Tetracistronic system containing homing receptors or cytokines and chimeric antigen receptors for genetic modification of immunotherapy

Abstract

Provided herein are modified cells comprising one or more nucleic acids encoding: i) homing receptors, ii) antigen binding proteins (ABPs) or chimeric antigen receptors (CARs) that specifically bind to a target antigen, iii) Fc receptors such as CD16 or CD16-158V, and / or iv) cytokines, wherein the nucleic acid sequence is operably linked to a promoter. Further provided herein are modified cells comprising one or more nucleic acids encoding: i) IL-12 and / or TGF-β traps, ii) antigen binding proteins (ABPs) or chimeric antigen receptors (CARs) that specifically bind to a target antigen, iii) Fc receptors such as CD16 or CD16-158V, and / or iv) cytokines, wherein the nucleic acid sequence is operably linked to a promoter. Compositions and kits comprising these modified cells are also provided, as well as methods for treating cancer using these modified cells.

Description

[0001] 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.

[0002] Sequence Listing

[0003] An ASCII text file of the sequence listing named 104077_0007PCT_Seq_listing_rev004_ST25, 97 KB in size, was created on August 1, 2019, was submitted electronically with this application via EFS-Web, and is incorporated by reference in its entirety. Technical Field

[0004] 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

[0005] 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.

[0006] 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.

[0007] Cancer immunotherapy based on adoptively transferred tumor-specific cytotoxic lymphocytes is expected to be used to treat patients with malignant tumors. Despite early success in some cancers, the treatment of tumors remains a challenge, mainly due to the immunosuppressive nature of the tumor microenvironment. See Swarts et al. "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy," Cancer Res, Vol. 72, 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 usually account for about 10%-15% of circulating lymphocytes, bind and kill target cells that are not specific for antigens and have no previous immune sensitization (including virus-infected cells and many malignant cells). Herberman et al., Science 214: 24 (1981). is a cytolytic cancer cell line that was found in the blood of a subject with non-Hodgkin's lymphoma and then immortalized in vitro. The cells are derived from NK cells but lack the major inhibitory receptors displayed by normal NK cells while retaining most of the activating receptors. The cells will not attack normal cells or cause unacceptable immune rejection in humans.

[0008] 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.

[0009] 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

[0010] This article provides a modified Cells comprising nucleic acids encoding a plurality of functional elements. The functional elements are typically proteins or polypeptides that provide specific functions that improve the effectiveness of the cells as a cell line for immunotherapy. In one aspect, The cell comprises a nucleic acid construct encoding four functional elements. In some embodiments, the nucleic acid construct comprises a sequence encoding four functional elements operably linked to a promoter (referred to as a "tetracistronic construct").

[0011] In some embodiments, the first element encoded by the nucleic acid construct is a cytokine that provides for expression of the cytokine, such as IL-2 or IL-15. Selection of cells. Therefore, 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").

[0012] 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) (corresponding to position 176 of the full-length form of the polypeptide containing the signal sequence) of the mature form of the polypeptide (SEQ ID NO: 12). 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.

[0013] 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.

[0014] In some embodiments, the third element encoded by the nucleic acid construct is a homing receptor. In some embodiments, the homing receptor is a cytokine receptor, a G protein-coupled receptor, a chemokine receptor, a cell adhesion molecule, a selectin, or an integrin. In some embodiments, the homing receptor is operably linked to a promoter that allows transcription of the nucleic acid. The cells are able to migrate toward the source of chemokines, which are ligands for the receptors. Unlike normal blood-derived NK cells, the modified Cells can develop into cell lines associated with human clinical trials, thereby providing significant 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, homing receptors are cell adhesion molecules, such as LFA-1. In some embodiments, homing receptors are selectins, such as L-selectin (CD62L). In some embodiments, homing receptors are integrins, 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.

[0015] In some embodiments, the third element encoded by the nucleic acid construct is a secreted cytokine, whereby the cytokine enhances or improves The cell functions 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.

[0016] 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.

[0017] In one aspect, the third element encoded by the nucleic acid construct is IL-12.

[0018] 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.

[0019] In some embodiments, the NK cells described herein are administered with a TGF-β inhibitor to block TGF-β and help remove immunosuppression. In some embodiments, the NK cells described herein are administered 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, a TGF-β inhibitor is combined with IL-2.

[0020] 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 a 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 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 or 41BB.

[0021] On the other hand, the modified The cell comprises a nucleic acid encoding IL-12. In another aspect, the modified The cell comprises 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 a TGFβRII molecule.

[0022] On the other hand, the modified The cell contains a nucleic acid encoding a cytokine that provides a stimulus for expression of the cytokine. Selection of cells or allowing expression of the cytokine Cell survival. 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").

[0023] In some embodiments, the modified The cell comprises 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 an IgG antibody 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 a nucleic acid sequence of SEQ ID NO: 13 or an amino acid sequence of SEQ IID NO: 12.

[0024] In some embodiments, the modified The cell comprises 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 a 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 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 or 41BB.

[0025] On the other hand, the modified The cell comprises 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 The cell comprises a nucleic acid encoding a Toll-like receptor (TLR) agonist. In some embodiments, the modified The cells contain a nucleic acid encoding IL-12 or IFN-α. In some embodiments, the modified The cell comprises a nucleic acid encoding a TGF-β inhibitor (e.g., a peptide that inhibits TGF-β). In some embodiments, the modified The cell comprises a nucleic acid encoding a TGF-β trap. In some embodiments, the TGF-β trap comprises the extracellular domain of a TGFβRII molecule, or a single chain dimer of the extracellular domain of a TGFβRII molecule.

[0026] On the one hand, the modified The cell comprises one or more, or a plurality of, nucleic acid molecules encoding a homing receptor, an ABP or a CAR, an Fc receptor and / or a cytokine that provides a signaling pathway for expression of the cytokine. Selection of cells or allowing expression of the cytokine Therefore, in some embodiments, the modified The cells comprise nucleic acid molecules encoding chemokine receptors, CAR, CD16 and erIL-2. In some embodiments, the modified The cells contain nucleic acid molecules encoding CCR7 or CXCR2, CAR, CD16 and erIL-2. In some embodiments, the modified The cells contain nucleic acid molecules encoding IL-12 or TGF-β trap, CAR, CD16 and erIL-2.

[0027] In some embodiments, the CAR comprises an intracellular signaling domain from Fcε receptor γ (FcεRIγ). In one embodiment, the CAR is In one embodiment, the CAR is expressed transiently by Stable expression in cells.

[0028] To date, FcεRIγ-containing CARs have not been used in Cells, other NK cell lines or endogenous NK cells, because other signaling domains (e.g., CD3ζ) have been determined to be more effective, especially when combined with additional signaling domains (second-generation CARs and third-generation CARs). This article describes the unexpected and surprising discovery that a "first generation" CAR expressing an intracellular domain comprising FcεRIγ Cells and a CAR expressing a CD3ζ signaling domain alone or in combination with other signaling domains (i.e., a second generation or third generation CAR) The cell has the same or higher cytotoxic activity against cancer cells expressing an antigen recognized by the CAR. 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.

[0029] On the one hand, a Cells or cell lines, which A chimeric antigen receptor (CAR) is expressed on the surface of a 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.

[0030] 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.

[0031] In some embodiments, the CAR comprises a hinge region from CD8. In some embodiments, the CAR comprises a transmembrane domain from CD28.

[0032] In some embodiments, the nucleic acid construct comprising SEQ ID NO: 31 (FcεRIγ intracellular cytoplasmic domain), SEQ ID NO: 32 (FcεRIγ intracellular signaling domain minus the 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) is used to construct the antibody. Cells or cell lines are genetically modified. In one embodiment, the amino acid sequence of the CD8 hinge region, CD28 transmembrane and FceRIγ 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 the 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 equimolar levels of polypeptides 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 that provides a response to expression of the cytokine (such as IL-2). Cell selection or allowing the Cell survival. In one embodiment, the cytokine targets the endoplasmic reticulum. In one embodiment, the CAR scFv may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 39.

[0033] In some embodiments, the construct comprises Fig.10 In some embodiments, The cell or cell line is genetically modified to express CD16 on the cell surface. The cells or cell lines are genetically modified to express high affinity CD16 (F158V) on the cell surface.

[0034] 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, CD33B7-H4 and 41BB. In one embodiment, the tumor-associated antigen is CD19. In another embodiment, the tumor-associated antigen is CD33.

[0035] In one aspect, the present disclosure relates to a A cell line transformed with a nucleic acid encoding a chimeric antigen receptor (CAR) having a cytoplasmic domain of FcεRIγ, wherein the CAR In one embodiment, the nucleic acid is expressed on the surface of a cell. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is DNA.

[0036] In some embodiments, The cells are further modified to express at least one cytokine or a variant thereof that provides a cytokine for expressing the cytokine. Selection of cells or allowing expression of the cytokine In one embodiment, the at least one cytokine is In one embodiment, the at least one cytokine is expressed transiently by Stable expression in cells.

[0037] In some embodiments, the modified The cell comprises 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 nucleic acid molecule in the multiple nucleic acid molecules is operably connected to a separate, differential and / or different promoter. In some embodiments, one or more nucleic acid molecules in these nucleic acid molecules are operably connected to the same promoter. In one embodiment, nucleic acid molecules encoding homing receptors, CAR, 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, nucleic acid molecules encoding cytokines are located downstream or 3' of nucleic acid molecules encoding homing receptors, CAR and Fc receptors (e.g., CD16 or high-affinity CD16).

[0038] In some embodiments, The cell expresses the protein encoded by the nucleic acid molecule described herein on the cell surface. For example, in some embodiments, the modified The cells express a homing receptor, ABP or CAR and an Fc receptor (e.g., CD16 or high affinity CD16) on the cell surface.

[0039] Also provided are modified Compositions and kits of cells. Methods of making modified cells and methods of using these cells to treat cancer are provided.

[0040] In another aspect, methods for treating cancer or reducing tumor size are described. In some embodiments, the method of treating cancer or reducing tumor size comprises administering to a subject in need thereof a therapeutically effective amount of a modified Cells, wherein administration treats cancer in a subject or reduces tumor size in a subject. In some embodiments, these methods comprise administering to a subject a therapeutically effective amount of a modified Cells, modified The cells contain 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 comprise administering to a subject a therapeutically effective amount of a modified Cells, modified The cells contain 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).

[0041] In some embodiments, the NK cells described herein are administered with a TGF-β inhibitor to block TGF-β and help remove immunosuppression. In some embodiments, the NK cells described herein are administered 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, a TGF-β inhibitor is combined with IL-2.

[0042] In another aspect, the use of the compositions described herein for treating a disease is provided. In some embodiments, the modified Cells are used as drugs for treating diseases. In some embodiments, the modified Cells are used to treat disease. In some embodiments, the modified The cell comprises a nucleic acid encoding a homing receptor, an ABP or CAR that specifically binds to a target antigen, an Fc receptor (such as CD16 or CD16-158V) and / or a cytokine (such as erIL-2 or erIL-15). In some embodiments, the modified The cell comprises a nucleic acid encoding a secreted cytokine, an ABP or CAR that specifically binds to a target antigen, an Fc receptor (such as CD16 or CD16-158V) and / or a cytokine (such as erIL-2 or erIL-15). In some embodiments, the disease is cancer.

[0043] 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

[0044] Figure 1 Is displayed for insertion Schematic representation of the CCR7 receptor-containing plasmid pNKAT-CCR7-LP3 at the AAVS1 locus in cells.

[0045] Figure 2 It is a schematic diagram showing the plasmid pCRENFAT-CCL21 containing the NFAT-responsive CCL21 gene.

[0046] Figure 3 Is displayed with Cell-related phenotypic markers in wild type Cells and modified CCR7 expressing Graph showing the expression of NK cells. (Lane 1: aNK (wild type); Lane 2: modified cells (MA3); Lane 3: modified Cells (MB4); Lane 4: modified Cells (MB6); Lane 5: modified Cells (ME6); Lane 6: modified Cells (MH3); A: isotype (APC); B: CD54 (ICAM-1); C: NKp30; D: NKG2D

[0047] Figure 4 is a modified gene expressing CCR7 Graph of the cytotoxic activity of cells against K562 cells.

[0048] Figure 5 is a modified gene expressing CCR7 Graph of the cytotoxic activity of cells against HL-60 cells.

[0049] 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.

[0050] Figure 7 is a modified gene expressing CCR7 Graph of cell (Mi-aNK) migration toward chemokines CCL19 and CCL21.

[0051] Figure 8 Figure 2 shows a schematic diagram of a modified protein used for in vitro testing of the modified protein described herein. Schematic diagram of an exemplary method for activating The cells (aNK) are modified to express a chemokine receptor (e.g., CCR7), and the target cells are modified to express a chemokine (e.g., CCL19 or CCL21) that binds to the receptor. As shown in the figure, the modified aNK cells are assayed in a modified Boyden chamber Transwell assay. Cells were tested.

[0052] Fig. 9 The use of the modified Representative cytotoxicity assay of cells. Figure 8 modified Cytotoxicity of cells against K562 target cells expressing and secreting one or both chemokine ligands. The ML4 clone showed the highest percentage of target cell lysis, and this percentage increased when K562 target cells simultaneously expressed CCL19 and CCL21.

[0053] 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.

[0054] Fig.11 Is displayed from Fig.10 Schematic diagram of the linearized plasmid.

[0055] Fig.12 Shows Cell surface expression of CCR7, CD16 and CD19 CAR. "aNK" is wild type Cell lines. "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).

[0056] 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 determined by one-way ANOVA followed by multiple comparisons by Tukey's test. The last figure presents the time course curve.

[0057] 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.

[0058] 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.

[0059] Fig.16The weight changes in the IV Raji-19.5 tumor model are illustrated. The weight change curves of IV 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 0). Data are mean ± SEM. Red arrows indicate the days of administration. Weight measurements made on the day of administration were performed before dose administration. For all time points before day 20, the curves of the NK cell-treated group were statistically significantly different (P < 0.05) from those of the vehicle control group by two-way ANOVA followed by multiple comparisons by Tukey's test.

[0060] Fig.17 The size of SC Raji-19.5 tumors at randomization is illustrated. The size of individual tumors at randomization is shown. Black boxes enclose sizes >200 mm 3 The blue box encloses a large tumor of less than 200 mm 3 Group mean ± SEM is also shown.

[0061] 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.

[0062] Fig.19 Tumor growth of a small tumor subset 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 two groups at any time point. (B) Individual curves. Red arrows indicate dosing days. Tx: treatment.

[0063] 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). The data are mean ± SEM. The red arrow indicates the number of days of administration. The body weight measurements made on the day of administration were performed before the dose was administered. For all time points before day 16, the curves of the NK cell-treated group were compared with the vehicle control group by two-factor mixed effects analysis, followed by multiple comparisons by Tukey's test, and statistically significant differences were achieved.

[0064] Fig.21 An example of a PD-L1 CAR construct equipped with a tetracistronic TGFβ-trap is illustrated.

[0065] Fig. 22 Expression analysis of PD-L1 CAR and CD16 in PD-L1 (TGFβ-trap) t-haNK clones is illustrated.

[0066] Fig.23 It is demonstrated that TGFβ trap is secreted into the culture supernatant of TGFβ trap / PD-L1 t-haNK clones.

[0067] Fig.24 The cytotoxicity of the tetracistronic TGFβ-trap construct towards K562 target cells is demonstrated.

[0068] Fig.25 CAR killing of SUP-B15 target cells expressing PD-L1 by a tetracistronic TGFβ-trap construct is demonstrated.

[0069] Fig.26 CAR killing of MDA-MB 231 target cells by a tetracistronic TGFβ-trap construct is demonstrated.

[0070] Fig. 27 Description of the tetracistronic TGFβ-trap construct for SUP-B15 CD19-CD20+ ADCC.

[0071] Fig.28 TGFβ / SMAD luciferase reporter HEK293 cells induced by TGFβ are shown.

[0072] Fig.29 Secreted TGFβ-trap sequestered TGFβ and suppressed luciferase expression in a HEK293T reporter assay are illustrated.

[0073] Fig.30 IL-12 viral transduction IL-12 secretion by cell lines.

[0074] Fig.31 An example of a tetracistronic IL-12 / PD-L1 t-haNK construct is described.

[0075] Fig.32 Cytotoxicity data for CCR7CD19 t-haNK cells are illustrated.

[0076] Fig.33 IL-12 / PD-L1 t-haNK TM IL-12 secretion by cell lines.

[0077] definition

[0078] 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.

[0079] 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:

[0080] 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.

[0081] 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%.

[0082] 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.

[0083] "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.

[0084] The term "comprising" is intended to mean that compositions and methods include the recited 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 would 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 recited method steps. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0085] The term "homing receptor" refers to a receptor that activates a cell pathway that directly or indirectly causes a cell to migrate to a target cell or target tissue. For example, a homing receptor expressed by a leukocyte is used by a leukocyte and a lymphocyte to enter secondary lymphoid tissue via a high endothelial venule. Homing receptors can also be used by cells to migrate to a chemical gradient source such as a chemokine gradient source. 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).

[0086] As used herein, "immunotherapy" refers to a modified or unmodified immunotherapy agent, alone or in combination, that is capable of inducing cytotoxicity upon contact with target cells. Use of cells, naturally occurring or modified NK cells or T cells.

[0087] 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.

[0088] The term "endogenous NK cells" is used to refer to NK cells that originate from a donor (or patient), Different cell lines. Endogenous NK cells are usually a heterologous cell population in which NK cells have been enriched. Endogenous NK cells can be used for autologous or allogeneic treatment of patients.

[0089] The term "NK-92" refers to a unique cell line derived from the highly potent NK cell line described by Gong et al. (1994) (the rights to which are All) natural killer cells (hereinafter referred to as “ cells”). Immortalized NK cell lines were originally obtained from patients with non-Hodgkin lymphoma. Unless otherwise specified, the term It means the original Cell lines and those that have been modified (e.g., by the introduction of foreign genes) Cell lines. 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 (F176V), and Cells are known to those of ordinary skill in the art, and such cells can be obtained from NantKwest, Inc. to those of ordinary skill in the art.

[0090] The term "aNK" refers to unmodified natural killer cells (hereinafter referred to as "aNK cells") derived from a highly efficient unique cell line described by Gong et al. (1994) (the rights to which are owned by Nantes West). The term "haNK" refers to natural killer cells (hereinafter referred to as "haNK cells") derived from a highly efficient unique cell line described by Gong et al. (1994) (the rights to which are owned by Nantes West) that have been modified to express CD16 on the cell surface. Cells" or " In some embodiments, CD16+ The cells contain a high affinity CD16 receptor on the cell surface. The term "taNK" refers to natural killer cells (hereinafter referred to as "CAR-modified" cells) derived from a highly efficient unique cell line described by Gong et al. (1994) (the rights to which are owned by Nant West) that have been modified to express a chimeric antigen receptor. Cells" or " The term "t-haNK" refers to natural killer cells derived from a highly efficient unique cell line described by Gong et al. (1994) (the rights to which are owned by Nant West), modified to express CD16 on the cell surface and to express a chimeric antigen receptor (hereinafter referred to as "CAR-modified CD16+ Cells” or “t-haNK cells”). In some embodiments, t-haNK cells express the high affinity CD16 receptor on the cell surface.

[0091] 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.

[0092] As used herein, the terms "cytotoxic" and "cytolytic" are used to describe effector cells (such as Cytotoxic activity is intended to be synonymous with cytotoxic activity. Generally, cytotoxic activity involves killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of the effector to cleave the plasma membrane of the target cell, thereby destroying its physical integrity. This results in the killing of the target cell. Without wishing to be bound by theory, it is believed that The cytotoxic effect on cells is due to cell lysis.

[0093] 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.

[0094] 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 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 typically found on NK cells. The cells do not express FCγRIII-A. The Fc-ε receptor (FcεR) binds to the Fc region of the IgE antibody.

[0095] 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, cells expressing CARs Cell targeting cells that express certain antigens on their cell surface. The scFv domain can be engineered to recognize any antigen, including tumor-specific antigens. For example, the CD19CAR recognizes CD19, a cell surface marker expressed by certain cancers.

[0096] 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.

[0097] 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 include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, nucleotide structure modification 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.

[0098] 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.

[0099] "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.

[0100] In the case of two or more nucleic acid sequences or polypeptide sequences, the term identical or percentage identity refers to two or more sequences or subsequences that are identical, or have a specified percentage of identical amino acid residues or nucleotides (i.e., when comparing and aligning maximum correspondence on a comparison window or a specified region, 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 region) as measured using the default parameters described below using BLAST or BLAST 2.0 sequence comparison algorithms, or by manual alignment and visual inspection measurement methods (see, e.g., NCBI website, etc.). Such sequences are then said 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 take into account spaces, 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.

[0101] 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.

[0102] 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 achieved, 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). Biology [Current Protocols in Molecular Biology] (Ausubel et al., ed., 1995 supplement)).

[0103] Preferred examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used to determine percent sequence identity for nucleic acids or proteins using the parameters described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, as is 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 that, when aligned with a word of the same length in a database sequence, either match or satisfy some positive-valued threshold score T. 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 until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score can be calculated using the parameters M (a 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 following occurs: the cumulative alignment score drops 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 score residue alignments; or the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The expected value (E) represents the number of different alignments, and its score is equal to or higher than the expected score that occurs by chance in a database search. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and a comparison of two chains by default. 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 an alignment (B) of 50, an expectation (E) of 10, M=5, and N=-4.

[0104] 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 constituting 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. The method of transformation includes, but is 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; magnetic perforation (magnetoporation); biolistic delivery; retroviral infection; lipofection; and direct microinjection of DNA into cells.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] As used herein, the term "exogenous" refers to a substance, such as a nucleic acid (e.g., a nucleic acid including regulatory sequences and / or genes) or a polypeptide, that is artificially introduced into a cell or organism and / or does not naturally occur in the cell in which it is located. In other words, a substance, such as a nucleic acid or polypeptide, originates from outside the cell or organism into which it is introduced. An exogenous nucleic acid can have the same nucleotide sequence as a nucleic acid naturally occurring in the cell. For example, Cells are engineered to include Optionally, an endogenous The heparanase sequence is operably linked to a gene having regulatory sequences that are not involved in nature. The heparanase sequence can be naturally present in the host cell, but according to the present disclosure, the introduced nucleic acid is exogenous. Exogenous nucleic acid can have a nucleotide sequence different from any nucleic acid naturally present in the cell. For example, the exogenous nucleic acid can be a heterologous nucleic acid, i.e., a nucleic acid from a different species or organism. Therefore, the exogenous nucleic acid can have a nucleic acid sequence that is identical to the nucleic acid naturally found in the source organism, but different from the cell into which the exogenous nucleic acid is introduced. As used herein, the term "endogenous" refers to a nucleic acid sequence that is natural to the cell. As used herein, the term "heterologous" refers to a nucleic acid sequence that is not natural to the cell, i.e., 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 the cell, but has a sequence 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 the cell, but has a sequence that is non-natural to the cell, such as a sequence from a different organism.

[0109] As described 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 minimum level of Fc receptor activity (e.g., a standard control cell). The skilled person will recognize that a standard control can be designed to evaluate many parameters (e.g., RNA levels, polypeptide levels, specific cell types, etc.).

[0110] The term "expression" refers to the production of a gene product (e.g., a protein). When referring to expression, the term "transient" means that the polynucleotide is not 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.

[0111] The term "cytokine" refers to a general class of biomolecules 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.

[0112] The term "cytokines that modulate the tumor microenvironment" refers to A molecule expressed by a cell and acting to increase the anti-tumor response. 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 cytokine inhibitors 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).

[0113] As used herein, the term "vector" refers to a non-chromosomal nucleic acid containing a complete replicon so that when placed in a permissive cell, for example, by a transformation process, the vector can be replicated. The 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). The 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.

[0114] 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 cleaved extracellularly and localized to the plasma membrane, Golgi apparatus, endosomes, endoplasmic reticulum, and other cell 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.

[0115] As used herein, the term "targeting" when referring to the targeting of a tumor means The ability of a cell to recognize and kill tumor cells (i.e., target cells). The term "targeted" in this context refers to, for example, The ability of a cell-expressed CAR to recognize and bind to a cell surface antigen expressed by the tumor.

[0116] 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.

[0117] 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

[0118] 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, The cells are engineered to express homing receptors known to direct lymphocytes to lymph nodes when expressed. The cells are engineered to express secreted cytokines that modulate the tumor microenvironment or inhibitors that block cytokines that modulate the tumor microenvironment.

[0119] 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.

[0120] In a proof-of-concept example, the four genes used to generate a modified gene expression profile in a therapeutic cell line were: CCR7, CD19 chimeric antigen receptor, a high affinity variant of CD16, and endoplasmic reticulum-bound IL-2. ER-bound IL-2 is expressed in addition to its ability to be incorporated into the endoplasmic reticulum upon stimulation. In addition to its role in the cytotoxic capacity of the cell line, it 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 element most likely 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 the cells). However, if all elements are successfully integrated into the genome, the 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 four elements-can be further verified by flow cytometric staining analysis of other components (see Examples).

[0121] The constructs described herein provide the advantages of reducing the development time of generating new therapeutic cell lines, as well as reducing the stress and adverse effects of multiple rounds of genome manipulation and subsequent selection on cells. In addition, by placing the selection agent (in this case ER-IL-2) at the end of the construct, it is expected that the cell will be difficult to silence any given component of the construct without causing IL-2 starvation due to the nature of RNA transcription and processing. Therefore, the stable cell line constructed in this way should maintain the expression of all components already contained in the vector as long as they continue to produce their own IL-2.

[0122] To demonstrate proof of concept, four specific targets were addressed by the 4 components that were part of this tetracistronic construct. IL-2 acted as a selector and was The CCR7 element (CC chemokine receptor type 7) is a chemokine receptor responsible for migrating immune cells expressing it in the direction of the chemokine gradient of the ligands CCL19 and CCL21, which are usually expressed in lymph nodes. In one embodiment, the CCR7 element considered herein may include a polynucleotide sequence with 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, the CCL21 considered herein may include a polynucleotide sequence with 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, the 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).

[0123] The CD19 CAR (chimeric antigen receptor) is a construct designed to increase the cytotoxicity of cells when they encounter the CD19 differentiation population (which is commonly expressed on normal and malignant B cells) on the surface of the cells they encounter, and has shown efficacy in targeted immunotherapy trials against B-cell lymphomas. The high affinity CD16 receptor allows the modified Cells 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 a CD16 receptor encounters a cell coated in one of these antibodies, it triggers ADCC (antibody-dependent cellular toxicity) and attempts to destroy the cell. In fact, this allows cells that have been armed in this way to be used in combination treatment regimens with monoclonal antibodies against cancer neoantigens, etc. Although each of these components has its own practicality, it is suggested that this particular combination will produce an effective therapy for B cell lymphomas that can migrate to common sites of tumor growth (lymph nodes), where the B cell antigen CD19 is recognized and CAR-mediated cytotoxicity is initiated, or it works with monoclonal antibodies (such as Rituximab) to avoid the possibility of antigen escape. It should be understood that this proof-of-concept example is non-limiting and can be modified using the methods described herein. cells to express other homing receptors and / or CARs targeting other target antigens in order to generate effective immunotherapeutic cell lines.

[0124] As described herein, modified Cells, after electroporation with a linearized gene construct containing a CCR7 expression cassette together with a removable selection cassette with a selection marker, have stable long-term expression of the CCR7 lymph node homing receptor driven by the elongation factor 1a (EF1a) promoter. 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 comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3 (EF1a promoter sequence).

[0125] 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 (IL-8 amino acid sequence), or SEQ ID NO: 57 (CXCL2 nucleotide sequence), or SEQ ID NO: 58 (CXCL2 nucleotide sequence), or SEQ ID NO: 59 (CXCL14 nucleotide sequence), or SEQ ID NO: 60 (CXCL14 amino acid sequence), or SEQ ID NO: 61 (CD62L nucleotide sequence), or SEQ ID NO: 62 (CD62L amino acid sequence), or SEQ ID NO: 63 (IL-8 nucleotide sequence), or SEQ ID NO: 64 (IL-8 amino acid sequence), or SEQ ID NO: 65 (CXCL14 nucleotide sequence), or SEQ ID NO: 66 (CXCL14 nucleotide sequence), or SEQ ID NO: NO:56 (CXCL1 amino acid sequence) has a polypeptide sequence or polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

[0126] Through the activation of NFAT transcription factors and their nuclear translocation, it was confirmed that Target engagement involving FceRIg or CD3ζ pathways (including ADCC or CAR-mediated target recognition) is sufficient to identify susceptible cell lines in cells. NFAT activation was induced in cells. This was demonstrated by insertion of a reporter gene cassette containing three terminator regions flanking the NFAT binding domain and a minimal promoter driving firefly luciferase. NFAT was activated through the CD3ζ pathway 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.

[0127] In one embodiment, the NFAT response element sequence (binding site for activating NFAT) 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] The cell line is a human IL-2-dependent NK cell line established from peripheral blood mononuclear cells (PBMC) of a 50-year-old male diagnosed with non-Hodgkin's lymphoma (Gong et al., Leukemia. 8:652-8 (1994)). 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 fraction of NK cells in peripheral blood that have immunomodulatory functions as cytokine producers. Unlike normal NK cells, Lack of expression of most killer cell inhibitor receptors (KIR) (Maki et al., J Hematother Stem Cell Res. [Journal of Hematopoietic Stem Cell Research] 10: 369-83 (2001)). Only KIR2DL4 was detected on the surface of NK-92, which is a KIR receptor expressed by all NK cells with activation function and inhibitory potential. 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 of cells is through the perforin / esterase pathway; Expresses high levels of perforin and granzyme B (Maki et al., J Hematother Stem Cell Res. 10:369-83 (2001)).

[0133] The 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 evaluation in severe combined immunodeficient (SCID) mice did not show any significant difference with the Treatment-related effects, such as acute toxicity or long-term carcinogenicity (Tam et al., J Hemaother. 8:281-90 (1999), Yan et al., Clin Cancer Res. 4:2859-68 (1998)). Administration of the 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. 8:281-90 (1999), Yan et al., Clin Cancer Res. 4:2859-68 (1998)). Phase I clinical trials have demonstrated safety. The invention is disclosed in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044. Characterization of cell lines, both of which are incorporated herein by reference in their entirety.

[0134] Optionally, the modified Cells can also express 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. The 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 typically found on NK cells. A representative amino acid sequence encoding CD16 is shown in SEQ ID NO: 12. A representative polynucleotide sequence encoding CD16 is shown in SEQ ID NO: 13. The complete sequence of CD16 can be found in the SwissProt database according to entry P08637.

[0135] 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 can affect 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.

[0136] Optionally, the modified The cell comprises a nucleic acid sequence that is 70%, 80%, 90% or 95% identical to SEQ ID NO: 13. Optionally, the modified The cell comprises 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 The cell comprises a polypeptide having 70%, 80%, 90% or 95% identity to SEQ ID NO: 12 (with valine at position 176 of the full-length polypeptide). Optionally, the modified The cell comprises a polypeptide that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12.

[0137] The cytotoxicity of cells depends on the presence of cytokines (e.g., interleukin 2 (IL-2)). The 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 endoplasmic reticulum confines autocrine growth stimulation to Targeting IL-2 to the endoplasmic reticulum restricts autocrine growth stimulation to Cells]" Exp Hematol. [Experimental Hematology] 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.

[0138] Optionally, the modified The cell comprises a nucleic acid sequence encoding IL-2 that is 70%, 80%, 90% or 95% identical to SEQ ID NO: 14. Optionally, the modified The cell comprises 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 The cell comprises an IL-2 polypeptide having 70%, 80%, 90% or 95% identity to SEQ ID NO: 15. Optionally, the modified The cells contain an IL-2 polypeptide that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15. The cells are advantageously capable of being maintained in the absence of IL-2 without secreting amounts of IL-2 that would cause clinical side effects.

[0139] In one aspect, the subject matter of the present invention includes modified Cells. Modified The cell preferably comprises 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, respectively, Fig.21 , Fig.30 and Fig.31 Tetracistronic vectors contemplated herein are described in. 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). 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).

[0140] 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.

[0141] 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.

[0142] In addition, the nucleic acid constructs of the subject matter of the present invention may also comprise a sequence encoding a 2A peptide, such as a T2A, P2A, E2A or F2A peptide, 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.

[0143] 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).

[0144] Chimeric Antigen Receptor

[0145] Optionally, the modified The cell is further engineered to express a chimeric antigen receptor (CAR) on the cell surface. Optionally, CAR is specific to a tumor-specific antigen. As a non-limiting example, 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 a polynucleotide sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 41.

[0146] Homing receptors

[0147] Provided herein are modified nucleic acids comprising a homing receptor cells. 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 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Optionally, the homing receptor is in a modified Optionally, the modified The cell further comprises a CAR. Optionally, the CAR is CD19. Optionally, the modified The cell further comprises an Fc receptor. Optionally, the Fc receptor is CD16. Optionally, the modified The 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.

[0148] Expression vector

[0149] Provided herein are expression vectors comprising a nucleic acid operably linked to a promoter. 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 a nucleic acid having SEQ ID NO: 1 or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1. Optionally, the nucleic acid is operably linked to a promoter. Optionally, the promoter is selected from the group consisting of SEQ ID NO: 3, 9, 10 or 11 or promoters having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3, 9, 10 or 11. Optionally, the nucleic acid is operably linked to a promoter. Optionally, the promoter comprises SEQ ID NO: 4 and / or SEQ ID NO: 5. Optionally, the promoter comprises SEQ ID NO: 6, or a nucleic acid that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6.

[0150] In some embodiments, an expression vector is provided that comprises SEQ ID NO: 1, or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1; SEQ ID NO: 25 (CD19CAR) or a nucleic acid having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 (CD16158V), 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 or polypeptide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: NO: 14 A nucleic acid with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, an expression vector is provided that comprises 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: NO: 14 has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 67. Optionally, the expression vector is a plasmid.

[0151] 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, as 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 the IL-12 viral transduction IL-12 secretion by cell lines.

[0152] Preparation of modified Cell Methods

[0153] Provided herein are methods for preparing modified nucleic acid molecules comprising the nucleic acid molecules described herein. These methods include transforming cells with expression vectors Cells, the expression vector comprising a nucleic acid described herein operably linked to a promoter.

[0154] 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 expression of the selected polynucleotide sequence in a 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 that control 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 1α1 (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.

[0155] 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 gives a significant growth or survival advantage or disadvantage to the cell or organism under certain limited culture conditions (selective conditions). As used herein, the phrase selection agent refers to the introduction of selection pressure to a cell or cell colony to favor or resist the reagent of 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. The example of a suitable selection marker for mammalian cells is dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin and puromycin.

[0156] 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 encompasses 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 nucleotide 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.

[0157] 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.

[0158] Treatment

[0159] 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.

[0160] 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 at a second location or locations (e.g., in the breast).

[0161] As used herein, "treating" a condition, disease or disorder and symptoms associated with the condition, disease or disorder, or "treatment" of a condition, disease or disorder and symptoms associated with the 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 remission. "Treatment" may also mean prolonging 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 a method for reducing one or more symptoms of a disease or condition characterized by protease expression or the impact of a symptom of a disease or condition characterized by protease expression. Therefore, in the disclosed method, treatment can refer to a determined disease, condition, or symptom of the disease or condition that is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in severity. For example, if one or more symptoms of a disease in a subject are alleviated by 10% compared to a control, it is considered that the method for treating a disease is treatment. Therefore, compared to 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.

[0162] The terms subject, patient, individual, etc. are not intended to be restrictive 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 restrictive. 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 humans and beast subjects.

[0163] As used herein, "administering" refers to providing, contacting and / or delivering a compound or compounds by any appropriate route to achieve the 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, The cells are administered parenterally. Optionally, The cells are administered intravenously. Optionally, Cells were administered peritumorally.

[0164] Thus, provided herein are methods of reducing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of a modified cells, thereby reducing cancer metastasis in a subject. Also provided are methods of treating cancer in a subject, comprising selecting a subject having cancer and administering to the subject a therapeutically effective amount of a modified The method further comprises administering to the subject an additional therapeutic agent.

[0165] In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of a modified Cells, wherein administration treats cancer in a subject or reduces tumor size in a subject. In some embodiments, these methods comprise administering to a subject a modified 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, these methods comprise administering to a subject a modified Cells comprising nucleic acids encoding i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor such as CD16 or CD16-158V, and / or iv) a cytokine such as erIL-2 or erIL-15.

[0166] 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-chelated 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.

[0167] Cells can be administered to a subject by a variety of routes. For example, cells can be administered by infusion (e.g., intravenous infusion) over a period of time. The cells are administered to a subject. Typically, for a single dose of Cells, the time period is 5 to 130 minutes. Optionally, the time period is between 90 and 120 minutes. Optionally, the time period is between 15 and 30 minutes.

[0168] The 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, a subject can be administered daily Cells once 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, once a day for a period of two days After this period, one or more hours, one day or several days, or one week or several weeks of no use Cell therapy. As used herein, the term "cycle" refers to a treatment that is repeated on a regular schedule with rest periods in between (e.g., no treatment or treatment with other agents). For example, one week of treatment followed by two weeks of rest is one treatment cycle. Such treatment cycles can be repeated one or more times. Thus, the treatment may 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. cell.

[0169] The absolute number of cells can be administered to the subject Cells, for example, can be administered to the subject from about 1000 cells / injection to up to about 10 billion cells / injection, 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) cells, or any range between any two of these numbers (including the endpoints). Optionally, 1×10 8 Up to 1×10 10The 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.

[0170] 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) cells, or any range (including endpoints) between any two of these numbers. Optionally, administer to the subject every m 2 Apply 1×10 3 Up to 1×10 10 indivual Optionally, the subject is administered 2 Apply 2×10 9 indivual cell.

[0171] Optionally, the relative number of cells may be administered to such individuals. Cells, for example, can be administered to the individual at about 1000 cells / kg of the individual 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) cells / kg individual, or any range between any two of these numbers (including the endpoints).

[0172] Optionally, the total dose can be calculated based on body surface area m 2 Calculations are performed, including every m 2 About 1×10 11 pcs, 1×10 10 pcs, 1×10 9 pcs, 1×10 8个 , 1×10 7 or any range (including endpoints) between any two of these numbers. Optionally, about 1 billion to about 3 billion of Optionally, each dose of The amount of cells can be calculated from the body surface area m2, including the number of cells 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.

[0173] Optionally, to include The composition of the cells and culture medium (such as human serum or its equivalent) is administered cells. 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 use in human therapeutics is used in place of human serum. Such serum substitutes may be known in the art. Optionally, a serum substitute comprising The composition is administered in the form of a cell and an isotonic liquid solution that supports cell viability Cells. Optionally, administered in the form of a composition reconstituted from a cryopreserved sample cell.

[0174] According to the methods provided herein, an effective amount of one or more medicaments 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 a medicament. The dosage range administered is those dosage ranges that are large enough to produce the desired effect, in which one or more symptoms of a 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 scheme, 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 "by a factor." 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 one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 22nd ed., Gennaro ed. (2012), and Pickar, Dosage Calculations (1999)).

[0175] Pharmaceutically acceptable compositions can include a variety of carriers and excipients. Various aqueous carriers can be used, for example, buffered saline, etc. These solutions are sterile and generally do not contain unwanted substances. Suitable carriers and their formulations 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 mean materials that are not biologically or otherwise unsuitable, that is, 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, the 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.

[0176] 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.

[0177] Combination therapy

[0178] Optionally, The cells are administered to a subject along with one or more other treatments for the cancer being treated. Without being bound by theory, it is believed that Treating subjects with cells and another cancer therapy will Cellular and replacement therapies provide the endogenous immune system with an opportunity to clear cancers that have heretofore overwhelmed such endogenous effects. Optionally, the two or more other treatments for the cancer being treated include, for example, antibodies, bispecific adaptors, radiation, chemotherapeutic agents, stem cell transplantation, or hormone therapy.

[0179] Optionally, the antibody is mixed with The cells are administered to a patient together. Optionally, Cells and antibodies are administered to a subject together, e.g., in the same formulation; administered to a subject separately, e.g., in separate formulations, at the same time; or may be administered separately, e.g., in different dosing schedules or at different times of the day. When administered separately, the antibody may be administered by any suitable route, such as intravenous or oral administration.

[0180] 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.

[0181] 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.

[0182] Optionally, the additional therapeutic agent is a chemotherapeutic agent. The chemotherapy treatment regimen may include administering a chemotherapeutic agent or a combination of chemotherapeutic agents to the subject. 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.

[0183] 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. Therapies, such as those disclosed herein, may 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. Therapies may be administered alone or in combination with any other treatment disclosed herein or known in the art. Additional treatments may be administered concurrently with the first therapy, at different times, or on a completely different treatment schedule (e.g., the first therapy may be administered once a day, while the additional treatment is administered once a week).

[0184] Reagent test kit

[0185] Provided herein are modified In some embodiments, the kit comprises a modified Cells comprising nucleic acids encoding the following substances operably linked to a promoter: i) a homing receptor, ii) an ABP or CAR that specifically binds to a target antigen, iii) an Fc receptor (such as CD16 or CD16-158V), and / or iv) a cytokine (such as erIL-2). Optionally, one or more proteins encoded by the nucleic acid sequence are expressed in the modified In some embodiments, the kit comprises a modified A cell comprising a nucleic acid encoding a CC chemokine receptor type 7 (CCR7), CXCR2 or CXCL14 receptor operably linked to a promoter. Optionally, the nucleic acid encoding CCR7 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. Optionally, the homing receptor is in a modified Optionally, the promoter comprises one or more NFAT binding elements and a minimal promoter. Optionally, the promoter is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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 Expressed on the cell surface of cells.

[0186] Optionally, the modified Optionally, the kit may contain other compounds, such as therapeutically active compounds or compounds to be used in administering the modified The instructions for use of the kit will optionally include instructions for using the kit components in cancer treatment. The instructions may further contain instructions on how to prepare the antibody (e.g., dilute or reconstitute in the case of freeze-dried protein) and The instructions may further include guidance on dosage and frequency of administration.

[0187] Disclosed are the following materials, compositions and components, 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 disclosing combinations, subsets, interactions, groupings, etc. of these materials, although 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 to be disclosed.

[0188] Publications cited herein and the materials for which they are cited are hereby incorporated by reference in their entirety.

[0189] 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.

[0190] Examples

[0191] Example 1. Modified NK cell line expressing the cytokine CCR7 that modulates the tumor microenvironment.

[0192] The NEON transfection system (ThermoFisher Scientific, Waltham, MA) was used to transfect the cells with the linearized pNKAT-CCR7-LP3 plasmid ( Figure 1 )right Cells were electroporated to prepare modified 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 cells contain the EF1α promoter, the CCR7 gene with a poly A tail, and a LoxP-flanked puromycin resistance gene driven by an ubiquitin promoter, all wrapped in homology arms targeting the AAVS1 locus (SEQ ID NO: 7).

[0193] To verify that the expression of CCR7 does not affect Cells, measured Expression of cell markers. Figure 3 shown. Figure 3 All data in are generated using Intellicyt iQue screener plus. Cells were incubated at 4°C for 30 minutes with APC-conjugated antibodies for the phenotypic markers or appropriate isotypes used as negative controls. The cells were then rinsed in PBS+1% BSA, precipitated, and resuspended in 30uL 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 percentages above the "positive" threshold and the "strong 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.

[0194] In order to determine the modified Cell cytotoxicity, the effector cells ( Cells and modified 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)) are seeded at 10,000 per well into all wells containing effector cells, as well as into control wells whose goal is simply to measure background death. The plate is then centrifuged briefly and incubated for 4 hours at 37°C and 5% CO2. The plate is then centrifuged, the supernatant is aspirated, and the cells are resuspended in PBS containing propidium iodide to measure cell death. The cells are then run through the Intellicyt iQuescreener plus, and the proportion of target cells that also stain positive for PI (distinguished from effectors by their staining) is measured. The percentage of dead cells is then compared to the number of naturally dying cells in the control wells, and the percentage of cells specifically killed by effector cells is 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.

[0195] In vitro testing consisted of using the Boyden chamber assay and a Matrigel layer to prevent 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 an 8uM pore. Cells (25,000 / well) were placed in low serum media (supplemented with 1% human serum and 500U / mL IL-2) in the upper chamber, and the same low serum media was used in the lower chamber alone or containing the target chemokine. In this case, CCL21 was used at 15ng / mL, CCL19 was used at 15ng / mL, and SDF-1a was used at 20ng / mL. For Cells or modified cells expressing CCR7 Cells were added and each test was performed in triplicate. The plate was then placed in an incubator overnight for an 18-hour invasion assay, after which the upper chamber was removed and 150 μL (total volume 750 μL) was sampled from the lower well after thorough mixing and read on a MacsQuant FACS analyzer. The live cells in the lower chamber were counted, and the cell number was then compared to the wells without chemokines, 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 still attached to the lower part of the ECM would not be represented in these numbers, which could result in differences between CCL19 and CCL21. The results are shown in Table 1. Figure 7 Specifically, Figure 7 The modified CCR7 expressing Statistically significant increase in cell invasiveness to CCL19 (a CCR7 chemokine). The lack of 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 CCL19 gradient migration. CCL21, while inducing migration, does not induce detachment from the matrix, requiring additional steps to demonstrate statistically significant invasive potential.

[0196] Example 2. Generation of NFAT-responsive constructs for controlled expression of CCL21.

[0197] To identify NFAT response elements, linearized constructs were electroporated into Cell lines stably expressing the NFAT-based luciferase expression cassette (NR2.2) were generated from The cells contain 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 will drive the production of firefly luciferase in the presence of activated NFAT. Then, Sup-B15 cells were also inoculated with an anti-CD19 CAR (an antigen present on Sup-B15 cells that is otherwise A subset of these cells were electroporated with mRNA that confer resistance to cell killing (ENR2.2). These cells are denoted as 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 presented as means with standard deviations calculated in Microsoft Excel. The results are shown in Figure 2. 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.

[0198] Example 3. Modified NK cell line expressing CCR7 and CCL21.

[0199] The pCRENFAT-CCL21 plasmid was incorporated into the CCR7-containing construct 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 CCR7 and CCL21 expressing cell.

[0200] To evaluate the expression of CCR7 and CCL21 in cells, unstained modified The cells were co-cultured with cells known to cause NFAT activation (K562 or other cell lines) in the lower well of the Boyden chamber and the stained modified The cells are placed in the upper chamber. If migration is demonstrated to be induced by co-culturing with sensitive cell lines, then the system is working.

[0201] Example 4: In vitro cytotoxicity assay using a modified NK cell line expressing CCR7

[0202] Fig. 9 It is shown that, as described in Example 1, the modified Cells remained cytotoxic to target cells after migration in a modified Boyden chamber transwell assay.

[0203] Example 5: CCR7, CD16 and CD19 CAR in cells transfected with nucleic acid constructs Cell surface expression in cells

[0204] Fig.12 It is shown that modified cells transfected with nucleic acid constructs encoding CCR7, CD16 and CD19 CAR The cells express high levels of the corresponding proteins on the cell surface.

[0205] Example 6: Biodistribution of CCR7-expressing chemokine-responsive NK cells in NSG mice bearing CCL19-positive subcutaneous K562 tumors

[0206] 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.

[0207] Experimental methods (Table 1):

[0208] a. Animals:

[0209] i. Animal type: NSG mice (JAX), female, 7-8 weeks old

[0210] 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)

[0211] b. Tumor Model:

[0212] i. Cell lines: K562, parental and CCL19-expressing subline, K-19

[0213] ii. Inoculation route: subcutaneous; parent K562 was inoculated in the left flank, K-19 was inoculated in the right flank

[0214] iii. Inoculum: 1E6 cells in 100 μL serum-free medium / Matrigel (v / v 1:1)

[0215] iv. Tumor burden at the start of treatment: K-19 average 107 mm 3 ; Parent K562 average 135mm 3

[0216] v. Random grouping: main Animals were randomized into groups based on the volume of K-19 tumors.

[0217] c. Test sample:

[0218] i.NK cells:

[0219] 1. CD19 t-haNK (non-CR) (NantKwest Torrey Pines)

[0220] 2. Tetracistronic Mi-aNK R7-19.1 (NantKwest Woburn)

[0221] ii. Fluorescent labeling:

[0222] 1. According to the manufacturer's manual, both types of NK cells were labeled with CFSE immediately prior to in vivo administration.

[0223] 2. For each time point, cultured CFSE-labeled cells were harvested to serve as a positive control for flow cytometry.

[0224] iii. Administration method: intravenous

[0225] iv. Dosage:

[0226] 1. 1E7 cells / mouse

[0227] v.Dosing frequency: Single dose

[0228] d. Tumor Collection:

[0229] i. Time point: 3, 24 and 48 hours after administration (± 2 hours)

[0230] ii. N = 4 mice / group / time point

[0231] iii. Tumor processing: Harvested tumors were dissociated into single-cell suspensions according to in-house protocols (supplemented) and flow cytometric enumeration of CFSE-positive NK cells was performed.

[0232] e. Formula and statistical analysis:

[0233] i. Tumor volume = length × width 2 / 2 (length and width are the longest and shortest diameters of the tumor, respectively)

[0234] 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.

[0235] Table 1. Experimental settings

[0236]

[0237] result:

[0238] a. Security:

[0239] 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).

[0240] iv. Within 24 hours after injection, 2 animals (total 14) in the R7-19.1 group were found dead, while there were no deaths in the CD19 t-haNK group.

[0241] After 24 hours, 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 hours (G0), but still looked rough-coated and breathed rapidly.

[0242] b.NK cell homing:

[0243] 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 .

[0244] 1. The four CCR7 receptor-CCL19 ligand combinations are:

[0245] a.CD19 K562 tumor: no receptor - no ligand [- -];

[0246] b.CD19 K-19 tumor: no receptor - ligand present [- +];

[0247] c. R7-19.1 cells, K562 tumor: receptor present - ligand absent [+ -]; and

[0248] d. R7-19.1 cells, K-19 tumors: receptor present - ligand present [+ +]

[0249] 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 vs. positive tumors in the same animal was not possible.

[0250] 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 Animals with NK cells showed similar levels of NK infiltration into both tumors, regardless of their CCL19 expression ( Fig.14 ).

[0251] 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.

[0252] 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 indicated.

[0253]

[0254] *, N=2.

[0255] This example demonstrates that the coexistence of the CCR7 receptor and the CCL19 ligand results in more effective NK cell infiltration 3 hours after administration. At 24 hours after administration, 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 effectively in 3 of 4 animals compared to parental controls that did not express the ligand. Regardless of receptor or ligand expression, the number of tumor-infiltrating NK cells decreased at 48 hours. In particular, there was a significant trend of R7-19.1 cells homing more to CCL19 tumors in the early hours (<48 hours after administration), suggesting that if the cells are used in a therapeutic setting, exposure is higher and cytotoxicity may also be stronger.

[0256] Example 7: Comparative Efficacy Evaluation of R7-19.1 Cells Expressing CCR7 in NSG Mice Bearing Intravenous CCL19-Positive RAJI Tumors

[0257] CCR7 is a chemokine receptor that induces cell migration toward gradients of chemokines CCL19 and CCL21, which are normally 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 t-haNK cells expressing CD19-CAR, CCR7, CD16.158V, and ERIL-2 based on the t-haNK platform. Cells (i.e., R7-19.1 cells). In addition to CCR7 expression, these cells also have chimeric antigen receptors (CARs) targeting cancer for CD19 cancer antigens, 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.

[0258] 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, Raji human Burkitt lymphoma cells engineered to express CCL19, in NSG mice. CD19 t-haNK cells ( [anti-CD19-CAR, CD16.158V, ERIL-2]) was used as a control NK cell line. A vehicle control group was also included.

[0259] 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.

[0260] Research rationale and purpose: In vivo distribution data showed that IV-administered R7-19.1 cells exhibited increased homing to SC tumors expressing CCL19. In this study, the anti-tumor effects of repeated IV administration of R7-19.1 cells were evaluated in an IV xenograft model of Raji-19.5 (a subline of Raji expressing CCL19) in NSG mice. Note that the original study protocol contained other animal groups (AC groups) not included in this report. They are not related to the efficacy determination of R7-19.1 cells in this tumor model (see Table 3 for a simplified experimental design).

[0261] Research Materials:

[0262] 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. Regarding CD19t-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.

[0263] 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 tmIWjl / 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.

[0264] Raji-19.5 tumor model (cancer cell line)

[0265] Cell Culture Medium: Raji-19.5 cancer cells were grown in ATCC modified RPMI-1640 medium supplemented with 10% fetal bovine serum.

[0266] Cell harvesting: Raji-19.5 cells (passage 16) in the exponential phase were collected by centrifugation according to Nantes West's SOP_Suspension Cancer Cell Collection for In Vivo Studies. The cells were then washed and plated 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 of animals. Cells used in in vivo studies had a viability of 97%.

[0267] Inoculation: 30 mice were inoculated via the lateral tail vein with 1×10 5 This was defined as day 0.

[0268] Experimental Procedure

[0269] 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 according to IACUC institutional policy and necropsy was performed at the discretion of the study director.

[0270] 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 director.

[0271] Random grouping: 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.

[0272] 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 in serum-free growth medium at a concentration of 1 × 10 cells / mL per mouse in a 200 μL injection volume. 7 The dose of 10 cells was administered IV. All cell handling and formulation procedures were performed at room temperature. Cell viability was above 80% for all dose formulations. 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.

[0273] 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.

[0274] Table 3: Study design (abbreviated)

[0275]

[0276] BIW: twice weekly; IACUC: Institutional Animal Care and Use Committee; IV: intravenous.

[0277] Data Analysis:

[0278] 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.

[0279] Survival curves: Survival curves were analyzed by log-rank (Mantel-Cox) test.

[0280] Statistical analysis: All statistical analyses were performed using GraphPad Prism version 8. P < 0.05 was considered statistically significant.

[0281] result

[0282] Efficacy: The primary readout for efficacy was animal survival. Mortality events were counted when animals were euthanized due to morbidity, paralysis, or weight loss >20%. No animal mortality was observed. Fig.15 As shown in Table 4, treatment with R7-19.1 and control CD19 t-haNK cells was able to significantly prolong the survival of Raji-19.5 IV tumor-bearing animals compared to vehicle controls (by log-rank test, for R7-19.1, P < 0.0001; for CD19 t-haNK, P = 0.0002), with median survival increased by 6.5 days and 2.5 days, respectively. This corresponds to an increase of 30% and 12% relative to vehicle controls, respectively. More importantly, the expression of CCR7 chemokine responsive receptors in R7-19.1 cells increased the median survival by another 4 days (increased by 17%) compared to CD19 t-haNK cells (P < 0.0001).

[0283] 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.

[0284]

[0285] NA: Not applicable.

[0286] Security

[0287] like Fig.16 As shown, animals treated with NK cells consistently demonstrated 5%-10% weight loss after each treatment administration. In most cases, animals were able to recover from cell injections and exhibited weight restoration, causing oscillations in the weight change curve between doses. However, the initial dose appeared to cause the most severe reaction and was associated with the longest recovery time for clinical signs 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 restoration of body weight suggests that the weight loss is temporary and reversible.

[0288] Notably, at the end of the study, the animals showed a dramatic loss of body weight ( Fig.16 ), which may be due to disease progression). Autopsies 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 a weight loss of >20% on the day after the sixth dose. No visible tumor nodules were identified during autopsy in this animal. Therefore, the exact reason for weight loss that warranted euthanasia could not be determined.

[0289] in conclusion

[0290] In the IV Raji-19.5 xenograft model, 1×10 7 IV 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.

[0291] 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.

[0292] 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.

[0293] Example 8: Comparative Efficacy Evaluation of R7-19.1 Cells Expressing CCR7 in NSG Mice Bearing Subcutaneous CCL19-Positive RAJI Tumors

[0294] CCR7 is a chemokine receptor that induces cell migration toward gradients of chemokines CCL19 and CCL21, which are normally 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 t-haNK cells expressing aCD19-CAR, CCR7, CD16.158V, and ERIL-2 based on the t-haNK platform. Cells (i.e., R7-19.1 cells). In addition to CCR7 expression, these cells also have chimeric antigen receptors (CARs) targeting cancer for CD19 cancer antigens, 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.

[0295] 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, Raji human Burkitt lymphoma cells engineered to express CCL19, in NSG mice. CD19 t-haNK cells ( [anti-CD19-CAR, CD16.158V, ERIL-2]) was used as a control NK cell line. A vehicle control group was also included.

[0296] 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.

[0297] 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.

[0298] Research Materials

[0299] 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.

[0300] R7-19.1 cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68.

[0301] CD19 t-haNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 0.05% Pluronic F68.

[0302] Experimental system: Thirty NOD.Cg-Prkdc mice were used in the study. They were 10-11 weeks old at the beginning of the study (after quarantine and acclimatization) and weighed 19-28 g on the day of tumor implantation. scid Iq tmIWjl / SzJ (NSG) female mice. The supplier was the Jackson Laboratory (610 Main Street, Bar Harbor, ME 04609, USA). Animals were identified using ear tags, cage numbers, and tail numbers.

[0303] Raji-19.5 tumor model (cancer cell line)

[0304] Cell culture medium: Raji-19.5 cancer cells were cultured in ATCC modified RPMI-1640 medium supplemented with 10% fetal bovine serum.

[0305] Cell harvesting: Raji-19.5 cells (passage 16) in the exponential phase were harvested by centrifugation according to Nantes West's SOP_Suspension Cancer Cell Collection 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 reach 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%.

[0306] Inoculation: 30 animals were inoculated unilaterally in the right flank with 2.5 × 10 5 The skin was shaved before the injection.

[0307] Experimental Procedure

[0308] 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 tumors with a volume of more than 2000mm 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.

[0309] 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) body weight were euthanized with subsequent necropsy in accordance with IACUC institutional policy.

[0310] Clinical observations: Animals were observed daily for mortality / morbidity (G0 to G4; see Table 5). Moribund or paralyzed animals were euthanized and subsequently autopsied.

[0311] Randomization: When the average tumor volume reaches 190 mm 3 At 14:00 pm, 30 mice were pseudo-randomly divided 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 (<200mm 3 ) Tumors (see Fig.17 ).

[0312] 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 in serum-free growth medium at a concentration of 1 × 10 cells / mL per mouse in a 200 μL injection volume. 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 5, while Group A received vehicle control, Groups B and C received CD19 t-haNK and R7-19.1 cells, respectively.

[0313] 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.

[0314] Table 5: Study design (abbreviated)

[0315]

[0316] BIW: twice weekly; IACUC: Institutional Animal Care and Use Committee; IV: intravenous; TGI: tumor growth inhibition.

[0317] 1 , IACUC-defined tumor burden endpoint: tumor volume greater than 2000mm 3 ; ulcerated tumors; and tumors that interfere with normal gait.

[0318] Data analysis

[0319] Tumor volume calculation: Tumor volume = length × width 2 / 2 (length and width are the longest and shortest diameters of the tumor, respectively)

[0320] Tumor growth inhibition (TGI) calculation: 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 practice point, and ΔT C is the change in mean tumor volume in the control group.

[0321] Statistical analysis of tumor growth and body weight curves: Tumor growth and body weight curves were analyzed by two-way ANOVA (or mixed effects analysis when there were missing values; see revision 2 of Appendix 1) followed by Tukey's test for multiple comparisons. All statistical analyses were performed using GraphPad Prism version 8. P < 0.05 was considered statistically significant.

[0322] result

[0323] 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. In addition, small and large tumors may respond to NK cell treatment in different ways. For these reasons, the two subpopulations (i.e., large and small tumors) were analyzed separately.

[0324] 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 tumor volume exceeding 2000 mm at day 9. 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, on 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).

[0325] 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) In these already small cohorts, there were some "outliers" in tumor growth, further precluding the possibility of statistical analysis.

[0326] like Fig. 20 As shown, animals treated with NK cells typically exhibited a 5%-10% weight loss after each treatment administration. In most cases, animals were able to recover from the cell injection and exhibited weight restoration, causing oscillations in the weight change curve between doses. However, the initial dose appeared to cause the most acute and severe reactions 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 restoration of body weight indicates that the weight loss is temporary and reversible.

[0327] in conclusion

[0328] 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.

[0329] Example 9: Production a TGFβ / PD-L1 CAR modified cell

[0330] 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. cells, 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 The cells co-expressed high levels of PD-L1 CAR and CD16.

[0331] Limiting dilution cloning

[0332] The polyclonal aTGFβ / PD-L1 t-haNK TM Aliquots of the library culture were diluted to a density of 3 cells / ml in growth medium without IL-2 addition. 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 containers for further expansion and characterization.

[0333] Bioanalytical methods

[0334] Cell culture:

[0335] Polyclonal and clonal aTGFβ / PD-L1 t-haNK TM Cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and without IL-2.

[0336] aNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and 500 IU / ml recombinant human IL-2.

[0337] HaNK cells were cultured in growth medium supplemented with 5% heat-inactivated human AB serum and without IL-2.

[0338] 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.

[0339] 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.

[0340] Antibody staining for flow cytometry analysis:

[0341] 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 and then incubated with streptavidin-APC antibodies. Samples were analyzed on a MACSQuant flow cytometer.

[0342] Cytotoxicity:

[0343] The cell lines grown in suspension were resuspended by pipetting the cell culture up and down. Cell viability was determined by automatic counting (trypan blue exclusion). Target cells were labeled with CFSE dye and diluted to the desired cell concentration in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotics / antimycotics. Effector cells and target cells were mixed in 96-well plates with different effectors and targets (E: T was 20: 1, 10: 1, 5: 1, 2.5: 1, 1.25: 1, 0.62: 1, 0.31: 1 and 0.15: 1) ratios and incubated in a 5% CO2 atmosphere and 37°C incubator for 4 h. PI was then added to fluorescently label dead cells and the assay was analyzed on a MACSquant flow cytometry device.

[0344] ADCC:

[0345] 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 the 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 pre-incubated with monoclonal antibodies trastuzumab, rituximab or without antibodies for 30 min at room temperature. Antibody-labeled target cells (and no antibody controls) 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 in an incubator at 5% CO2 atmosphere and 37°C for 4 h. PI was then added to fluorescently label dead cells and the assay was analyzed on a MACSquant flow cytometry device.

[0346] Quantification of TGFβ trap secreted by aTGFβ / PD-L1t-haNK cells

[0347] The sample supernatant for analysis was 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. The sample supernatant was frozen at -80 °C until analysis. The cell pellet from the 500 x g centrifugation step was resuspended, pooled in triplicate and the 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 Details between cells).

[0348] aTGFβ / PD-L1 t-haNK TM Cell cytotoxicity against target cell lines

[0349] aTGFβ / PD-L1 t-haNK TM The cytotoxicity of cells was investigated 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 could kill K562 cells (target cells). TM The cells maintained cytotoxicity equivalent to that of parental aNK cells.

[0350] Fig.25 Display aTGFβ / PD-L1 t-haNK TM Cells display aNK TM Enhanced specific killing of the resistant, PD-L1-positive SUP-B15 cell line - at an effector to target ratio of 8, approximately 70% of cells were killed by aTGFβ / PD-L1 t-haNK cells compared to only approximately 10% of cells killed by aNK cells TM Cell killing.

[0351] Fig.26 Display aTGFβ / PD-L1 t-haNK TM The cells demonstrated 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 TM Cell killing.

[0352] Fig. 27 Display aTGFβ / PD-L1 t-haNK TM SUP-B15 cells were treated with anti-CD20 rituximab monoclonal antibody or anti-Her2-neu trastuzumab monoclonal antibody in combination CD19KO / CD20+ In a 4-h cytotoxicity assay, aTGFβ / PD-L1t-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, and aTGFβ / PD-L1 t-haNK TM None of the clones could kill the target SUP-B15 CD19KO / CD20+ cell.

[0353] Example 10: Modified Secreted by cells TGFβ Well Suppression TGFβ activity

[0354] HEK293 cells engineered to express a TGFβ-responsive element (SMAD-binding promoter) of a luciferase reporter gene showed a dose-dependent increase in luciferase activity when treated with TGFβ ( Fig.28 ).

[0355] 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.

[0356] Example 11: Modified Cells produce IL-12

[0357] NK-92 cells were transduced with lentiviral constructs encoding functional IL-12 p70 dimers as single-chain polypeptides in either the p35-p40 or p40-p35 orientations. 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.

[0358] IL-12 / PD-L1 CAR modified Cell production

[0359] 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. The modified NK-92 cells were selected by IL-2-deficient medium cells, 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 The cells were able to secrete large amounts of scIL-12 p70 cytokine.

[0360] 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.

[0361] 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 to the scope of the appended claims. In addition, when interpreting this specification and claims, all terms should be interpreted in a manner as broad as possible 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. A modified NK-92 cell comprising a tetracistronic nucleic acid encoding the following four functional elements: (i) a TGFβ trap operably linked to a promoter, wherein the amino acid sequence of the TGFβ trap is shown in SEQ ID NO: 66; (ii) an antigen binding protein operably linked to a promoter, wherein the antigen binding protein comprises a chimeric antigen receptor (CAR), and wherein the CAR specifically binds to programmed cell death 1 ligand 1 (PD-L1), and the amino acid sequence of the PD-L1 CAR is as shown in SEQ ID NO: 69; (iii) an Fc receptor operably linked to a promoter; and (iv) a cytokine operably linked to a promoter.

2. The modified NK-92 cell of claim 1, wherein the cytokine is IL-2, erIL-2, IL-15, erIL-15 or a combination thereof.

3. The modified NK-92 cell of claim 1, wherein the cytokine is erIL-2, and the nucleic acid encoding erIL-2 is SEQ ID NO:

14.

4. The modified NK-92 cell of claim 1, wherein the cytokine is erIL-15, and the nucleic acid encoding erIL-15 is SEQ ID NO:

67. 5 . The modified NK-92 cell of claim 1 , wherein the Fc receptor is CD16 or high affinity CD16 (SEQ ID NO: 12), or a nucleic acid having the sequence of SEQ ID NO:

13.

6. The modified NK-92 cell of any one of claims 1-3, wherein the TGFβ trap, antigen binding protein, CAR and / or Fc receptor are expressed on the cell surface of the modified NK-92 cell.

7. A method for producing the modified NK92 cell line as described in claim 1, comprising targeted homologous recombination at a specific locus, wherein the locus is AAVS1, and wherein the amino acid sequence of the AAVS1 locus is shown in SEQ ID NO:7.

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