Modified NK-92 cells for treating cancer

Abstract

Provided are genetically modified NK-92 cells engineered to express multiple transgenes for improved therapies and methods of treating cancer cells. The present invention provides NK-92 cells that have been modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) so that the NK-92 cells present the at least one Fc receptor and the at least one CAR on their cell surface. The present invention also provides a method for treating a patient having or suspected of having a disease treatable with NK-92 cells, such as cancer, comprising administering an NK-92 cell line to the patient.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 173,701, filed June 10, 2015, and U.S. Provisional Patent Application No. 62 / 337,044, filed May 16, 2016, each of which is incorporated herein by reference in whole. [Background technology]

[0002] background Natural killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells generally make up about 10–15% of circulating lymphocytes and bind to and kill target cells, including virus-infected cells and many malignant cells, nonspecifically with respect to antigens and without prior immune sensitization. (Herberman et al., Science 214:24 (1981) (Non-patent Literature 1)). Target cell death occurs by inducing cytolysis. NK cells used for this purpose are isolated from the peripheral blood lymphocyte ("PBL") fraction of the target's blood, augmented in cell culture to obtain a sufficient number of cells, and then reinjected into the target. NK cells have been shown to be somewhat effective in both ex vivo and in vivo treatments. However, such therapies are complicated by the fact that not all NK cells are cytolytic and the therapy is specific to the treated patient.

[0003] In cancer, phenotypic changes that distinguish tumor cells from normal cells of the same tissue often involve one or more changes in the expression of specific gene products, including the loss of normal cell surface components or the acquisition of other components (i.e., antigens not detected in the corresponding normal non-cancerous tissue). Antigens that are expressed in neoplastic or tumor cells but not in normal cells, or are expressed in neoplastic cells at levels substantially exceeding those seen in normal cells, are referred to as "tumor-specific antigens" or "tumor-associated antigens." Such tumor-specific antigens may serve as markers of the tumor phenotype. Tumor-specific antigens can be assigned to three main groups: cancer / testis-specific antigens (e.g., MAGE, BAGE, GAGE, PRAME, and NY-ESO-1), melanocyte differentiation antigens (e.g., tyrosinase, Melan-A / MART, gp100, TRP-1, and TRP-2), and mutated or aberrantly expressed antigens (e.g., MUM-1, CDK4, β-catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V).

[0004] Tumor-specific antigens are used as targets for cancer immunotherapy. One such therapy uses chimeric antigen receptors (CARs) expressed on the surface of immune cells, including T cells and NK cells, to improve cytotoxicity against cancer cells. A CAR contains a single-chain variable fragment (scFv) linked to at least one intracellular signaling domain. The scFv recognizes and binds to an antigen on the target cell (e.g., a cancer cell), triggering effector cell activation.

[0005] In addition, cancer treatment with monoclonal antibodies (mAbs) has significantly improved the clinical outcomes of patients with cancer, particularly when combined with chemotherapy. However, despite antigen presentation by malignant cells and the presence of immune cells, cancer cells are known to evade immune-mediated rejection. One mechanism by which cancer cells evade immune elimination is by interfering with detection. For example, tumor evasion mechanisms include defective or reduced antigen presentation (e.g., mutation or downregulation of tumor antigens) that reduces the effectiveness of targeted therapies such as CAR-expressing immune cells and mAbs alone. Thus, improved treatments and methods for treating cancer cells are still needed.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

[0007] Brief Summary Genetically modified NK-92 cells or cell lines engineered to express multiple transgenes are provided herein. For example, NK-92 cells are modified to simultaneously express at least one Fc receptor and at least one chimeric antigen receptor (CAR) such that at least one Fc receptor and at least one CAR are presented on the cell surface of the NK-92 cells.

[0008] Thus, the present disclosure provides an NK-92 cell line in which the cells of the NK-92 cell line are modified to express at least one Fc receptor and at least one CAR such that the Fc receptor and the chimeric antigen receptor (CAR) are presented on the cell surface of the NK-92 cells.

[0009] [Invention 1001] NK-92 cells modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) so as to present the at least one Fc receptor and at least one chimeric antigen receptor (CAR) on the cell surface of the NK-92 cells. [Invention 1002] The cell of the present invention 1001, wherein the Fc receptor is FcγRIII-A(CD16) or a CD16 polypeptide having valine at position 158 of the mature form of said CD16. [Invention 1003] The cell of the present invention 1001, wherein the Fc receptor comprises a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, and contains valine at position 158. [Invention 1004] The cell of the present invention 1001, wherein the Fc receptor contains the amino acid sequence of SEQ ID NO:2. [Invention 1005] A cell according to any of the present invention 1001 to 1004, wherein the CAR has at least 90% identity with SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. [Invention 1006] The CAR targets a tumor-associated antigen selected from the group consisting of CD19, CSPG-4, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY-ESO1, and CD33, according to any of the cells described in items 1001 to 1004 of the present invention. [Invention 1007] A cell according to any of the present invention 1001 to 1006, which further expresses cytokines. [Invention 1008] The cell of the present invention 1007, wherein the cytokine is interleukin-2 or a variant thereof. [Invention 1009] A cell according to the present invention 1007 or 1008, wherein the cytokine targets the endoplasmic reticulum. [Invention 1010] A cell according to any of the inventions 1001 to 1009, wherein the Fc receptor and the CAR are encoded on different vectors. [Invention 1011] An NK-92 cell line in which cells of an NK-92 cell line are modified to express at least one Fc receptor and at least one CAR so as to present the Fc receptor and a chimeric antigen receptor (CAR) on the cell surface of the NK-92 cells. [Invention 1012] The NK-92 cell line of the present invention 1011, wherein the Fc receptor is FcγRIII-A(CD16) or a CD16 polypeptide having valine at position 158 of the mature form of said CD16. [Invention 1013] The method of the present invention 1011, wherein the Fc receptor comprises a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, and comprises valine at position 158. [Invention 1014] The method of the present invention 1011, wherein the Fc receptor contains the amino acid sequence of SEQ ID NO:2. [Invention 1015] An NK-92 cell line according to any of the present invention 1011 to 1014, wherein the CAR has at least 90% identity with SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13. [Invention 1016] An NK-92 cell line according to any of the invention 1011 to 1014, wherein the CAR targets a tumor-associated antigen selected from the group consisting of CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY-ESO1, and CD33. [Invention 1017] An NK-92 cell line according to any of the invention 1011-1016, which further expresses cytokines. [Invention 1018] The NK-92 cell line of the present invention 1017, wherein the cytokine is interleukin-2 or a variant thereof. [Invention 1019] An NK-92 cell line according to invention 1017 or 1018, wherein the cytokine targets the endoplasmic reticulum. [Invention 1020] A cell according to any of the invention 1011 to 1019, wherein the Fc receptor and the CAR are encoded on different vectors. [Invention 1021] A cell line according to any of the present invention 1011 to 1020, wherein the cells of the cell line undergo less than 10 collective doublings. [Invention 1022] A cell line according to any of the present invention 1011 to 1020, wherein the cells are cultured in a medium containing less than 10 U / ml of IL-2. [Invention 1023] A composition comprising a certain amount of cells from any of Invention 1001-1010 or Invention 1011-1022. [Invention 1024] The composition of the present invention 1023, further comprising at least one monoclonal antibody. [Invention 1025] The composition of the present invention 1024, wherein the at least one monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody. [Invention 1026] The composition of the present invention 1024, wherein the monoclonal antibody is selected from the group consisting of alemtuzumab, rituxumab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adtrastuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab. [Invention 1027] A method for treating cancer in a patient in need, comprising the step of administering to the patient an effective amount of any one of the cells of the present invention 1001 to 1010 or any one of the cell lines of the present invention 1011 to 1022, thereby treating the cancer. [Invention 1028] The method of the present invention 1027, wherein the cells are administered to the patient by a route selected from the group consisting of intravenous, intraperitoneal, and subcutaneous. [Invention 1029] The method of the present invention 1027, further comprising the step of administering an effective amount of at least one monoclonal antibody to the patient. [Invention 1030] The method of the present invention 1029, wherein the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody. [Invention 1031] The method of the present invention 1029, wherein the monoclonal antibody is selected from the group consisting of alemtuzumab, rituxumab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adtrastuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab. [Invention 1032] A method according to any of items 1029 to 1031 of the present invention, wherein the monoclonal antibody and the cells are administered simultaneously. [Invention 1033] Any method 1029 to 1031 of the present invention, wherein the monoclonal antibody and the cells are mixed before being administered to the patient. [Invention 1034] A method according to any one of items 1029 to 1031 of the present invention, wherein the monoclonal antibody and the cells are administered sequentially. [Invention 1035] The method according to any one of items 1027 to 1034 of the present invention, wherein the cancer is selected from the group consisting of leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenström hypergammaglobulinemia, heavy chain disease, sarcoma, and carcinoma. [Invention 1036] The patient's body surface area 1 m² 2 Approximately 1 x 10 8 From each piece, approximately 1 x 10 11 A method according to any of items 1027 to 1035 of the present invention, wherein individual cells are administered to the patient. [Invention 1037] A kit for treating cancer comprising (a) NK-92 cells modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) on the cell surface, and (b) instructions for use. [Invention 1038] (c) A kit of the present invention 1037 further comprising at least one monoclonal antibody. [Invention 1039] The kit of the present invention 1038, wherein the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody. [Invention 1040] The kit of the present invention 1038, wherein the monoclonal antibody is selected from the group consisting of alemtuzumab, rituxumab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adtrastuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab. [Invention 1041] Modified NK-92 cells comprising a multidentate ligand-binding element selected from the group consisting of one or more Fc receptors and one or more CARs, such that the modified NK-92 cells have at least two ligand-binding elements on their cell surface. [Invention 1042] A method for enhancing the binding affinity of NK-92 cells to cancer cells, comprising the step of using polydentate coordination elements on the cell surface of modified NK-92 cells, wherein the modified NK-92 cells include polydentate coordination elements for one or more Fc receptors and one or more CARs such that they have at least two ligand-binding elements on the cell surface of the NK-92 cells. The general descriptions above and the detailed descriptions below are illustrative and illustrative, and are intended to provide further information about this disclosure. Other purposes, advantages, and novel features will be readily apparent to those skilled in the art. [Brief explanation of the drawing]

[0010] The purpose, features, and benefits will be more readily understood by referring to the following disclosures in conjunction with the attached drawings. [Figure 1A] This graph shows the in vitro cytotoxicity assay. Figure 1A shows the death of the target cell line by unelectroporated parental NK-92 cells. [Figure 1B] This graph shows the in vitro cytotoxicity assay. Figure 1B shows the death of target cell lines by parental NK-92 cells expressing CD19-CAR. [Figure 1C] This graph shows the in vitro cytotoxicity assay. Figure 1C shows the death of a target cell line by CD16(158V)-ERIL2 NK-92 cells expressing CD19-CAR. [Modes for carrying out the invention]

[0011] Detailed explanation NK-92 cells are provided herein that have been modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) so as to present at least one Fc receptor and at least one chimeric antigen receptor (CAR) on the cell surface of the NK-92 cells. Optionally, the Fc receptor may include FcγRIII-A (CD16). Optionally, NK-92 cells are genetically modified to express an Fc receptor encoding a polypeptide having at least 90% sequence identity with SEQ ID NO:1 (FcγRIII-A or CD16 (F-158) with phenylalanine at position 158); or at least 90% identity with SEQ ID NO:2 (CD16 (F158V) with valine at position 158, a more affinity form). In a typical embodiment, the CD16 polypeptide has valine at position 158. Optionally, NK-92 cells are genetically modified to express a CAR encoding a polypeptide having at least 90% sequence identity with SEQ ID NO:8(CD19), SEQ ID NO:9(CD19), SEQ ID NO:10(CD33), SEQ ID NO:11(CD33), SEQ ID NO:12(CSPG-4), or SEQ ID NO:13(CSPG-4). Optionally, the CAR targets tumor-associated antigens, such as CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY-ESO1, and CD33. In some embodiments, the NK-92 cell line undergoes fewer than 10 population doublings.

[0012] Optionally, NK-92 cells further express cytokines, such as interleukin-2 or its variants. In some embodiments, NK-92 cells are modified to express polypeptides having the sequence SEQ ID NO:6 or SEQ ID NO:7. In further embodiments, the cytokines target the endoplasmic reticulum. Optionally, NK-92 cells of the cell line are cultured in a medium containing less than 10 U / ml of IL-2.

[0013] This disclosure provides compositions comprising any of the NK-92 cells described herein. Optionally, this disclosure provides compositions comprising any of the above embodiments of NK-92 cells and at least one antibody, such as alemtuzumab, rituxumab, trastuzumab, ibritumomab, gemtuzumab, brentuximab, adtrastuzumab, blinatumomab, daratumumab, or elotuzumab. In some embodiments, the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody.

[0014] This disclosure provides a method for treating cancer in a patient in need, comprising the step of administering an effective amount of cells of any of the above embodiments to the patient. In some embodiments, the cells are administered to the patient by a route selected from the group consisting of intravenous, intraperitoneal, and subcutaneous. In some embodiments, the patient's body surface area is 1 m² 2 Approximately 1 x 10 8 From each piece, approximately 1 x 10 11Individual cells are administered to the patient. Optionally, the method of this disclosure provides a step of administering to the patient an effective dose of at least one monoclonal antibody, e.g., alemtuzumab, rituxumab, trastuzumab, ibritumomab, gemtuzumab, brentuximab, adtrastuzumab, blinatumomab, daratumumab, or elotuzumab. In some embodiments, the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody. In some embodiments, the monoclonal antibody is administered to the patient by a route selected from the group consisting of intravenous, intraperitoneal, and subcutaneous. In one embodiment, the monoclonal antibody and cells are administered simultaneously. In some embodiments, the monoclonal antibody and cells are mixed together before administration to the patient. In other embodiments, the monoclonal antibody and cells are administered sequentially. In another embodiment, subjects are administered a monoclonal antibody, and subsequently, within, for example, 24 hours after administration of the monoclonal antibody, or within 24 to 72 hours, they are administered modified NK-92 cells.

[0015] In one embodiment, cancer is, for example, leukemia (e.g., chronic B-cell leukemia, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL)), lymphoma (e.g., non-Hodgkin lymphoma (NHL)), polycythemia vera, multiple myeloma, Waldenström hypergammaglobulinemia, heavy chain disease, sarcoma, or carcinoma.

[0016] This disclosure provides a kit for use in any of the above methods for treating cancer, wherein the kit comprises (a) a certain amount of NK-92 cells modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) on the cell surface, and (b) at least one instruction manual describing at least one of the methods of this disclosure. In some embodiments, the kit further comprises at least one monoclonal antibody.

[0017] After reading this specification, it will become clear to those skilled in the art how to carry out various other embodiments and uses. However, not all embodiments are described herein. It will be understood that the embodiments shown herein are for illustrative purposes only and not to limit. Accordingly, the detailed descriptions of these various other embodiments should not be construed as limiting the scope and breadth of the disclosure set forth herein. It should be understood that the aspects described below are not limited to specific compositions, methods for preparing such compositions, or uses thereof, and are of course subject to change.

[0018] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0019] In this specification and the appended claims, several terms are used as to be defined as having the following meanings:

[0020] The terms used herein are intended solely to describe and not to limit to specific aspects. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context explicitly indicates otherwise. Therefore, for example, a reference to “natural killer cells” includes multiple natural killer cells.

[0021] All numerical notations, including those involving ranges, such as pH, temperature, time, concentration, quantity, and molecular weight, are approximations that change (+) or (-) in increments of 0.1 or 1.0 as needed. It should be understood that, although not always explicitly stated, all numerical notations may be preceded by the term "approximately."

[0022] As will be understood by those skilled in the art, for any and all purposes, and especially with respect to providing a detailed description of the invention, all scopes described herein also encompass any and all possible sub-scopes and combinations thereof. Any described scope can be readily recognized as sufficient to describe and enable the same scope which is divided at least equally into two, three, four, five, ten, etc. As a non-limiting example, each scope described herein can be readily divided into the bottom third, the middle third, the top third, etc. As will also be understood by those skilled in the art, all words such as “at most,” “at least,” “greater than,” and “less than” refer to scopes which include the number described and can subsequently be divided into sub-scopes as described above. Finally, as will be understood by those skilled in the art, a scope includes each individual element. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0023] Although not always explicitly stated, it should be understood that the reagents described herein are merely examples, and that equivalents of such reagents may be known in the art.

[0024] "Optional" or "optional" means that the event or situation described thereafter may or may not occur, and that such description includes both cases in which the event or situation occurs and cases in which it does not occur.

[0025] The term “contains” is intended to mean that the compositions and methods described include the elements, but do not exclude other elements. “Essentially consisting of” when used to define compositions and methods shall mean excluding any other elements that are essentially important to the combination. For example, a composition essentially consisting of elements as defined herein does not exclude other elements that do not substantially affect the basic and novel features of the claims. “Consists of” shall mean excluding other components and substantial steps of the method in amounts greater than trace amounts. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0026] As used herein, “simultaneous” or “at the same time” means administering at least two active agents (e.g., NK-92-Fc-CAR cells and a monoclonal antibody) at the same time or approximately at the same time.

[0027] As used herein, the term “effective dose” refers to the amount of a composition sufficient to achieve the desired therapeutic effect, for example, the amount that results in remission of cancer cells or one or more signs associated with cancer. In the context of therapeutic applications, the amount of NK-92 cells or antibodies administered to a subject depends on the type and progression of cancer, as well as individual characteristics such as overall health, age, sex, weight, and drug tolerance. It also depends on the stage, severity, and type of the disease. Those skilled in the art can determine the appropriate dosage in accordance with these and other factors. NK-92 cells may also be administered in combination with one or more additional therapeutic compounds (e.g., antibodies).

[0028] As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed into mRNA, and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. If the polynucleotide originates from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. The level of gene expression can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the level of gene expression from a sample can be directly compared to the level of that gene expression from a control or reference sample. In another aspect, the level of gene expression from a sample can be directly compared to the level of that gene expression from the same sample after administration of NK-92 cells.

[0029] As used herein, “immunotherapy” refers to the use of modified or unmodified NK-92 cells, naturally occurring or modified NK cells or T cells, whether alone or in combination, which can induce cytotoxicity upon contact with target cells.

[0030] As used herein, “natural killer (NK) cells” are immune system cells that kill target cells in the absence of specific antigen stimulation and without restriction by major histocompatibility complex (MHC) class. Target cells may be cancer or tumor cells. NK cells are characterized by the presence of the CD56 surface marker and the absence of the CD3 surface marker.

[0031] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), distinct from NK-92 cell lines. Endogenous NK cells are generally a heterogeneous population of cells in which NK cells are enriched. Endogenous NK cells can be used for autologous therapy or allogeneic therapy.

[0032] "NK-92 cells" refers to the immortal NK cell line, NK-92, originally obtained from patients with non-Hodgkin lymphoma. The term "NK-92" is intended to refer to the original NK-92 cell line as well as NK-92 cell lines that have been modified (e.g., by the introduction of exogenous genes). NK-92 cells and their exemplary and non-exclusive modifications are described in U.S. Patents 7,618,817; 8,034,332; and 8,313,943, all of which are incorporated herein by reference in their entirety.

[0033] "Modified NK-92 cells" refers to NK-92 cells further comprising a vector encoding a transgene, which includes the Fc receptor, CAR, IL-2, and / or suicide genes. In a preferred embodiment, modified NK-92 cells express at least one transgene protein.

[0034] As used herein, "unirradiated NK-92 cells" refers to NK-92 cells that have not been irradiated. Irradiation prevents the cells from growing and proliferating. To maintain optimal activity, the time between irradiation and injection should not exceed 4 hours; therefore, it is assumed that NK-92 cells are irradiated at the treatment facility or elsewhere before the patient's treatment. Alternatively, NK-92 cells may be inactivated by other mechanisms.

[0035] As used herein, “inactivation” of NK-92 cells renders them unable to grow. Inactivation may also be associated with the death of NK-92 cells. It is assumed that NK-92 cells may be inactivated after they have effectively purged ex vivo samples of cells associated with pathology in therapeutic applications, or after they have been in the mammalian body for a sufficient time to effectively kill many or all target cells present in the body. Inactivation can be induced, in non-limiting examples, by administering an inactivating agent to which NK-92 cells are sensitive.

[0036] As used herein, the terms “cytotoxic” and “cytolytic” are intended to be synonymous when used to describe the activity of effector cells such as NK cells. Generally, cytotoxic activity relates to the killing of target cells by any of the following biological, biochemical, or biophysical mechanisms. More specifically, cytolytic activity refers to the activity of the effector in which the plasma membrane of the target cell is dissolved, thereby destroying its physical integrity. This results in the death of the target cell. While we do not wish to be bound by theory, the cytotoxic effect of NK cells is considered to be due to cytolysis.

[0037] The term “to kill” in relation to cells / cell populations is intended to include any type of operation that leads to the death of those cells / cell populations.

[0038] The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the defense function of immune cells by binding to a portion of an antibody known as the Fc region. Binding of an antibody's Fc region to a cell's Fc receptor (FcR) stimulates phagocytic or cytotoxic activity in the cell via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fc-γ receptors (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. NK-92 cells do not express FcγRIII-A. A representative polynucleotide sequence encoding the native form of CD16 is shown in SEQ ID NO:5.

[0039] The term “chimeric antigen receptor” (CAR), as used herein, refers to an extracellular antigen-binding domain that fuses with an intracellular signaling domain. CARs can be expressed on T cells or NK cells and can enhance cytotoxicity. Generally, the extracellular antigen-binding domain is an scFv specific to the antigen found on the cell of interest. CAR-expressing NK-92 cells target cells that express a particular antigen on their cell surface, based on the specificity of the scFv domain. The scFv domain can be manipulated to recognize any antigen, including tumor-specific antigens.

[0040] As used herein, the term "tumor-specific antigen" refers to an antigen that is present on cancer or neoplastic cells but is undetectable on normal cells originating from the same tissue or lineage as the cancer cells. As used herein, tumor-specific antigens also refer to tumor-associated antigens, i.e., antigens that are expressed at higher levels on cancer cells compared to normal cells originating from the same tissue or lineage as the cancer cells.

[0041] The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably and refer to polymeric forms of any length of nucleotides, either deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. Where present, modifications to the nucleotide structure may be conjugated before or after the assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or requested, polynucleotides encompass both a double-stranded form and two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0042] Polynucleotides consist of specific sequences of the following four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) is used instead of thymine. Therefore, the term "polynucleotide sequence" is the alphabetical representation of the polynucleotide molecule.

[0043] As used herein, “identity percentage” refers to sequence identity between two peptides or between two nucleic acid molecules. The identity percentage can be determined by comparing the positions in each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. As used herein, the terms “homologous” or “variant” nucleotide sequence, or “homologous” or “variant” amino acid sequence, refer to sequences characterized by at least a specified percentage of identity at the nucleotide or amino acid level. Homologous nucleotide sequences include sequences encoding naturally occurring allele variants and mutants of nucleotide sequences described herein. Homologous nucleotide sequences include nucleotide sequences encoding proteins of non-human mammalian species. Homologous amino acid sequences include amino acid sequences containing conserved amino acid substitutions and whose polypeptides have the same binding and / or activity. In some embodiments, homologous nucleotides or amino acid sequences have at least 60% or more, e.g., at least 70%, at least 80%, at least 85% or more, compared to the comparison sequence. In some embodiments, homologous nucleotides or amino acid sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a comparison sequence. In some embodiments, homologous amino acid sequences have 15 or fewer, 10 or fewer, 5 or fewer, or 3 or fewer conserved amino acid substitutions. The identity percentage can be determined, for example, using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.) with default settings, using the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489).

[0044] The term "expression" refers to the production of a gene product. When referring to expression, the term "transient" means that the polynucleotide is not yet integrated into the cell's genome.

[0045] The term "cytokine" refers to a general class of biological molecules that act on 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.

[0046] As used herein, the term “vector” refers to a non-chromosomal nucleic acid containing an intact replicon that can replicate when placed in a suitable cell, for example, through a transformation process. A vector may be able to replicate in one cell type, such as bacteria, but may have limited ability to replicate in another cell type, such as mammalian cells. A vector may be viral or nonviral. Exemplary nonviral vectors for delivering nucleic acids include: naked DNA; DNA complexed with cationic lipids, either alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles containing DNA condensed with cationic polymers such as heterologous polylysines, oligopeptides of a certain length, and polyethyleneimine, sometimes contained within liposomes; and the use of ternary complexes containing viruses and polylysine-DNA.

[0047] As used herein, the term “targeting” is intended to include, but is not limited to, directing a protein or polypeptide to a suitable intracellular or extracellular destination. Targeting is typically achieved via a signal peptide or targeting peptide, which is a sequence of amino acid residues in a polypeptide chain. Such signal peptides can be located anywhere within the polypeptide sequence, but are often located at the N-terminus. Polypeptides can also be manipulated to have a signal peptide at their C-terminus. Signal peptides can direct polypeptides to extracellular regions, placement on the plasma membrane, Golgi, endosomes, endoplasmic reticulum, or other intracellular compartments. For example, a polypeptide having a specific amino acid sequence (e.g., KDEL) at its C-terminus may be retained in or returned to the ER lumen.

[0048] The term "suicide gene" refers to a gene that enables negative selection of cells. Suicide genes are used as a safety system, allowing cells expressing the gene to be killed by the introduction of a selective agent. This is desirable when recombinant genes induce mutations that lead to uncontrolled cell proliferation. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene (see, for example, Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9). In one embodiment, the suicide gene is inducible caspase 9 (iCas9) (Di Stasi, (2011) “Inducible apoptosis as a safety switch for adoptive cell therapy.” N Engl J Med 365: 1673-1683. See also Morgan, “Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic” Molecular Therapy (2012); 20: 11-13). The TK gene can be wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir.

[0049] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal, or its cells, whether in vitro or in situ, that is suitable for the methods described herein. In preferred embodiments, the patient, subject, or individual is a mammal. In certain preferred embodiments, the patient, subject, or individual is a human.

[0050] The term “to treat” or “treatment” extends to the treatment of a disease or disorder described herein in a subject such as a human, and includes (i) suppressing the disease or disorder, i.e., halting its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) suppressing, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. The term “to administer” or “administer” monoclonal antibodies or natural killer cells to a subject includes any route for introducing or delivering the antibodies or cells to perform the intended function. Administration may be carried out by any route suitable for the delivery of the cells or monoclonal antibodies. Thus, the route of delivery may include intravenous, intramuscular, intraperitoneal, or subcutaneous delivery. In some embodiments, monoclonal antibodies and / or NK-92 cells are administered directly to a tumor, for example, by injection into the tumor. Administration includes self-administration and administration by another person.

[0051] As used herein, effective dose or effective amount means the dose of an active substance or a composition containing said active substance that produces the desired effect (e.g., to treat or prevent a disease). The exact dose and formulation of nanoparticles depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington (2005); and Pickar, Dosage Calculations (9th edition) (1999)). For example, with respect to a given parameter, a therapeutically effective dose shows an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic effectiveness can also be expressed as an increase or decrease of "--times". For example, a therapeutically effective dose may have an effect at least 1.2 times, 1.5 times, 2 times, 5 times, or more than that of a standard control. A therapeutically effective dose or therapeutically effective dose may relieve one or more symptoms of the disease. A therapeutically effective dose or therapeutically effective dose may prevent or delay the onset of the disease or one or more symptoms of the disease if the effect it is administered to treats a person at risk of developing the disease.

[0052] As used herein, the term “antibody” refers to an immunoglobulin or a fragment thereof. An antibody may be of any type (e.g., IgG, IgA, IgM, IgE, or IgD). Preferably, the antibody is IgG. An antibody may be non-human (e.g., derived from a mouse, goat, or any other animal), fully human, humanized, or chimeric. An antibody may be polyclonal or monoclonal. Optionally, the antibody is monoclonal.

[0053] As used herein, the term “monoclonal antibody” refers to a pure, target-specific antibody produced from a single clone of a cell that has the ability to grow and proliferate indefinitely in culture. Possible monoclonal antibodies include naked antibodies that attach to and block antigens on cancer cells. In one embodiment, a naked monoclonal antibody is alemtuzumab, which binds to the CD52 antigen in lymphocytes. Possible monoclonal antibodies also include conjugated monoclonal antibodies, such as tagged, labeled, or loaded antibodies. Specifically, antibodies may be tagged or loaded with drugs or toxins, or radiolabeled. Examples of such antibodies include, but are not limited to, ibritumomab targeting the CD20 antigen, brentuximab targeting the CD30 antigen, and trastuzumab targeting the HER2 protein. Other possible monoclonal antibodies are bispecific monoclonal antibodies, such as blinatumomab, which targets CD19 on lymphoma cells and CD3 on T cells.

[0054] As used herein, the term “antibody fragment” refers to any portion of an antibody that recognizes an epitope. Antibody fragments may be glycosylated. In non-limiting examples, antibody fragments may include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, rIgG fragments, functional antibody fragments, and the aforementioned single-chain recombinant forms. F(ab')2, Fab, Fab', and Fv are antigen-binding fragments that can be prepared from the variable regions of IgG and IgM, respectively. They differ in size, valence, and Fc content. Fragments may be prepared by any method, including the expression of components (e.g., heavy chain and light chain portions) by one cell or cell line, or by multiple cells or cell lines. Preferably, antibody fragments contain a portion of the Fc region that has sufficient ability to recognize an epitope and bind to an Fc receptor.

[0055] As used herein, the term “cancer” refers to all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, carcinomas, and sarcomas. Exemplary cancers include cancers of the brain, breast, cervix, colon, head and neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovarian, sarcoma, stomach, uterus, and medulloblastoma. Further examples include Hodgkin’s disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical cancer, endocrine and exocrine neoplasms of the pancreas, and prostate cancer.

[0056] Titles or subtitles may be used in this specification for the convenience of the reader and are not intended to affect the scope of this disclosure. In addition, some terms used herein are defined more specifically below.

[0057] NK-92 cells The NK-92 cell line is a unique cell line discovered to proliferate in the presence of interleukin-2 (IL-2). Gong et al., Leukemia 8:652-658 (1994). These cells exhibit high cytolytic activity against various cancers. The NK-92 cell line is a homogeneous population of cancerous NK cells with broad antitumor cytotoxicity, obtainable in predictable yields after growth. Its safety profile has been confirmed in Phase I clinical trials. NK-92 was discovered in the blood of subjects with non-Hodgkin lymphoma and subsequently immortalized ex vivo. Although NK-92 cells are derived from NK cells, they lack the main inhibitory receptors exhibited by normal NK cells and retain most of the activating receptors. However, NK-92 cells do not attack normal cells and do not induce unacceptable immune rejection in humans. The characterization of the NK-92 cell line is described in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044.

[0058] The NK-92 cell line is CD56 bright It has been found to exhibit the CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers. Furthermore, it does not exhibit the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. Growth of NK-92 cells in culture is dependent on the presence of recombinant interleukin-2 (rIL-2), and even low doses of around 1 IU / mL are sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term growth, nor do other cytokines tested, including IL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ. NK-92 exhibits high cytotoxicity even at a low effector:target (E:T) ratio of 1:1. Gong, et al., op. cit. NK-92 cells are deposited in the American Type Culture Collection (ATCC) under the name CRL-2407.

[0059] Previous studies on endogenous NK cells have shown that IL-2 (1000 IU / mL) is important for NK cell activation during transport, but that it is not necessary to maintain these cells at 37°C and 5% carbon dioxide. Koepsell, et al., Transfusion 53:398-403 (2013).

[0060] Modified NK-92 cells are known and not limited to those described in, for example, U.S. Patent Nos. 7,618,817, 8,034,332, and 8,313,943, U.S. Patent Application Publication No. 2013 / 0040386, all of which are incorporated herein by reference in their entirety, including, for example, wild-type NK-92, NK-92-CD16, NK-92-CD16-γ, NK-92-CD16-ζ, NK-92-CD16(F157V), NK-92mi, and NK-92ci.

[0061] NK-92 cells possess almost all of the activating receptors and cytolytic pathways associated with NK cells, but they do not express CD16 on their cell surface. CD16 is an Fc receptor that recognizes and binds to the Fc portion of antibodies, activating NK cells in response to antibody-dependent cell-mediated cytotoxicity (ADCC). Due to the absence of the CD16 receptor, NK-92 cells cannot lyse target cells via the ADCC mechanism and therefore cannot enhance the antitumor effects of endogenous or exogenous antibodies (i.e., rituxumab and herceptin).

[0062] Studies on endogenous NK cells have shown that IL-2 (1000 IU / mL) is important for NK cell activation during transport, but that these cells do not need to be maintained at 37°C and 5% carbon dioxide. Koepsell, et al., Transfusion 53:398-403 (2013). However, endogenous NK cells differ significantly from NK-92 cells, mainly due to the difference in their cell origin: NK-92 is a cancer-derived cell line, while endogenous NK cells are collected from donors (or patients) and processed for infusion into patients. Endogenous NK cell preparations are heterogeneous cell populations, while NK-92 cells are homogeneous clonal cell lines. NK-92 cells readily proliferate in culture while maintaining cytotoxicity, whereas endogenous NK cells do not. In addition, the heterogeneous endogenous population of NK cells does not aggregate at high density. Furthermore, endogenous NK cells express Fc receptors, including the CD-16 receptor, which is not expressed by NK-92 cells.

[0063] Fc receptor Fc receptors bind to the Fc portion of antibodies. Several Fc receptors are known, and they differ in their preferred ligand, affinity, expression, and effects after binding to antibodies.

[0064] (Table 1) Exemplary Fc receptors TIFF0007836780000001.tif255160TIFF0007836780000002.tif88160

[0065] In some embodiments, NK-92 cells are modified to express the Fc receptor protein on their cell surface.

[0066] In some embodiments, the Fc receptor is CD16. For the purposes of this disclosure, specific amino acid residues of CD16 are specified relative to SEQ ID NO:2, or relative to SEQ ID NO:1, which differs from SEQ ID NO:2 by one position. Thus, the amino acid residue at position 158 of the CD16 polypeptide is the amino acid residue corresponding to position 158 of SEQ ID NO:2 (or SEQ ID NO:1) when the CD16 polypeptide is maximally aligned with SEQ ID NO:2. In some embodiments, NK-92 cells are modified to express human CD16, e.g., SEQ ID NO:1, which has phenylalanine at position 158 of the mature form of this protein. In a typical embodiment, NK-92 cells are modified to express a high-affinity form of human CD16, e.g., SEQ ID NO:2, which has valine at position 158 of the mature form of this protein. Position 158 of the mature protein corresponds to position 176 of the CD16 sequence containing the native signal peptide. In some embodiments, the CD16 polypeptide is encoded by a polynucleotide encoding a precursor polypeptide sequence of SEQ ID NO:3 or SEQ ID NO:4 (i.e., having a native signal peptide).

[0067] In some embodiments, the polynucleotide encoding the CD16 polypeptide has at least about 70% polynucleotide sequence identity with a full-length, naturally occurring CD16-encoding polynucleotide sequence containing a signal peptide, having phenylalanine at position 176 of full-length CD16 (corresponding to position 158 of the mature CD16 protein). In some embodiments, the polynucleotide encoding the CD16 polypeptide has at least about 70% polynucleotide sequence identity with a full-length, naturally occurring CD16-encoding polynucleotide sequence containing a signal peptide, having valine at position 176 (corresponding to position 158 of the mature protein). In some embodiments, the CD16-encoding polynucleotide has at least 70% identity with SEQ ID NO:5 and contains a valine-encoding codon at the position of the polynucleotide encoding position 176 of the full-length CD16 polypeptide containing the signal peptide. In some embodiments, the CD16-encoding polynucleotide has at least 90% identity with SEQ ID NO:5 and contains a valine-encoding codon at position 176 of full-length CD16. In some embodiments, the polynucleotide encoding CD16 contains SEQ ID NO:5 but has a codon encoding valine at position 176 of the full-length CD16.

[0068] In some embodiments, the CD16 polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity with SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 70% or at least 80% identity with SEQ ID NO:2 and contains valine at position 158, determined based on SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 90% identity with SEQ ID NO:2 and contains valine at position 158, determined based on SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 95% identity with SEQ ID NO:2 and contains valine at position 2, determined based on SEQ ID NO:2. In some embodiments, the polynucleotide encodes SEQ ID NO:2. In some embodiments, the CD16 polynucleotide encodes a chimeric receptor comprising the extracellular domain of CD16 with or without a signal sequence, any other fragment of full-length CD16, or at least a partial sequence of CD16 fused to the amino acid sequence of another protein. In other embodiments, epitope tag peptides, such as FLAG, myc, polyhistidine, or V5, are attached to the amino-terminal domain of a mature polypeptide and can be used to aid in cell surface detection by using anti-epitope tag peptide monoclonal or polyclonal antibodies.

[0069] In some embodiments, CD16 variants having more than 700 to 800 polynucleotides are within the scope of this disclosure, although homologous CD16 polynucleotides may be about 150 to about 700, about 750, or about 800 polynucleotides in length.

[0070] Homologous polynucleotide sequences include those encoding polypeptide sequences that encode variants of CD16. Homologous polynucleotide sequences also include naturally occurring allelic variants associated with SEQ ID NO:5. Transfection of NK-92 cells with any polynucleotide encoding an amino acid sequence shown in either SEQ ID NO:1 or SEQ ID NO:2, its naturally occurring variant, or a polypeptide having a sequence that is at least 70% identical or at least 80%, 90%, or 95% identical to SEQ ID NO:1 or SEQ ID NO:2 is within the scope of this disclosure. In some embodiments, homologous polynucleotide sequences encode conserved amino acid substitutions in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, NK-92 cells are transfected with a degenerate homologous CD16 polynucleotide sequence that encodes the same polypeptide, but is different from the naturally occurring polynucleotide sequence.

[0071] In other examples, cDNA sequences with polymorphisms that alter the CD16 amino acid sequence are used to modify NK-92 cells, for example, by creating allelic variants between individuals exhibiting genetic polymorphisms in the CD16 gene. In yet another example, CD16 genes from other species with polynucleotide sequences different from the sequence of SEQ ID NO:5 are used to modify NK-92 cells.

[0072] In some cases, variant polypeptides are produced using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985), or other known techniques can be applied to cloned DNA to generate CD16 variants (Ausubel, 2002; Sambrook and Russell, 2001).

[0073] In some embodiments, the polynucleotide encoding CD16 is mutated to alter the amino acid sequence encoding CD16 without changing the function of CD16. For example, polynucleotide substitutions resulting in amino acid substitutions at "non-essential" amino acid residues can be made in SEQ ID NO:1 or SEQ ID NO:2.

[0074] Conservative substitutions in SEQ ID NO:1 or SEQ ID NO:2, in which an amino acid of one class is substituted for another amino acid of the same class, fall within the range of disclosed CD16 variants, provided that the substitution does not substantially alter the activity of the polypeptide. Conservative substitutions are well known to those skilled in the art. Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, e.g., β-sheet or α-helix conformation, (2) charge, (3) hydrophobicity, or (4) the bulkiness of the side chain at the target site, may alter the function or immunological uniqueness of the CD16 polypeptide. Non-conservative substitutions involve exchanging one member of one of these classes for another. Substitutions may be introduced at a conservative substitution site, more preferably at a non-conservative site.

[0075] In some embodiments, the CD16 polypeptide variant is at least 200 amino acids long and has at least 70% amino acid sequence identity, or at least 80%, or at least 90% identity, to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the CD16 polypeptide variant is at least 225 amino acids long and has at least 70% amino acid sequence identity, or at least 80%, or at least 90% identity, to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the CD16 polypeptide variant has a valine at position 158, determined based on SEQ ID NO:2.

[0076] In some embodiments, nucleic acids encoding a CD16 polypeptide can encode a CD16 fusion protein. A CD16 fusion polypeptide contains any portion or the entirety of CD16 fused with a non-CD16 polypeptide. Fusion polypeptides are readily constructed using recombinant methods. For example, a polynucleotide encoding a CD16 polypeptide, such as SEQ ID NO:1 or SEQ ID NO:2, is fused in-frame with a non-CD16 encoding polynucleotide (e.g., a polynucleotide sequence encoding a signal peptide of a heterologous protein). In some embodiments, fusion polypeptides can be constructed in which the heterologous polypeptide sequence is fused to the C-terminus of CD16 or positioned within CD16. Typically, up to approximately 30% of the CD16 cytoplasmic domain can be replaced. Such modifications can enhance expression or increase cytotoxicity (e.g., ADCC responsiveness). In other examples, chimeric proteins, including but not limited to Ig-α, Ig-B, CD3-e, CD3-d, DAP-12, and DAP-10, have domains derived from other lymphocyte-activating receptors that replace a portion of the CD16 cytoplasmic domain.

[0077] Fusion genes can be synthesized using conventional techniques, which include automated DNA synthesizers and PCR amplification using anchor primers that create a complementary overhang between two consecutive gene fragments, which can then be annealed and re-amplified to generate a chimeric gene sequence (Ausubel, 2002). Many vectors are commercially available that facilitate the in-frame subcloning of CD16 into the fusion region.

[0078] Chimeric antigen receptor Where described herein, NK-92 cells are further manipulated to express a chimeric antigen receptor (CAR) on their cell surface. Optionally, the CAR is specific to a tumor-specific antigen. Tumor-specific antigens are described, as non-limiting examples, in US 2013 / 0189268;WO 1999024566 A1;US 7098008; and WO 2000020460 A1, each of which is incorporated herein by reference in whole. Tumor-specific antigens include, but are not limited to, NKG2D, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, CD19, and CD33. Further non-limited tumor-associated antigens and their associated malignancies can be found in Table 1.

[0079] (Table 1) Tumor-specific antigens and associated malignancies TIFF0007836780000003.tif121164TIFF0007836780000004.tif238164

[0080] In some embodiments, the CAR targets CD19, CD33, or CSPG-4. Representative polynucleotide and polypeptide sequences of CD19, CD33, and CSPG-4 CARs are provided in SEQ ID NO:8 (CD19 CAR polynucleotide), SEQ ID NO:9 (CD19 CAR polypeptide), SEQ ID NO:10 (CD33 CAR polynucleotide), SEQ ID NO:11 (CD33 CAR polypeptide), SEQ ID NO:12 (CSPG-4 CAR polynucleotide), and SEQ ID NO:13 (CSPG-4 CAR polypeptide). In some embodiments, the CD19 CAR polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity with respect to SEQ ID NO:9. Optionally, the CD19 CAR polypeptide has at least 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with respect to SEQ ID NO:9. In some embodiments, the CD33 CAR polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity with SEQ ID NO:11. Optionally, the CD33 CAR polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:11. In some embodiments, the CSPG-4 CAR polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity with SEQ ID NO:13. Optionally, the CSPG-4 CAR polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:13. In some embodiments, epitope tag peptides, such as FLAG, myc, polyhistidine, or V5, are attached to the amino-terminal domain of a polypeptide and can be used to aid in cell surface detection by using anti-epitope tag peptide monoclonal or polyclonal antibodies.

[0081] In some cases, variant polypeptides are produced using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985), or other known techniques can be applied to cloned DNA to generate CD16 variants (Ausubel, 2002; Sambrook and Russell, 2001).

[0082] In some embodiments, the polynucleotide encoding the CAR is mutated to alter the amino acid sequence encoding the CAR without changing the function of the CAR. For example, polynucleotide substitutions resulting in amino acid substitutions at "non-essential" amino acid residues can be made at SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13.

[0083] Conservative substitutions in SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13, in which an amino acid of one class is substituted for another amino acid of the same class, fall within the scope of the disclosed variants, provided that the substitution does not substantially alter the activity of the polypeptide. Conservative substitutions are well known to those skilled in the art. Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, e.g., β-sheet or α-helix conformation, (2) charge, (3) hydrophobicity, or (4) the bulkiness of the side chain at the target site, may alter the function or immunological uniqueness of the polypeptide. Non-conservative substitutions involve exchanging one member of one of these classes for another. Substitutions may be introduced at a conservative substitution site, more preferably at a non-conservative site.

[0084] Optionally, CARs target antigens associated with specific cancer types. Optionally, cancers include leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström hypergammaglobulinemia, heavy chain disease, and, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovoma, The group is comprised of solid tumors, including sarcomas and carcinomas such as mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminocarcinoma, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0085] CARs can be operated as described, for example, in patent publications WO 2014039523; US 20140242701; US ​​20140274909; US 20130280285; and WO 2014099671, each of which is incorporated herein by reference in whole. Optionally, a CAR is a CD19 CAR, a CD33 CAR, or a CSPG-4 CAR.

[0086] Further modifications - Cytokines The cytotoxicity of NK-92 cells depends on the presence of cytokines (e.g., interleukin-2 (IL-2)). Adding IL-2 externally to maintain and increase NK-92 cells in commercial-scale cultures is considerably costly. Administering IL-2 to human subjects in amounts sufficient to sustain NK92 cell activation may cause adverse side effects.

[0087] In some embodiments, FcR-expressing NK-92 cells are further modified to express at least one cytokine and a suicide gene. In certain embodiments, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. In preferred embodiments, the cytokine is IL-2 (SEQ ID NO:6). In certain embodiments, IL-2 is an endoplasmic reticulum-targeting variant, and the suicide gene is iCas9.

[0088] In one embodiment, IL-2 is expressed with a signaling sequence that directs IL-2 to the endoplasmic reticulum. In several embodiments, the polynucleotide encoding IL-2 encodes a polypeptide having the sequence SEQ ID NO:7. Although not bound by theory, directing IL-2 to the endoplasmic reticulum allows for IL-2 expression at levels sufficient for autocrine activation without releasing IL-2 extracellularly. See Konstantinidis et al “Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells” Exp Hematol. 2005 Feb;33(2):159-64. Sequential activation of FcR-expressing NK-92 cells can be prevented, for example, by the presence of a suicide gene.

[0089] Further modification - suicide gene The term "suicide gene" refers to a gene that enables negative selection of cells. Suicide genes are used as a safety system, allowing cells expressing the gene to be killed by the introduction of a selective agent. This is desirable when recombinant genes induce mutations that lead to uncontrolled cell proliferation. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the Escherichia coli Deo gene (see, for example, Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9). As used herein, suicide genes are active in NK-92 cells. Typically, suicide genes encode proteins that do not have adverse effects on cells but kill them in the presence of specific compounds. Therefore, suicide genes are typically part of a system.

[0090] In one embodiment, the suicide gene is a thymidine kinase (TK) gene. The TK gene may be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir.

[0091] In another embodiment, the suicide gene is cytosine deaminase, which is toxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez et al. “Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation.” Blood 1998 Jul 15;92(2):672-82.

[0092] In another embodiment, the suicide gene is a cytochrome P450 that is toxic in the presence of ifosfamide or cyclophosphamide. See, for example, Touati et al. “A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response.” Curr Gene Ther. 2014;14(3):236-46.

[0093] In another embodiment, the suicide gene is iCas9. See Di Stasi, (2011) “Inducible apoptosis as a safety switch for adoptive cell therapy.” N Engl J Med 365: 1673-1683. See also Morgen, “Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic” Molecular Therapy (2012); 20: 11-13. The iCas9 protein induces apoptosis in the presence of the small molecule AP1903. AP1903 is a biologically inactive small molecule that has been shown to be well-tolerated in clinical trials and is used in adoptive cell therapy.

[0094] In one embodiment, modified NK-92 cells are irradiated prior to administration to a patient. Irradiation of NK-92 cells is described, for example, in U.S. Patent No. 8,034,332, which is incorporated herein by reference in its entirety. In one embodiment, modified NK-92 cells that have not been engineered to express a suicide gene are irradiated.

[0095] Transgene expression Transgenes (e.g., CD19 CAR and CD16) can be manipulated within an expression vector by any mechanism known to those skilled in the art. Transgenes can be manipulated within the same expression vector or different expression vectors. In a preferred embodiment, transgenes are manipulated within the same vector.

[0096] In some embodiments, the vector enables the uptake of a transgene into the cell's genome. In some embodiments, the vector has a positive selection marker. A positive selection marker includes any gene that can grow cells under conditions that would kill cells that do not express that gene. A non-limiting example is antibiotic resistance, e.g., Genethecin (the Neo gene derived from Tn5).

[0097] Any number of vectors can be used to express the Fc receptor and / or CAR. In some embodiments, the vector is a plasmid. In one embodiment, the vector is a viral vector. Viral vectors include, but are not limited to, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, and poxvirus vectors.

[0098] Transgenes can be introduced into NK-92 cells using any transfection method known in the art, including, but not limited to, infection, electroporation, lipofection, nucleofection, or “gene guns.”

[0099] antibody Optionally, antibodies can be used to target cancer cells or cells expressing cancer-related markers. Some antibodies are approved for cancer treatment on their own.

[0100] (Table 2) Examples of FDA-approved therapeutic monoclonal antibodies TIFF0007836780000005.tif141170TIFF0007836780000006.tif104170

[0101] Antibodies can treat cancer through several mechanisms. Antibody-dependent cell-mediated cytotoxicity (ADCC) occurs when immune cells, such as NK cells, bind to antibodies that bind to target cells via Fc receptors such as CD16.

[0102] Therefore, in some embodiments, NK-92 cells expressing CD16 and / or CAR are administered to the patient together with an effective dose of at least one monoclonal antibody against specific cancer-associated proteins, such as alemtuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, rituximab, and trastuzumab. In some embodiments, the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody. In one embodiment, a bispecific antibody can be used that binds to cancer cells and also to cell surface proteins present on the surface of NK-92 cells.

[0103] Cancer-specific antibodies bind to specific protein antigens expressed on the surface of cancer cells. NK-92 cells can be modified to associate antibodies on their surface. In a preferred embodiment, the antibody is cancer-specific. In this way, NK-92 cells can specifically target cancer. Neutralizing antibodies can also be isolated. For example, the secreted glycoprotein, YKL-40, is elevated in several types of advanced human cancer. It is conceivable that antibodies against YKL-40 could be used to suppress tumor growth, angiogenesis, and / or metastasis. Faibish et al., (2011) Mol. Cancer Ther. 10(5):742-751.

[0104] Antibodies can be administered in combination with NK-92 cell administration. Antibodies specific to the cancer to be treated can be administered before, simultaneously with, and / or after the administration of NK-92 cells.

[0105] Antibodies against cancer can be purchased from commercial suppliers or produced by any method known in the art. For example, antibodies can be produced by obtaining B cells, bone marrow, or other samples from one or more patients who had previously had and recovered from cancer or were recovering at the time of sample collection. Methods are known for identifying, screening, and amplifying antibodies (e.g., monoclonal antibodies) from these samples. For example, a phage display library can be prepared by isolating RNA from a sample or cells of interest, preparing cDNA from the isolated RNA, enriching the cDNA for heavy and / or light chain cDNA, and creating a library using a phage display vector. Libraries can be prepared and screened, for example, as described in Maruyama, et al., which is incorporated herein by reference in its entirety. Antibodies can be produced by recombinant methods or any other method. The isolation, screening, characterization, and production of human monoclonal antibodies are also described in Beerli, et al., PNAS(2008) 105(38):14336-14341, which is incorporated herein by reference in its entirety.

[0106] treatment Methods for treating a patient with modified NK-92 cells described herein are also provided. In one embodiment, the patient has cancer, and the CAR expressed by the NK-92 cells is specific to an antigen expressed on the surface of the tumor. In addition to the antigen-specific CAR expressed on the surface of the tumor, the NK-92 expresses an Fc receptor (i.e., NK-92-Fc-CAR). For example, the NK-92 cells can express CD16 and MAGE on their cell surface (i.e., NK-92-CD16-MAGE). Optionally, the patient is treated with modified NK92 cells and even antibodies.

[0107] NK-92 cells can be administered to an individual in terms of the absolute number of cells. For example, the individual can be administered from about 1000 cells / injection up to a maximum of about 10 billion cells / injection, such as about, at least about, or at most about 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 cells (etc.) of NK-92, or any range including the endpoints between any two numbers can be administered.

[0108] In other embodiments, the individual can be administered from about 1000 cells / injection / m 2 up to a maximum of about 10 billion cells / injection / m 2 up to, such as about, at least about, or at most about 1×10 8 / m 2 、1×10 7 / m 2 、5×10 7 / m 2 、1×10 6 / m 2 、5×10 6 / m 2 、1×10 5 / m 2 、5×10 5 / m 2 、1×10 4 / m 2 、5×10 4 / m 2 、1×10 3 / m 2 、5×10 3 / m 2 (etc.) of NK-92 cells, or any range including the endpoints between any two numbers can be administered.

[0109] In another embodiment, NK-92 cells can be administered to such an individual in a relative number of cells, for example, from about 1,000 cells per kilogram of the individual to a maximum of about 10 billion cells, for example, about, at least about, or at most about 1 × 10¹⁶ cells per kilogram of the individual. 8 , 1 x 10 7 , 5×10 7 , 1 x 10 6 , 5×10 6 , 1 x 10 5 , 5×10 5 , 1 x 10 4 , 5×10 4 , 1 x 10 3 , 5×10 3 Individual (or similar) NK-92 cells, or any range including the endpoints between any two numbers, may be administered.

[0110] In other embodiments, the total dose is calculated based on the body surface area m². 2 It can be calculated by 1 m 2 Approximately 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 , or any range including the endpoints between any two numbers. The average person is about 1.6 m 2 From approximately 1.8 m 2 In a preferred embodiment, approximately 1 billion to 3 billion NK-92 cells are administered to the patient. In another embodiment, the amount of NK-92 cells injected per dose is approximately m² of the body surface area. 2 It can be calculated by 1 m 2 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 This includes the average person's height of 1.6-1.8 m. 2 That is the case.

[0111] NK-92 cells, and optionally other anticancer agents, may be administered once to a patient with cancer, or multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours throughout the course of treatment, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or more, or once every range including the endpoints between any two numbers.

[0112] In some embodiments, NK-92 cells are administered in a composition comprising NK-92 cells and a medium, such as human serum or its equivalent. In some embodiments, the medium comprises human serum albumin. In some embodiments, the medium comprises human plasma. In some embodiments, the medium comprises about 1% to about 15% human serum or its equivalent. In some embodiments, the medium comprises about 1% to about 10% human serum or its equivalent. In some embodiments, the medium comprises about 1% to about 5% human serum or its equivalent. In preferred embodiments, the medium comprises about 2.5% human serum or its equivalent. In some embodiments, the serum is human AB serum. In some embodiments, a serum substitute acceptable for use in human therapy is used instead of human serum. Such serum substitutes are known in the art or may be developed in the future. Human serum at concentrations greater than 15% may be used, but concentrations greater than about 5% may be too expensive. In some embodiments, NK-92 cells are administered in a composition comprising NK-92 cells and an isotonic solution to support cell viability. In some embodiments, NK-92 cells are administered with a composition reconstituted from cryopreserved samples.

[0113] Pharmacovigilant compositions may include a variety of carriers and excipients. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. Suitable carriers and excipients, as well as their formulations, are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). A pharmaceutically acceptable carrier means a material that is not biologically or otherwise undesirable; that is, the material is administered to a subject without causing undesirable biological effects or interacting in an adverse manner with other components of the pharmaceutical composition containing it. When administered to a subject, the carrier may be selected to minimize the degradation of the active ingredient and minimize adverse side effects in the subject. As used herein, the term pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions generally contain an active ingredient for buffering and preservation during storage, and may also contain buffers and carriers for appropriate delivery depending on the route of administration.

[0114] These compositions for in vivo or in vitro use can be sterilized by common, well-known sterilization techniques. The compositions may contain acceptable adjuncts required for the appropriate physiological state, such as pH adjusters and buffers, as well as toxicity modifiers, such as sodium acetate, sodium hydrochloride, potassium hydrochloride, calcium hydrochloride, and sodium lactate. The concentrations of cells and / or other active ingredients in these formulations may vary and are primarily selected based on factors such as liquid volume, viscosity, and body weight, according to the specific dosage form and target needs of the chosen individual.

[0115] In one embodiment, NK-92 cells are administered to a patient in combination with one or more other treatments for the cancer being treated. While not bound by theory, it is thought that the simultaneous treatment of a patient with NK-92 cells and other therapies for the cancer allows NK-92 cells and alternative therapies to give such endogenous immune systems a chance to eliminate cancer that had previously suppressed its endogenous action. In some embodiments, two or more other treatments for the cancer being treated may include, for example, antibodies, radiation, chemotherapy, stem cell transplantation, or hormone therapy.

[0116] In one embodiment, the antibody is administered to the patient in combination with NK-92 cells. In one embodiment, NK-92 cells and the antibody may be administered to the patient together, for example, in the same formulation; separately, for example, in separate formulations, simultaneously; or separately, for example, on different dosing schedules or at different times of the day. When administered separately, the antibody may be administered by any suitable route, such as intravenous or oral administration.

[0117] While not bound by theory, NK-92 cells expressing a combination of the Fc receptor and CAR, when administered with a monoclonal antibody, are thought to more readily prevent escape variants, and may even avoid selecting escape variants. In addition, the patient's own effector cells may be involved in ADCC mediated by monoclonal antibodies and target cancer cells. This dual system (both Fc receptor and CAR) may also be more selective to cancer cells than non-cancerous cells (off-tumor on-target). While there are few tumor-associated antigens that are exclusively expressed on cancer cells, it is extremely rare for non-cancerous cells to overexpress two tumor-associated / specific antigens. For example, lymphocytes typically express both CD19 and CD20, and often one is upregulated while the other is downregulated, and vice versa. NK-92-CD16-CD19, in combination with ibritumomab tiuxetan or rituximab, may be effective in treating certain lymphomas.

[0118] kit Also disclosed is a kit for treating cancer, comprising a composition containing a certain amount of NK-92 cells modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) on their cell surface, and instructions for use in treating cancer. In some embodiments, the kit of this disclosure may also include at least one monoclonal antibody.

[0119] The components of the kit may be contained in one or more vials or other containers. The antibody may be in liquid or solid form (e.g., after lyophilization) to improve shelf life. In liquid form, the components may include additives such as stabilizers and / or preservatives, e.g., proline, glycine, or sucrose, or other additives to improve shelf life.

[0120] In certain embodiments, the kit may include additional compounds, such as therapeutically active compounds or drugs, to be administered before, simultaneously with, or after the administration of modified NK-92 cells or NK-92 cells and antibodies. Examples of such compounds include vitamins, minerals, fludrocortisone, ibuprofen, lidocaine, quinidine, and chemotherapeutic agents.

[0121] In various embodiments, the instructions for use of the kit include instructions for using the kit components in the treatment of cancer. The instructions may further include information on how to prepare the antibodies and NK-92 cells (e.g., thawing and / or culturing the lyophilized proteins). The instructions may further include guidance on dosage and frequency of administration.

[0122] Materials, compositions, and components that can be used in, in combination with, or in preparation thereof, or are products thereof, are disclosed herein. Where these and other materials are disclosed herein and combinations, subsets, interactions, groups, etc., of these materials are described, specific descriptions of various individual and collective combinations and permutations of these compounds may not be explicitly stated, but it is understood that each is specifically contemplated and described herein. For example, where a method is disclosed and discussed and several modifications that can be made to some molecules including the method are discussed, each and all combinations and permutations of the method, as well as possible modifications, are specifically contemplated unless otherwise specifically indicated. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure, including steps in methods using the compositions disclosed, but is not limited to these. Therefore, where there are various additional steps that can be implemented, it is understood that each of these additional steps can be implemented by any particular method step or combination of method steps of the method of disclosure, and that each of such combinations or subsets of combinations should be considered to be specifically contemplated and disclosed. [Examples]

[0123] The following embodiments are for illustrative purposes only and should not be construed as limiting. Various alternative techniques and methods are available to those skilled in the art and will similarly enable the successful implementation of the following embodiments.

[0124] Example 1: Extension of survival time after treatment with NK-92-Fc-CAR CD19-positive leukemia cells derived from patients with T-lineage acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and pre-B-ALL were adopted and enlarged in NSG mice by SC inoculation. Leukemia cells (passage 1) were collected from leukemia nodules of these mice. 5 × 10⁶ cells from passage 1 were administered to each group of NSG mice in 0.2 mL of PBS. 6 Individual leukemia cells are inoculated into IP. All human leukemia cells grow actively in NSG mice. After 24 hours, mice are treated weekly for 4 months with either (a) rituximab, (b) NK-92-CD16-CD19 cells, or (c) rituximab and NK-92-CD16-CD19 cells. Treatment with either NK-92-CD16-CD19 cells or a combination of rituximab and NK-92-CD16-CD19 cells significantly extends lifespan and is thought to prolong the survival period of mice compared to treatment with rituximab alone.

[0125] Example 2. NK-92 cells can express Fc receptors and CARs. To analyze NK-92 cells expressing the Fc receptor and CAR, in vitro cytotoxicity assays were performed on NK-92 cells electroporated with CD19-CAR-encoding mRNA against cell lines K562 (NK-92 sensitive, CD19 negative), SUP-B15 (NK-92 resistant, CD19 positive), and SR-91 (NK-92 resistant, CD19 negative). The results are shown in Figures 1A, 1B, and 1C. Figure 1A shows the death of target cell lines by unelectroporated parental NK-92 cells. Figure 1B shows the death of target cell lines by parental NK-92 cells expressing CD19-CAR. Figure 1C shows the death of target cell lines by CD16(158V)-ERIL2 NK-92 cells expressing CD19-CAR. NK-resistant CD19-positive SUP-B15 cells become sensitive to CD19-CAR-expressing NK-92 cells and CD16(158V)-ERIL2 NK-92 cells, but NK-resistant CD19-negative SR-91 cells remain resistant. K562 death is not affected by CD19-CAR expression.

[0126] Example 3. Electroporation of chimeric antigen receptor (CAR) mRNA into human NK cell lines results in high transfection efficiency and target-specific cytotoxicity. This study provides mRNA transfection, expression, and cytotoxicity data for three different CARs based on first-generation CAR constructs: CD19, CD33, and CSPG-4. Target cell lines for mRNA transfection were aNK (parental NK-92 cells) and haNK (high-affinity FcR-expressing NK-92 cells). scFv sequences were custom-made using GeneArt (codon optimization), and taNK (target-activated NK cells) were generated by transfecting the mRNA using MaxCyte GT. Expression was determined by immunofluorescence using corresponding antibodies, and cytotoxicity was measured using standard flow cytometry assays.

[0127] After optimizing the transfection protocol with respect to electrical pulse voltage and duration, it was determined that all three mRNA CAR constructs could be effectively transfected into both aNK and haNK cells. The viability of transfected NK cells after transfection consistently exceeded 80%, and the expression of the corresponding CARs was 55-60% at 6 hours, 80-95% at 24 hours, and over 80% at 48 hours. Specific cytotoxicity was assessed against aNK-resistant cell lines (SUP-B15 for CD19, SR-91 for CD33, and SK-MEL for CSPG-4). Cytotoxicity against aNK-resistant cell lines at 24 hours after transfection consistently exceeded 80%.

[0128] This technology allows for reliable and consistent transfection of both aNK and haNK cells with mRNA from various CAR constructs, maintaining high viability of transfected NK cells, excellent CAR expression, and target cell-specific cytotoxicity for at least 48 hours. This technique can be easily scaled to the clinical-grade production of CAR-expressing NK cell lines. The fact that haNK cells can be effectively transfected (becoming t-haNK cells) opens up the possibility of cross-reactive dual receptor targeting of malignant lesions (i.e., CD19 CAR with CD20 antibody).

[0129] The examples and embodiments described herein are for illustrative purposes only, and it will be understood that various modifications or changes in that regard should be suggested to those skilled in the art and should be included in the spirit and scope of this application and in the claims attached herein. All publications, sequence accession numbers, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0130] Example sequence SEQ ID NO:1 Amino acid sequence (mature form) of low-affinity immunoglobulin γFc region receptor III-A. Phenylalanine at position 158 is underlined. TIFF0007836780000007.tif77158 SEQ ID NO:2 Amino acid sequence (mature form) of the high-affinity variant F158V immunoglobulin γFc domain receptor III-A. The valine at position 158 is underlined. TIFF0007836780000008.tif77158 SEQ ID NO:3 Amino acid sequence of low-affinity immunoglobulin γFc domain receptor III-A (precursor form). Position 176 in the precursor form corresponds to position 158 in the mature form. The Phe at position 176 is underlined. TIFF0007836780000009.tif85159 SEQ ID NO:4 Amino acid sequence of high-affinity variant immunoglobulin γFc domain receptor III-A (precursor form). Position 176 in the precursor form corresponds to position 158 in the mature form. The "Val" at position 176 is underlined. TIFF0007836780000010.tif85159 SEQ ID NO:5 Polynucleotide encoding low affinity immunoglobulin γFc domain receptor III-A (precursor) (encoding phenylalanine at position 158) TIFF0007836780000011.tif70154 SEQ ID NO:6 Wild type IL-2 TIFF0007836780000012.tif55160 SEQ ID NO:7 IL-2-ER TIFF0007836780000013.tif56160 SEQ ID NO:8 CD19-CAR DNA sequence TIFF0007836780000014.tif215153 SEQ ID NO:9 CD19-CAR amino acid sequence TIFF0007836780000015.tif70150 SEQ ID NO:10 CD33-CAR DNA sequence TIFF0007836780000016.tif208154 SEQ ID NO:11 CD33-CAR amino acid sequence TIFF0007836780000017.tif70149 SEQ ID NO:12 CSPG4-CAR DNA sequence TIFF0007836780000018.tif222153 SEQ ID NO:13 CSPG4-CAR amino acid sequence TIFF0007836780000019.tif70151

[0131] Sequence information SEQUENCE LISTING <110> IMMUNITYBIO, INC. <120> MODIFIED NK-92 CELLS FOR TREATING CANCER <150> US 62 / 173,701 <151> 2015-06-10 <150> US 62 / 337,044 <151> 2016-05-16 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> synthetic Low Affinity Immunoglobulin Gamma Fc Region Receptor III-A amino acid sequence (mature form) <400> 1 Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp 1 5 10 15 Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala 20 25 30 Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu 35 40 45 Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp 50 55 60 Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp 65 70 75 80 Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro 85 90 95 Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His Ser 100 105 110 Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys 115 120 125 Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala 130 135 140 Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe Gly Ser 145 150 155 160 Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu 165 170 175 Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser 180 185 190 Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr 195 200 205 Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp 210 215 220 His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 225 230 235 <210> 2 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> synthetic High Affinity Variant F158V Immunoglobulin Gamma Fc Region Receptor III-A amino acid sequence (mature form) <400> 2 Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp 1 5 10 15 Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala 20 25 30 Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu 35 40 45 Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp 50 55 60 Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp 65 70 75 80 Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro 85 90 95 Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His Ser 100 105 110 Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys 115 120 125 Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala 130 135 140 Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser 145 150 155 160 Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu 165 170 175 Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser 180 185 190 Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr 195 200 205 Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp 210 215 220 His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 225 230 235 <210> 3 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> synthetic Low Affinity Immunoglobulin Gamma Fc Region Receptor III-A amino acid sequence (precursor form) <400> 3 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> 4 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> synthetic High Affinity Variant Immunoglobulin Gamma Fc Region Receptor III-A amino acid sequence (precursor form) <400> 4 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> 5 <211> 765 <212> DNA <213> Artificial Sequence <220> <223> synthetic Polynucleotide Encoding the Low Affinity Immunoglobulin Gamma Fc Region Receptor III-A (Precursor) <400> 5 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag aaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagcccta ttcacctgag gtgtcacagc tggaaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtt tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccata atttaatg gagaaggac cctcagaca atga 765 <210> 6 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide - Wild-Type IL-2 <400> 6 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Lew Lys Pro Lew Glu Glu Val Lew Asn Lew Wing Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 7 <211> 160 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide IL-2-ER <400> 7 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Lew Lys Pro Lew Glu Glu Val Lew Asn Lew Wing Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr Gly Ser Glu Lys Asp Glu Leu 145 150 155 160 <210> 8 <211> 1455 <212> DNA <213> Artificial Sequence <220> <223> synthetic CD19-CAR DNA sequence <400> 8 cccgggaatt cgccaccatg gactggatct gttcctcgtg ggagccgcca 60 caggcgccca ttctgcccag cccgccgaca tccagatgac ccagaccacc agcagcctga 120 gcgccagcct gggcgacaga gtgaccatca gctgccgggc cagccaggac atcagcaagt 180 acctgaactg gtatcagcag aaacccgacg gcaccgtgaa gctgctgatc taccacacca 240 gccggctgca cagcggcgtg cccagcagat tttctggcag cggcagcggc accgactaca 300 gcctgaccat ctccaacctg gaacaggaag atatcgctac ctacttctgt cagcaaggca 360 acaccctgcc ctacaccttc ggcggaggca ccaagctgga actgaagaga ggcggcggag 420 gctctggtgg aggcggatct gggggcggag gaagtggcgg gggaggatct gaagtgcagc 480 tgcagcagag cggccctggc ctggtggccc ctagccagag cctgtccgtg acctgtaccg 540 tgtccggcgt gtccctgccc gactacggcg tgtcctggat ccggcagccc cccagaaagg 600 gcctggaatg gctgggcgtg atctggggca gcgagacaac ctactacaac agcgccctga 660 agtcccggct gaccatcatc aaggacaaca gcaagagcca ggtgttcctg aagatgaaca 720 gcctgcagac cgacgacacc gccatctact actgcgccaa gcactactac tacggcggca 780 gctacgccat ggactactgg ggccagggca ccaccgtgac cgtgtccagc gccctgtcca 840 acagcatcat gtacttcagc cacttcgtgc ccgtgtttct gcccgccaag cccaccacca 900 cccctgcccc tagacctccc accccagccc caacaatcgc cagccagcct ctgtccctgc 960 ggcccgaagc tagcagacct gctgccggcg gagccgtgca caccagaggc ctggacccca 1020 agctgtgcta cctgctggac ggcatcctgt tcatctatgg cgtgatcctg accgccctgt 1080 tcctgagagt gaagttcagc agaagcgccg acgcccctgc ctaccagcag ggccagaacc 1140 agctgtacaa cgagctgaac ctgggcagac gggaagagta cgacgtgctg gacaagcgga 1200 gaggcaggga ccccgagatg ggcggcaagc ccagacggaa gaacccccag gaaggcctgt 1260 ataacgaact gcagaaagac aagatggccg aggcctacag cgagatcggc atgaagggcg 1320 agcggcggag gggcaagggc cacgatggac tgtaccaggg cctgagcacc gccaccaagg 1380 acacctacga cgccctgcac atgcaggccc tgccccccag atgacagcca gggcatttct 1440 ccctcgagcg gccgc 1455 <210> 9 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD19-CAR amino acids sequence <400> 9 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Asp Ile Gln Met Thr Gln Thr Thr Ser 20 25 30 Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala 35 40 45 Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp 50 55 60 Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu 85 90 95 Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110 Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Leu Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro 145 150 155 160 Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser 165 170 175 Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro 180 185 190 Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr 195 200 205 Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn 210 215 220 Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp 225 230 235 240 Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr 245 250 255 Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 260 265 270 Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu 275 280 285 Pro Ala Light Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 290 295 300 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg 305 310 315 320 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Pro Lys Leu 325 330 335 Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr 340 345 350 Ala Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 355 360 365 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 370 375 380 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 385 390 395 400 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 405 410 415 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 420 425 430 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 435 440 445 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 450 455 460 Leu Pro Pro Arg 465 <210> 10 <211> 1437 <212> DNA <213> Artificial Sequence <220> <223> synthetic CD33-CAR DNA sequence <400> 10 cccgggaatt cgccaccatg gactggatct ggcggatcct gttcctcgtg ggagccgcca 60 caggcgccca ttctgcccag cccgccgaca tccagatgac ccagagccct agcagcctga 120 gcgccagcgt gggcgacaga gtgaccatca cctgtcgggc cagcgagagc gtggacaact 180 acggcatcag cttcatgaac tggttccagc agaagcccgg caaggccccc aagctgctga 240 tctacgccgc cagcaatcag ggcagcggcg tgcccagcag attcagcggc tctggcagcg 300 gcaccgactt caccctgacc atcagcagcc tgcagcccga cgacttcgcc acctactact 360 gccagcagag caaagaggtg ccctggacct tcggccaggg caccaaggtg gaaatcaagg 420 gcggaggcgg cagcggaggt ggaggaagtg gcggcggagg atctcaggtg cagctggtgc 480 agtctggcgc cgaagtgaag aaacccggca gcagcgtgaa ggtgtcctgc aaggccagcg 540 gctacacctt caccgactac aacatgcact gggtccgcca ggccccaggc cagggactgg 600 aatggatcgg ctacatctac ccctacaacg gcggcaccgg ctacaaccag aagttcaaga 660 gcaaggccac catcaccgcc gacgagagca ccaacaccgc ctacatggaa ctgagcagcc 720 tgcggagcga ggacaccgcc gtgtactact gcgccagagg cagacccgcc atggactact 780 ggggccaggg aaccctggtg acagtgtcca gcgccctgag caacagcatc atgtacttca 840 gccacttcgt gcccgtgttt ctgcccgcca agcccaccac cacccctgcc cctagacctc 900 ccaccccagc cccaacaatc gccagccagc ctctgtccct gcggcccgaa gctagcagac 960 ctgctgccgg cggagccgtg cacaccagag gcctggaccc caagctgtgc tacctgctgg 1020 acggcatcct gttcatctac ggcgtgatcc tgaccgccct gttcctgaga gtgaagttca 1080 gcagaagcgc cgacgcccct gcctaccagc agggccagaa ccagctgtac aacgagctga 1140 acctgggcag acgggaagag tacgacgtgc tggacaagcg gagaggcagg gaccccgaga 1200 tgggcggcaa gcccagacgg aagaaccccc aggaaggcct gtataacgaa ctgcagaaag 1260 acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg aggggcaagg 1320 gccacgatgg actgtaccag ggcctgagca ccgccaccaa ggacacctac gacgccctgc 1380 acatgcaggc cctgcccccc agatgacagc cagggcattt ctccctcgag cggccgc 1437 <210> 11 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> synthetic CD33-CAR amino acid sequence <400> 11 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Asp Ile Gln Met Thr Gln Ser Pro Ser 20 25 30 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 35 40 45 Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln 50 55 60 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn 65 70 75 80 Gln Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr 100 105 110 Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly 115 120 125 Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly 195 200 205 Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser 210 215 220 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Leu Ser Asn Ser Ile Met 260 265 270 Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr 275 280 285 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 290 295 300 Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala 305 310 315 320 Val His Thr Arg Gly Leu Asp Pro Lys Leu Cys Tyr Leu Leu Asp Gly 325 330 335 Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala Leu Phe Leu Arg Val 340 345 350 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 355 360 365 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 370 375 380 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 385 390 395 400 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 405 410 415 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 420 425 430 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 435 440 445 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460 <210> 12 <211> 1509 <212> DNA <213> Artificial Sequence <220> <223> synthetic CSPG4-CAR DNA sequence <400> 12 cccgggaatt cgccaccatg gactggatct ggcgcatcct cttcctcgtc ggcgctgcta 60 ccggcgctca ttcggcccag ccggccgata tcgagctcac ccaatctcca aaattcatgt 120 ccacatcagt aggagacagg gtcagcgtca cctgcaaggc cagtcagaat gtggatacta 180 atgtagcgtg gtatcaacaa aaaccagggc aatctcctga accactgctt ttctcggcat 240 cctaccgtta cactggagtc cctgatcgct tcacaggcag tggatctggg acagatttca 300 ctctcaccat cagcaatgtg cagtctgaag acttggcaga gtatttctgt cagcaatata 360 acagctatcc tctgacgttc ggtggcggca ccaagctgga aatcaaacgg gctgccgcag 420 aaggtggagg cggttcaggt ggcggaggtt ccggcggagg tggctctggc ggtggcggat 480 cggccatggc ccaggtgaag ctgcagcagt caggaggggg cttggtgcaa cctggaggat 540 ccatgaaact ctcctgtgtt gtctctggat tcactttcag taattactgg atgaactggg 600 tccgccagtc tccagagaag gggcttgagt ggattgcaga aattagattg aaatccaata 660 atttggaag atattatgcg gagtctgtga aagggaggtt caccatctca agagatgatt 720 ccaaaagtag tgcctacctg caaatgatca acctaagagc tgaagatact ggcatttatt 780 actgtaccag ttatggtaac tacgttgggc actattttga ccactggggc caagggacca 840 cggtcaccgt atcgagtgcc gcggttctag agctcttgag caactccatc atgtacttca 900 gccacttcgt gccggtcttc ctgccagcga agcccaccac gacgccagcg ccgcgaccac 960 caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc 1020 cagcggcggg gggcgcagtg cacacgaggg ggctggacct gctggatccc aaactctgct 1080 acctgctgga tggaatcctc ttcatctatg gtgtcattct cactgccttg ttcctgagag 1140 tgaagttcag caggagcgca gacgccccg cgtaccagca gggccagaac cagctctata 1200 acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga cgtggccggg 1260 accctgagat ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg 1320 aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc 1380 ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct 1440 acgacgccct tcacatgcag gccctgcccc ctcgctaaca gccagggcat ttctccctcg 1500 agcggccgc 1509 <210> 13 <211> 486 <212> PRT <213> Artificial Sequence <220> <223> synthetic CSPG4-CAR amino acid sequence <400> 13 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Ala Gln Pro Ala Asp Ile Glu Leu Thr Gln Ser Pro Lys 20 25 30 Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala 35 40 45 Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Gln Ser Pro Glu Pro Leu Leu Phe Ser Ala Ser Tyr Arg Tyr Thr Gly 65 70 75 80 Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 85 90 95 Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln 100 105 110 Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125 Ile Lys Arg Ala Ala Ala Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Met Ala Gln Val 145 150 155 160 Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Met 165 170 175 Lys Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Asn Tyr Trp Met 180 185 190 Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Ala Glu 195 200 205 Ile Arg Leu Lys Ser Asn Asn Phe Gly Arg Tyr Tyr Ala Glu Ser Val 210 215 220 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Ala Tyr 225 230 235 240 Leu Gln Met Ile Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys 245 250 255 Thr Ser Tyr Gly Asn Tyr Val Gly His Tyr Phe Asp His Trp Gly Gln 260 265 270 Gly Thr Thr Val Thr Val Ser Ser Ala Ala Val Leu Glu Leu Leu Ser 275 280 285 Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala 290 295 300 Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 305 310 315 320 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 325 330 335 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Leu Leu Asp Pro Lys 340 345 350 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 355 360 365 Thr Ala Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485

Claims

1. A pharmaceutical composition for treating B-cell malignancies in patients who require it, The solution contains an effective amount of the NK-92 cell line to be administered to the patient. The NK-92 cell line contains modified NK-92 cells, The modified NK-92 cells are modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR), respectively, so that at least one Fc receptor and at least one chimeric antigen receptor (CAR) are presented on the cell surface of the modified NK-92 cells. The CAR has the amino acid sequence of SEQ ID NO:

9. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the Fc receptor is FcγRIII-A(CD16) or a CD16 polypeptide having valine at position 158 of the mature form of CD16.

3. The pharmaceutical composition according to claim 1, wherein the Fc receptor has the amino acid sequence of SEQ ID NO:

2.

4. The pharmaceutical composition according to claim 1, wherein the modified NK-92 cells are modified to express cytokines.

5. The effective amount is at least about 1 × 10 8 The pharmaceutical composition according to claim 1, comprising individual cells.

6. The pharmaceutical composition according to claim 1, wherein the CAR targets the CD19 tumor-associated antigen.

7. Modified NK-92 cells, (1) The modified NK-92 cells are modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR), respectively, so that at least one Fc receptor and at least one chimeric antigen receptor (CAR) are presented on the cell surface of the modified NK-92 cells. (2) The CAR has the amino acid sequence of SEQ ID NO:9, Modified NK-92 cells.

8. The modified NK-92 cell according to claim 7, wherein the Fc receptor is FcγRIII-A(CD16) or a CD16 polypeptide having valine at position 158 of the mature form of CD16.

9. The modified NK-92 cell according to claim 7, wherein the Fc receptor has the amino acid sequence of SEQ ID NO:

2.

10. The modified NK-92 cells according to claim 7, wherein the modified NK-92 cells are further modified to express cytokines.

Images