Notably, at the end of the study, the animals showed a dramatic decrease in body weight ( Fig.16 ), which may be due to disease progression). Necropsy revealed tumor nodules in the liver, ovaries, and occasionally spleen of nearly all animals examined. One exception was the first mouse euthanized in the CD19 t-haNK cell group, which achieved >20% weight loss on the day after the sixth dose. No visible tumor nodules were identified during necropsy in this animal. Therefore, the exact reason for weight loss that warranted euthanasia could not be determined.

[0286] in conclusion

[0287] In the IV Raji-19.5 xenograft model, 1 × 10 7IV administration of freshly prepared R7-19.1 cells (twice a week for 4 weeks) at a dosing level of 10 cells / dose showed noteworthy and statistically significant anti-tumor efficacy. This treatment provided a 6.5-day or 30% increase in median survival relative to the vehicle control group and a 4-day or 17% increase relative to the CD19 t-haNK treated group.

[0288] Although clear treatment-related reactions were observed, these reactions were transient and surviving animals did show signs of recovery. It is speculated that these reactions are mouse-specific issues related to the administration of relatively high doses of human NK cells and are therefore unlikely to translate to humans.

[0289] Overall, in this IV model of Raji-19.5, CCR7-expressing R7-19.1 cells showed significant therapeutic efficacy compared to vehicle and its non-chemokine-responsive counterparts.

[0290] Example 8: Comparative Efficacy Evaluation of R7-19.1 Cells Expressing CCR7 in NSG Mice Bearing Subcutaneous CCL19-Positive RAJI Tumors

[0291] CCR7 is a chemokine receptor that induces cell migration toward gradients of chemokines CCL19 and CCL21, which are typically expressed in lymph nodes and other lymphoid organs, which are the primary sites of disease manifestation in B-cell lymphomas. We have created chemokine-responsive NK-92® cells (i.e., R7-19.1 cells) expressing aCD19-CAR, CCR7, CD16.158V, and ERIL-2 based on the t-haNK platform. In addition to CCR7 expression, these cells also have cancer-targeting chimeric antigen receptors (CARs) against the CD19 cancer antigen, CD16 variants, and ER-IL-2. Previous distribution studies have demonstrated that R7-19.1 cells preferentially home to subcutaneous (SC) tumors expressing CCL19 compared to their parental counterparts.

[0292] In this study, the antitumor effect of repeated intravenous (IV) administration of R7-19.1 cells was evaluated in a SC xenograft model of Raji-19.5 in NSG mice, which are Raji human Burkitt lymphoma cells engineered to express CCL19. CD19 t-haNK cells (NK-92® [anti-CD19-CAR, CD16.158V, ERIL-2]) that do not express CCR7 were used as a control NK cell line. A vehicle control group was also included.

[0293] In a subset of tumor-bearing animals, both NK cell treatments showed significant effects in inhibiting tumor growth, with R7-19.1 treatment demonstrating a stronger inhibitory effect than control CD19 t-haNK cells. Although significant treatment-related responses were observed in both NK cell lines, these responses are presumably mouse-specific issues related to the use of relatively high doses of human cells.

[0294] Study rationale and purpose : In vivo distribution data showed that IV-administered R7-19.1 cells exhibited increased homing to CCL19-expressing SC tumors. In this study, the anti-tumor effects of repeated IV administration of R7-19.1 cells were evaluated in both IV and SC xenograft models of Raji-19.5 (a CCL19-expressing subline of Raji) in NSG mice.

[0295] Research Materials

[0296] One or more test articles: R7-19.1 cells (clone: ​​and CD19 t-haNK cells (control NK cells not expressing CCR7; clone 6) were used as test articles, while growth medium was used as vehicle control.

[0297] R7-19.1 cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68.

[0298] CD19 t-haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68.

[0299] Experimental System: Thirty NOD.Cg-Prkdc mice that were 10-11 weeks old at the start of the study (after quarantine and acclimatization) and weighed 19-28 g on the day of tumor implantation were used in the study. scid Iq tm1Wjl / SzJ (NSG) female mice were obtained from the Jackson Laboratory (610 Main Street, Bar Harbor, ME 04609, USA). Animals were identified using ear tags, cage numbers, and tail numbers.

[0300] Raji-19.5 tumor model (cancer cell line)

[0301] Cell culture medium: Raji-19.5 cancer cells were cultured in ATCC modified RPMI-1640 medium supplemented with 10% fetal bovine serum.

[0302] Cell harvesting: Raji-19.5 cells (passage 16) in the exponential phase were harvested by centrifugation according to Nantes West SOP_Harvesting of Suspension Cancer Cells for In Vivo Studies. The cells were then washed and resuspended in serum-free medium and then mixed with an equal amount of Matrigel to a concentration of 2.5 × 10 6 The cells were stored on ice prior to inoculation of animals. The cells used in the in vivo studies had a viability of 97%.

[0303] Inoculation: 30 animals were inoculated unilaterally in the right flank with 2.5 × 10 5 The skin was shaved before the injection.

[0304] Experimental Procedure

[0305] Tumor volume measurement: After SC tumor implantation, animals were examined at least twice a week for tumor formation. When tumors became measurable, tumor volume (TV) was measured twice a week with a digital handheld caliper and calculated using the following formula: TV = length × width 2 / 2 [length is the largest diameter of the tumor, width is the smallest diameter of the tumor]. According to the institutional IACUC policy, for tumor volumes exceeding 2000 mm 3 Animals with ulcerated tumors were euthanized and subsequently autopsied. Tumor growth inhibition (TGI) was calculated as follows: TGI = (T C -T t ) / ΔT C × 100%, where T C and T t are the mean tumor volumes of the control and treatment groups at a specific time point, respectively; and ΔT C is the change in mean tumor volume in the control group.

[0306] Body Weight: Animals were weighed prior to enrollment (after quarantine / acclimatization), prior to randomization, on the day of each dosing (but prior to dosing), the day after each dosing, and prior to euthanasia. Animals that exhibited a weight loss of > 20% compared to baseline (Day 1) weight were euthanized and subsequently necropsy performed in accordance with IACUC institutional policy.

[0307] Clinical Observations: Animals were observed daily for mortality / morbidity (G0 to G4; see Table 5). Moribund or paralyzed animals were euthanized and subsequently autopsied.

[0308] Randomization: When the mean tumor volume reached 190 mm 3At day 1, 30 mice were pseudo-randomized into 3 study groups of 10 mice each to achieve similar tumor volumes between groups. This was defined as day 1. Of note, due to the large variability in tumor size at randomization, each group contained two subgroups: 4-5 animals with large (> 200 mm 3 ) tumors, 5-6 with small (< 200 mm 3 ) Tumors (see Fig.17 ).

[0309] Test article administration: R7-19.1 and control CD19 t-haNK cells growing in the exponential phase were harvested by centrifugation twice a week for 4 consecutive weeks (on days 1, 4, 9, 12, and 15) and cultured at 5 × 10 7 The cells were prepared at a concentration of 1 × 10 cells / mL in serum-free growth medium and injected at a volume of 200 μL per mouse. 7 The dose of 10 cells was administered IV. All cell handling and formulation procedures were performed at room temperature. The cell viability of all dose preparations was above 80%. As shown in Table 5, while Group A received vehicle control, Groups B and C received CD19 t-haNK and R7-19.1 cells, respectively.

[0310] Endpoints: Animals were euthanized when they reached any of the above endpoints as defined by the institutional IACUC. Euthanasia was performed via CO2 inhalation followed by cervical dislocation. At the discretion of the study director, some euthanized animals were necropsy-examined to identify visible tumor nodules on internal organs.

[0311] Table 5: Study design (abbreviated)

[0312]

[0313] BIW: twice weekly; IACUC: Institutional Animal Care and Use Committee; IV: intravenous; TGI: tumor growth inhibition.

[0314] 1 , IACUC-defined tumor burden endpoint: tumor volume greater than 2000 mm 3 ; ulcerated tumors; and tumors that interfere with normal gait.

[0315] Data analysis

[0316] Tumor volume calculation: Tumor volume = length × width 2 / 2 (length and width are the longest and shortest diameters of the tumor, respectively)

[0317] Tumor growth inhibition (TGI) calculation: TGI = (T C -Tt ) / ΔT C × 100%, where T C and T t are the mean tumor volumes of the control and treatment groups at a specific practice point, and ΔT C is the change in mean tumor volume in the control group.

[0318] Statistical analysis of tumor growth and body weight curves: Tumor growth and body weight curves were analyzed by two-way ANOVA (or by mixed-effects analysis when there were missing values; see revision 2 of Appendix 1) followed by multiple comparisons by Tukey's test. All statistical analyses were performed using GraphPad Prism version 8. P < 0.05 was considered statistically significant.

[0319] result

[0320] Efficacy: The primary readout in this study was tumor growth inhibition. SC tumors with different initial tumor volumes may have differently developed vasculature and CCL19 gradients, which may affect the distribution of the test article to the tumor. Additionally, small and large tumors may respond to NK cell treatment in different ways. For these reasons, both subpopulations (i.e., large and small tumors) were analyzed separately.

[0321] like Fig.18 As shown, in a subset of animals with large initial tumors, treatment with both NK cell lines showed significant tumor growth inhibition when compared to vehicle control, with the highest inhibition of tumor growth at day 9 due to tumor volumes exceeding 2000 mm 3 or the presence of ulcerated tumors, 3 of 4 animals had to be euthanized. In contrast, in the NK cell-treated groups, most animals survived until day 12 or 15. More importantly, at day 15, tumor growth inhibition was clearly enhanced in the R7-19.1 group compared with its counterparts that did not express CCR7, with a TGI of 35%. This difference failed to reach statistical significance, likely due to the small cohort size (N of 2 and 4 for the CD19 t-haNK and R7-19.1 groups, respectively).

[0322] However, in animals with small tumors at the start of treatment, neither NK cell therapy was effective in inhibiting tumor growth when compared to vehicle controls or to each other ( Fig.19). This may be attributed to three factors: 1) The vasculature of small tumors may not be fully developed, thus having lower exposure / accessibility to the test article, resulting in ineffectiveness of both NK cell therapies. 2) CCR7-mediated chemotaxis requires a chemokine gradient, rather than responding to the presence of chemokine alone. Such CCL19 gradients may not be fully established in small tumors, resulting in a lack of distinguishability between R7-19.1 cells and CD19 t-haNK controls. And 3) there were some “outliers” in tumor growth in these already small cohorts, further precluding the possibility of statistical analysis.

[0323] like Fig. 20 As shown, animals treated with NK cells typically show a 5%-10% weight loss after each treatment administration. In most cases, animals are able to recover from cell injections and show weight recovery, causing oscillations in the weight change curve between doses. However, the initial dose seems to cause the most acute and severe reactions and is associated with the longest recovery time for clinical symptoms and weight changes. Such reactions are not uncommon in animals receiving IV NK infusions and are certainly not R7-19.1 cell-specific. The recovery of body weight indicates that weight loss is temporary and reversible.

[0324] in conclusion

[0325] 1 × 10 7 IV administration of freshly prepared chemokine-responsive R7-19.1 cells twice a week for 4 weeks at a dosing level of 10 cells / dose resulted in significant inhibition of tumor growth in animals with large SC Raji-19.5 tumors, but such effects did not reach statistical significance, which may be due to the small size of the cohort. This treatment regimen did not show therapeutic efficacy in animals with smaller initial tumor volumes. This may be attributed to the inadequate development of the tumor vasculature and / or inadequate establishment of chemokine gradients in small tumors. Obvious treatment-related reactions were observed. However, these reactions were transient and the animals did show signs of recovery. It is speculated that these reactions are mouse-specific issues associated with the administration of relatively high doses of human NK cells and are therefore unlikely to be translated into humans.

[0326] Example 9: Production a TGFβ / PD-L1 CAR modified NK-92® cells

[0327] The TGFβ trap consisting of a single-chain dimer of the extracellular domain of TGFβRII was cloned into a tetracistronic plasmid vector that also contained the PD-L1 CAR, CD16, and erIL-2 transgenes ( Fig.21). The tetracistronic plasmid was electroporated into aNK cells to generate modified NK-92 cells. The modified NK-92® cells were selected by IL-2 deficient medium, because non-transformed aNK cells that are dependent on IL-2 cannot survive in IL-2 deficient medium. Fig. 22 Show a TGFβ / PD-L1 CAR-modified NK-92® cells co-express high levels of PD-L1 CAR and CD16.

[0328] Limiting dilution cloning

[0329] Aliquots of the polyclonal aTGFβ / PD-L1 t-haNK™ library culture were diluted to a density of 3 cells / ml in growth medium without IL-2 supplementation. This cell suspension was aliquoted in 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 and then visually inspected for cell growth. Clones were picked and transferred to larger vessels for further expansion and characterization.

[0330] Bioanalytical methods

[0331] Cell culture:

[0332] Polyclonal and clonal aTGFβ / PD-L1 t-haNK™ cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and without IL-2.

[0333] aNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 500 IU / ml recombinant human IL-2.

[0334] HaNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and without IL-2.

[0335] K562 and MDA-MB-231 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic / antimycotic cocktail. K562 cells were passaged every 2-5 days or whenever the culture medium developed a yellow color.

[0336] SUP-B15 PD-L1+ and SUP-B15 CD19KO / CD20+ Cells were cultured in RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum, 55 uM β-mercaptoethanol, and an antibiotic / antimycotic cocktail. Cells were otherwise passaged as above for K562 cells.

[0337] Antibody staining for flow cytometry analysis:

[0338] Cells were collected by centrifugation, washed twice in FACS buffer (5% FBS in 1X D-PBS), and resuspended in 1 ml FACS buffer. For direct fluorophore-conjugated antibody staining of surface proteins, cells were incubated with appropriate conjugated antibodies (or isotype controls) at 4°C in the dark for 20 minutes and then washed twice with FACS buffer. To detect CAR protein, cells were incubated with biotinylated anti-F(ab')2 fragment antibodies followed by streptavidin-APC antibodies. Samples were analyzed on a MACSQuant flow cytometer.

[0339] Cytotoxicity:

[0340] Cell lines grown in suspension were resuspended by pipetting the cell culture up and down. Cell viability was determined by automated counting (Trypan blue exclusion). Target cells were labeled with CFSE dye and target and effector cells were diluted to the desired cell concentration in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotics / antimycotics. Effector and target cells were mixed in 96-well plates at different effector to target ratios (E: T of 20: 1, 10: 1, 5: 1, 2.5: 1, 1.25: 1, 0.62: 1, 0.31: 1, and 0.15: 1) and co-incubated in an incubator at 5% CO2 atmosphere and 37°C for 4 h. PI was then added for fluorescent labeling of dead cells and the assay was analyzed on a MACSquant flow cytometry device.

[0341] ADCC:

[0342] Cell lines grown in suspension were resuspended by pipetting the cell culture up and down. Cell viability was determined by automated counting (Trypan blue exclusion). Target cells were labeled with PKH67-GL dye and target and effector cells were diluted to the desired cell concentration in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotics / antimycotics. Target cells were preincubated with monoclonal antibodies trastuzumab, rituximab, or without antibodies for 30 min at room temperature. Antibody-labeled target cells (and no antibody control) and effector cells were then mixed in 96-well plates at different effector to target ratios (E:T of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, 0.31:1, and 0.15:1) and co-incubated for 4 h in a 5% CO2 atmosphere and 37°C incubator. PI was then added to fluorescently label dead cells, and the assay was analyzed on a MACSquant flow cytometry device.

[0343] Quantification of TGFβ trap secreted by aTGFβ / PD-L1t-haNK cells

[0344] Sample supernatants for analysis were prepared by removing cells by a first centrifugation step at 500 x g for 5 min, followed by a second centrifugation step at 2000 x g for 5 min to remove cell debris. Sample supernatants were frozen at -80°C until analysis. Cell pellets from the 500 x g centrifugation step were resuspended, pooled in triplicate and cell density recorded. TGFβRII ELISA test kit was used to measure the TGFβ trap concentration and compare it with the standards provided. TGFβ trap The concentrations were normalized to the cell number and expressed as pg / ml / 10 6 cells. Fig.23 Show, all a TGFβ PD-L1 t-haNK clones all secreted large amounts of TGFβ trap (ranging from ~6 to ~13 ng / ml / 10 6 between cells).

[0345] Cytotoxicity of aTGFβ / PD-L1 t-haNK™ cells against target cell lines

[0346] The cytotoxicity of aTGFβ / PD-L1 t-haNK™ cells was demonstrated by combining them with target cells K562 cells, SUP-B15 PD -L1+ Cells were incubated with MDA-MB-231 cells for analysis. Fig.24 It was shown that aTGFβ / PD-L1 t-haNK™ cells maintained equivalent cytotoxicity to parental aNK cells when killing K562 cells (target cells).

[0347] Fig.25 aTGFβ / PD-L1 t-haNK™ cells were shown to exhibit enhanced specific killing of the aNK™-resistant, PD-L1-positive SUP-B15 cell line - at an effector to target ratio of 8, approximately 70% of the cells were killed by aTGFβ / PD-L1 t-haNK™ cells compared to only approximately 10% of the cells killed by aNK cells.

[0348] Fig.26 aTGFβ / PD-L1 t-haNK™ cells were shown to exhibit enhanced specific killing of the MDA-MB-231 cell line - at an effector to target ratio of 8, approximately 90% of the cells were killed by aTGFβ / PD-L1 t-haNK™ cells compared to only approximately 40% of the cells killed by aNK cells.

[0349] Fig. 27The combination of aTGFβ / PD-L1 t-haNK™ cells with anti-CD20 rituximab monoclonal antibody or anti-Her2-neu trastuzumab monoclonal antibody showed an inhibitory effect on SUP-B15 CD19KO / CD20+ In a 4 h cytotoxicity assay, aTGFβ / PD-L1 t-haNK cells were able to effectively target and kill resistant SUP-B15 cells when combined with the anti-CD20 antibody rituximab. CD19KO / CD20+ When combined with the anti-Her2 / neu control antibody trastuzumab, both haNK® and aTGFβ / PD-L1 t-haNK™ clones were unable to kill the target SUP-B15 CD19KO / CD20+ cell.

[0350] Example 10: Modified NK-92® cells secrete TGFβ Well Suppression TGFβ activity

[0351] HEK293 cells engineered to directly express the TGFβ-responsive element (SMAD-binding promoter) of the luciferase reporter gene showed a dose-dependent increase in luciferase activity when treated with TGFβ ( Fig.28 ).

[0352] Fig.29 showed that the induction of luciferase activity by TGFβ in HEK293 reporter cells could be inhibited by co-incubation with the culture supernatant of aTGFβ / PD-L1 t-haNK cells, whereas the culture supernatant from haNK control cells had limited effect on luciferase activity.

[0353] Example 11: Modified NK-92® cells produce IL-12

[0354] NK-92 cells were transduced with lentiviral constructs encoding functional IL-12 p70 dimers as single-chain polypeptides in either the p35-p40 orientation or the p40-p35 orientation. Fig.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.

[0355] IL-12 / Generation of PD-L1 CAR-modified NK-92® cells

[0356] The single-chain dimer of IL-12 (scIL-12 p70) was cloned into a tetracistronic plasmid vector that also contained the PD-L1 CAR, CD16, and erIL-2 transgenes. Fig.31 ) were electroporated into aNK cells to generate modified NK-92 cells. Modified NK-92® cells were selected by IL-2-deficient medium, because non-transformed aNK cells that are dependent on IL-2 cannot survive in IL-2-deficient medium. Fig.33 Display IL-12 / PD-L1 CAR-modified NK-92® cells are able to secrete large amounts of scIL-12 p70 cytokines.

[0357] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations in view thereof will be suggested to those skilled in the art and are included within the spirit and scope of the present application and within the scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0358] It should be clear to those skilled in the art that more modifications other than those already described are possible without departing from the inventive concept of this document. Therefore, the subject matter of the present invention is limited only by the scope of the appended claims. In addition, when interpreting this specification and claims, all terms should be interpreted in the broadest possible manner consistent with the context. Specifically, the terms "comprises" and "comprising" should be interpreted as referring to elements, components or steps in a non-exclusive manner, indicating that the elements, components or steps mentioned can exist, or utilize, or combine with other elements, components or steps not explicitly mentioned. In the case where the claims of this specification refer to at least one of something selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element in the group, rather than A plus N or B plus N, etc.

Claims

1. An inducible promoter for selective and targeted secretion of NK-92 cells, comprising a NFAT transcription factor response element.

2. The inducible promoter according to claim 1, wherein the NFAT transcription factor responsive gene is CCL21.

3. The inducible promoter according to claim 1, wherein NFAT is activated by the FcεRIγ pathway or the CD3ζ pathway through activation of CD19 CAR. The inducible promoter according to claim 1 , wherein the NFAT transcription factor response element has the polynucleotide sequence of SEQ ID NO:

13. The inducible promoter according to claim 1 , wherein the NFAT transcription factor response element has the polynucleotide sequence of SEQ ID NO:

5. The inducible promoter according to claim 1 , wherein the NFAT transcription factor response element has the polynucleotide sequence of SEQ ID NO:

6. 7 . The inducible promoter according to claim 1 , wherein the NFAT transcription factor response element has the polynucleotide sequence of SEQ ID NO:

8.

8. A modified NK-92 cell comprising a nucleic acid encoding a homing receptor operably linked to a promoter, wherein the homing receptor is selected from a G protein coupled receptor (GPCR), a chemokine receptor, a cytokine receptor, a cell adhesion molecule, a selectin or an integrin, and The promoter contains a NFAT transcription factor response element.

9. The modified NK-92 cell according to claim 8, wherein the NFAT transcription factor responsive gene is CCL21.

10. The modified NK-92 cell according to claim 8, wherein NFAT is activated by the FcεRIγ pathway or the CD3ζ pathway through activation of CD19 CAR.

11. The modified NK-92 cell according to claim 8, wherein the NFAT transcription factor response element has the polynucleotide sequence of SEQ ID NO:

13.

12. The modified NK-92 cell according to claim 8, wherein the NFAT transcription factor response element has a polynucleotide sequence of SEQ ID NO:

5.

13. The modified NK-92 cell according to claim 8, wherein the NFAT transcription factor response element has a polynucleotide sequence of SEQ ID NO:

6.

14. The modified NK-92 cell according to claim 8, wherein the NFAT transcription factor response element has a polynucleotide sequence of SEQ ID NO:

8.

15. The modified NK-92 cell according to claim 8, wherein the chemokine receptor is selected from CCR7, CXCR2 or CXCL14 receptor.

16. The modified NK-92 cell according to claim 8, wherein CCR7 is encoded by the nucleic acid according to SEQ ID NO:

1.

17. The modified NK-92 cell according to claim 8, wherein the cell adhesion molecule is LPAM-1 and / or LFA-1.

18. The modified NK-92 cell according to claim 8, wherein the selectin is L-selectin (CD62L).

19. The modified NK-92 cell according to claim 8, wherein the integrin is α4β7 integrin (VLA-4).

20. The modified NK-92 cell of claim 8, wherein the cytokine comprises IL2, IL-15, erIL-2, erIL-15, or a combination thereof.

21. The modified NK-92 cell of claim 8, wherein the cytokine is erIL-2.

22. The modified NK-92 cell of claim 21, wherein erIL-2 is encoded by the nucleic acid of SEQ ID NO:

14.

23. The modified NK-92 cell of claim 8, wherein the cytokine is erIL-15.

24. The modified NK-92 cell of claim 23, wherein erIL-15 is encoded by the nucleic acid of SEQ ID NO:

67.

25. The modified NK-92 cell according to claim 8, further comprising a TGFβ trap, wherein the amino acid sequence of the TGFβ trap is shown in SEQ ID NO:

66.

26. The modified NK-92 cell of claim 8, further comprising an Fc receptor.

27. The modified NK-92 cell of claim 26, wherein the Fc receptor is CD16 or high affinity CD16 (SEQ ID NO: 12).

28. The modified NK-92 cell of claim 8, further comprising a chimeric antigen receptor (CAR), wherein the CAR specifically binds to CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, IGF1R, CSPG4 or B7-H4.

29. The modified NK-92 cell of claim 28, wherein the CAR comprises an FcεRIγ signaling domain, a CD8 hinge domain, a CD28 transmembrane domain, and an antigen binding domain, wherein the antigen binding domain is selected from CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, IGF1R, CSPG4, or B7-H4.

30. The modified NK-92 cell of claim 29, wherein the FcεRIγ signaling domain has the amino acid sequence of SEQ ID NO:

37.

31. The modified NK-92 cell of claim 29, wherein the CD8 hinge domain has the amino acid sequence of SEQ ID NO:

33.

32. The modified NK-92 cell of claim 29, wherein the CD28 transmembrane domain has the amino acid sequence of SEQ ID NO:

35.

33. A method of generating the modified NK-92 cell line of any one of claims 8 to 32, comprising targeted homologous recombination at a specific locus, wherein the locus is AAVS1.

34. The method of claim 33, wherein the amino acid sequence of the AAVS1 locus is as shown in SEQ ID NO:

7.

35. Use of the modified NK-92 cells according to any one of claims 8 to 32 in the preparation of a medicament for treating cancer or reducing tumor size in a subject.

36. A kit comprising the modified NK-92 cells according to any one of claims 8 to 32 and instructions for use.

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