Modified NK-92 cells for treating cancer

Abstract

To provide genetically modified NK-92 cells engineered to express multiple transgenes, for improved therapeutics and methods of treating cancer cells.SOLUTION: The NK-92 cells are modified to each express at least one Fc receptor and at least one chimeric antigen receptor (CAR), such that the at least one Fc receptor and the at least one CAR are displayed on the cell surface of the NK-92 cells. Also provided are methods of treatment of a patient having or suspected of having a disease that is treatable with NK-92 cells, such as cancer, the methods comprising administering to the patient an NK-92 cell line.SELECTED DRAWING: Figure 1C

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 173,701, filed on Jun. 10, 2015, and U.S. Provisional Patent Application No. 62 / 337,044, filed on May 16, 2016, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background Natural killer (NK) cells are cytotoxic lymphocytes that form a major component of the innate immune system. NK cells generally account for about 10 - 15% of circulating lymphocytes and bind to and kill target cells, including virus - infected cells and many malignant cells, non - specifically with respect to antigens and without prior immunization. Herberman et al., Science 214:24 (1981) (Non - Patent Document 1). The death of target cells occurs by inducing cell lysis. NK cells used for this purpose are isolated from the peripheral blood lymphocyte (“PBL”) fraction of blood from a subject, expanded in cell culture to obtain a sufficient number of cells, and then reinjected into the subject. NK cells have been shown to be somewhat effective in both ex vivo therapy and in vivo treatment. However, such therapies are complicated by the fact that not all NK cells are cytolytic and that 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 that 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 being 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, especially 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 preventing detection. For example, tumor escape 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, there remains a need for improved treatments and methods for treating cancer cells.

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 co-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 them on the cell surface of the NK-92 cells. [Invention 1002] The cell of Invention 1001, wherein the Fc receptor is FcγRIII-A (CD16) or a CD16 polypeptide having valine at position 158 of the mature form of CD16. [Invention 1003] The cell of 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 Invention 1001, wherein the Fc receptor comprises the amino acid sequence of SEQ ID NO:2. [Invention 1005] The cell of any one of Inventions 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 cell of any one of Inventions 1001 to 1004, wherein 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. [Invention 1007] The cell of any one of Inventions 1001 to 1006, which further expresses a cytokine. [Invention 1008] The cell of Invention 1007, wherein the cytokine is interleukin-2 or a variant thereof. [Invention 1009] The cell of the present invention 1007 or 1008, wherein the cytokine targets the endoplasmic reticulum. [The present invention 1010] The cell of any one of the present inventions 1001 to 1009, wherein the Fc receptor and the CAR are encoded on different vectors. [The present invention 1011] The NK-92 cell line, wherein the cells of the NK-92 cell line are modified to express at least one of the Fc receptors and at least one of the CARs so as to present the Fc receptor and the chimeric antigen receptor (CAR) on the cell surface of the NK-92 cells. [The present 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 CD16. [The present 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 contains valine at position 158. [The present invention 1014] The method of the present invention 1011, wherein the Fc receptor comprises the amino acid sequence of SEQ ID NO:2. [The present invention 1015] The NK-92 cell line of any one of the present inventions 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. [The present invention 1016] The NK-92 cell line of any one of the present inventions 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. [The present invention 1017] Any NK-92 cell line of the present invention 1011-1016 that further expresses cytokines. [The present invention 1018] The NK-92 cell line of the present invention 1017, wherein the cytokine is interleukin-2 or a variant thereof. [The present invention 1019] The NK-92 cell line of the present invention 1017 or 1018, wherein the cytokine targets the endoplasmic reticulum. [The present invention 1020] Any cell of the present invention 1011-1019, wherein the Fc receptor and the CAR are encoded on different vectors. [The present invention 1021] Any cell line of the present invention 1011-1020, wherein the cells of the cell line have undergone less than 10 population doublings. [The present invention 1022] Any cell line of the present invention 1011-1020, wherein the cells are cultured in a medium containing less than 10 U / ml of IL-2. [The present invention 1023] A composition comprising an amount of cells from any of the present invention 1001-1010 or the present invention 1011-1022. [The present invention 1024] The composition of the present invention 1023, further comprising at least one monoclonal antibody. [The present 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. [The present 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, adotranstuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab. [The present invention 1027] A method for treating cancer in a patient in need thereof, comprising 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. [The present 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. [The present invention 1029] The method of the present invention 1027, further comprising administering to the patient an effective amount of at least one monoclonal antibody. [The present 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. [The present invention 1031] The method of the present invention 1029, wherein the monoclonal antibody is selected from the group consisting of alemtuzumab, rituximab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, ado-trastuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab. [The present invention 1032] The method according to any one of the present inventions 1029 to 1031, wherein the monoclonal antibody and the cells are administered simultaneously. [The present invention 1033] The method according to any one of the present inventions 1029 to 1031, wherein the monoclonal antibody and the cells are mixed before being administered to the patient. [The present invention 1034] The method according to any one of the present inventions 1029 to 1031, wherein the monoclonal antibody and the cells are administered sequentially. [The present invention 1035] The method according to any one of the present inventions 1027 to 1034, wherein the cancer is selected from the group consisting of leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, sarcoma, and carcinoma. [The present invention 1036] About 1×10 per 1 m of the body surface area of the patient 2 to about 1×10 8 cells to about 1×10 11 cells are administered to the patient, any method of the present invention from 1027 to 1035. [Invention 1037] (a) NK-92 cells modified to express at least one Fc receptor on the cell surface and at least one chimeric antigen receptor (CAR) on the cell surface, and (b) instructions for use, a kit for treating cancer. [Invention 1038] (c) The 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, rituximab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, ado-trastuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab. [Invention 1041] A modified NK-92 cell comprising a multidentate ligand-binding element selected from the group consisting of one or more Fc receptors and one or more CARs such that at least two ligand-binding elements are present on the cell surface of the modified NK-92 cell. [Invention 1042] A method for enhancing the binding affinity of NK-92 cells to cancer cells, comprising the step of using a multidentate binding element on the cell surface of the modified NK-92 cell, wherein the modified NK-92 cell comprises a multidentate binding element for one or more Fc receptors and one or more CARs such that at least two ligand-binding elements are present on the cell surface of the NK-92 cell. The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure. Other objects, advantages, and novel features will be readily apparent to those skilled in the art.

Brief Description of the Drawings

[0010] The objectives, features, and advantages will be more readily understood by referring to the following disclosure when considered in conjunction with the accompanying drawings.

Figure 1A

Figure 1B

Figure 1C

Modes for Carrying Out the Invention

[0011] Detailed Description NK-92 cells are provided herein that are modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) such that the at least one Fc receptor and the at least one CAR are presented on the cell surface of the NK-92 cells. Optionally, the Fc receptor may comprise FcγRIII-A (CD16). Optionally, the NK-92 cells have at least 90% sequence identity with SEQ ID NO:1 (FcγRIII-A or CD16 having phenylalanine at position 158 (F-158)); or are genetically modified to express an Fc receptor encoding a polypeptide having at least 90% identity with SEQ ID NO:2 (CD16 having valine at position 158 (F158V), a higher affinity form). In a typical embodiment, the CD16 polypeptide has valine at position 158. Optionally, the 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 a tumor-associated antigen 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 variants thereof. In some embodiments, NK-92 cells are modified to express a polypeptide having the sequence of SEQ ID NO:6 or SEQ ID NO:7. In further embodiments, the cytokine targets the endoplasmic reticulum. Optionally, the NK-92 cells of the cell line are cultured in a medium containing less than 10 U / ml of IL-2.

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

[0014] The present disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of any of the cells of the above embodiments. 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, about 1×10 2 per square meter of the patient's body surface area 8 from about 1×10 11Individual cells are administered to a patient. Optionally, the methods of the present disclosure provide for administering to the patient an effective amount of at least one monoclonal antibody, such as alemtuzumab, rituximab, trastuzumab, ibritumomab, gemtuzumab, brentuximab, ado-trastuzumab, 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 the cells are administered simultaneously. In some embodiments, the monoclonal antibody and the cells are mixed together prior to administration to the patient. In other embodiments, the monoclonal antibody and the cells are administered sequentially. In other embodiments, the subject is administered the monoclonal antibody and subsequently, modified NK-92 cells are administered, for example, within 24 hours; or within 24 to 72 hours after administration of the monoclonal antibody.

[0015] In one embodiment, the 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 macroglobulinemia, heavy chain disease, sarcoma, or carcinoma.

[0016] The present disclosure provides a kit for use in any of the above methods of treating cancer, wherein the kit comprises (a) an amount of NK-92 cells modified to express at least one Fc receptor on the cell surface and at least one chimeric antigen receptor (CAR) on the cell surface, and (b) at least one of a set of instructions that describe at least one method of the present disclosure. In some embodiments, the kit further comprises at least one monoclonal antibody.

[0017] After reading this specification, methods for practicing various alternative aspects and uses will become apparent to those of ordinary skill in the art. However, not all aspects are described herein. The aspects shown herein are presented for purposes of illustration only and are not intended to be limiting. Accordingly, a detailed description of these various alternative aspects should not be construed as limiting the scope or breadth of the disclosure described herein. It is to be understood that the aspects described below are not limited to a particular composition, method of preparing such composition, or its use, and may, of course, vary.

[0018] The term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0019] Reference is made to several terms that are defined to have the following meanings in this specification and the appended claims.

[0020] The terms used herein are for the purpose of describing particular aspects only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Thus, for example, reference to "natural killer cells" includes plural natural killer cells.

[0021] All numerical notations that include ranges, for example, pH, temperature, time, concentration, amount, and molecular weight, etc., are approximate values that change by increments of 0.1 or 1.0 in the (+) or (-) direction as necessary. Although not always explicitly stated, it should be understood that all numerical notations can be preceded by the term "about."

[0022] As will be understood by those skilled in the art, for any and all purposes, particularly with respect to providing a detailed description of the invention, all ranges described herein include any and all possible sub-ranges and combinations of those sub-ranges. Any recited range can be readily recognized as fully describing and enabling the same range which has been at least equivalently divided into two, three, four, five, ten, etc. sub-ranges. By way of non-limiting example, each range described herein can be readily decomposed into, for example, the lower one-third, the middle one-third, and the upper one-third. As will also be understood by those skilled in the art, all terms such as "up to", "at least", "more than", and "less than" refer to the recited number and include ranges that can subsequently be decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, ranges include 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 necessarily specified, it should also be understood that the reagents described herein are merely exemplary and that equivalents of such reagents are known in the art.

[0024] "Any" or "optionally" means that the event or circumstance recited thereafter may or may not occur and that the description includes both the case where the event or circumstance occurs and the case where it does not occur.

[0025] The term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. "Consisting essentially of" when used to define compositions and methods is intended to mean excluding any other elements that are essential to the combination. For example, a composition consisting essentially of the elements as defined herein does not exclude other elements that do not substantially affect the basic and novel characteristics of the claims. "Consisting of" is intended to mean excluding amounts of other components in excess of trace amounts and substantial method steps. The aspects defined by each of these transitional terms are within the scope of the present disclosure.

[0026] As used herein, "simultaneous" or "simultaneously" refers to administering at least two agents (e.g., NK-92-Fc-CAR cells and monoclonal antibodies) at the same time or approximately the same time.

[0027] As used herein, the term "effective amount" refers to an amount of a composition sufficient to achieve a desired therapeutic effect, e.g., an amount that results in remission of cancer cells or one or more symptoms associated with cancer. In the context of a therapeutic application, the amount of NK-92 cells or antibody administered to a subject will depend on the type and progression of cancer, as well as individual characteristics such as general health, age, gender, weight, and drug tolerance. It also depends on the degree, severity, and type of disease. One of ordinary skill in the art can determine the appropriate dosage according to these and other factors. NK-92 cells can 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 process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. When the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from a sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from a sample can be directly compared to the expression level of that gene 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, either alone or in combination, which can induce cytotoxicity upon contact with target cells.

[0030] As used herein, a "natural killer (NK) cell" is a cell of the immune system that kills target cells in the absence of specific antigen stimulation and without restriction by the major histocompatibility complex (MHC) class. The target cells can 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), which are distinct from the NK-92 cell line. Endogenous NK cells are generally a heterogeneous population of cells in which NK cells are enriched. Endogenous NK cells can be for autologous or allogeneic treatment of a patient.

[0032] "NK-92 cells" refers to the immortal NK cell line, NK-92, originally obtained from a patient with non-Hodgkin's 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 introduction of exogenous genes). NK-92 cells and their exemplary and non-limiting modifications are described in U.S. Patent Nos. 7,618,817; 8,034,332; and 8,313,943, all of which are hereby incorporated by reference in their entirety.

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

[0034] As used herein, "non-irradiated NK-92 cells" are NK-92 cells that have not been irradiated. Irradiation renders the cells unable to grow and proliferate. Since the time between irradiation and injection should not exceed 4 hours to maintain optimal activity, NK-92 cells are envisioned to be irradiated at the treatment facility or another location prior to treatment of the patient. Alternatively, NK-92 cells can be inactivated by another mechanism.

[0035] As used herein, "inactivation" of NK-92 cells renders them unable to grow. Inactivation can also be associated with the death of NK-92 cells. It is envisioned that NK-92 cells can be inactivated after they have effectively purged ex vivo samples of cells associated with the pathology in a therapeutic application or after they have been present in a mammalian body for a time sufficient to effectively kill many or all of the target cells present in the body. Inactivation can be induced, as a non-limiting example, by administration of 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. In general, cytotoxic activity is related to killing target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity by which the effector lyses the plasma membrane of the target cell, thereby destroying its physical integrity. This results in the death of the target cell. Without wishing to be bound by theory, the cytotoxic action of NK cells is thought to be due to cytolysis.

[0037] The term "kill" with respect to a cell / cell population is directed to include any type of manipulation that leads to the death of that cell / cell population.

[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 defensive functions of immune cells by binding to a portion of an antibody known as the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates the phagocytic or cytotoxic activity of the cell via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fc-gamma receptors (FcγRs) bind antibodies of the IgG class. FcγRIII-A (also called CD16) is a low-affinity Fc receptor that binds 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] As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen-binding domain that is fused to an intracellular signaling domain. CARs are expressed in T cells or NK cells and can enhance cytotoxicity. Generally, the extracellular antigen-binding domain is a scFv that is specific for an antigen found on the cells 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 engineered 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 cannot be detected on normal cells derived from the same tissue or lineage as the cancer cells. As used herein, the term "tumor-specific antigen" also refers to a tumor-associated antigen, i.e., an antigen that is expressed at a higher level on cancer cells compared to normal cells derived from the same tissue or lineage as the cancer cells.

[0041] The terms "polynucleotide", "nucleic acid", and "oligonucleotide" are used interchangeably and refer to a polymer form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide can assume any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs 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. A polynucleotide can contain modified nucleotides such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form

[0042] A polynucleotide is composed of a specific sequence of the following four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, in the case where the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule.

[0043] As used herein, "percent identity" refers to sequence identity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing positions in each sequence that may be aligned for purposes of comparison. A molecule is identical at a position if the position in the compared sequences is occupied by the same base or amino acid. As used herein, the phrases "homologous" or "variant" nucleotide sequences, or "homologous" or "variant" amino acid sequences, 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 allelic variants and mutants of the 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 that contain conservative amino acid substitutions and in which the polypeptide has the same bonds and / or activities. In some embodiments, homologous nucleotide or amino acid sequences have at least 60% or greater, such as at least 70%, at least 80%, at least 85% or greater, identity with the comparison sequence. In some embodiments, homologous nucleotide or amino acid sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the comparison sequence. In some embodiments, homologous amino acid sequences have 15 or fewer, 10 or fewer, 5 or fewer, or 3 or fewer conservative amino acid substitutions. Percent identity 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, by the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

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

[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 (IL), 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 an extrachromosomal nucleic acid containing an intact replicon that can replicate, for example, by the process of transformation, when the vector is placed within a permissive cell. A vector can replicate in one cell type, such as a bacterium, but has limited ability to replicate in another cell, such as a mammalian cell. A vector can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include: naked DNA; DNA complexed with cationic lipids, either alone or in combination with a cationic polymer; anionic and cationic liposomes; DNA-protein complexes and particles containing DNA condensed with cationic polymers such as heterologous polylysine, oligopeptides of a defined length, and polyethyleneimine, which in some cases are contained within liposomes; and the use of ternary complexes containing a virus and polylysine-DNA.

[0047] As used herein, the term "targeting" is intended, without limitation, to include directing a protein or polypeptide to an appropriate destination, either intracellularly or extracellularly. Targeting is typically accomplished via a signal peptide or targeting peptide, which is a contiguous series of amino acid residues in the polypeptide chain. Such signal peptides can be located anywhere within the polypeptide sequence, but are often located at the N-terminus. The polypeptide can also be engineered to have a signal peptide at the C-terminus. Signal peptides can direct the polypeptide to extracellular regions, to the plasma membrane, to the Golgi, to endosomes, to the endoplasmic reticulum, or to other intracellular compartments. For example, a polypeptide having a specific amino acid sequence (e.g., KDEL) at its C-terminus is retained within the ER lumen or returned to the ER lumen.

[0048] The term "suicide gene" enables negative selection of cells. Suicide genes are used as a safety system, and cells expressing the gene can be killed by the introduction of a selection agent. This is desirable when recombinant genes cause mutations leading to uncontrolled cell growth. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella-zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli gpt gene, and E. coli Deo gene (see also, for example, Yazawa K, Fisher W E, Brunicardi F C: 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 a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir.

[0049] Terms such as "patient," "subject," and "individual" are used interchangeably herein and refer to any animal suitable for the methods described herein, or to its cells, whether in vitro or in situ. In a preferred embodiment, the patient, subject, or individual is a mammal. In a particular preferred embodiment, the patient, subject, or individual is a human.

[0050] The term "treating" or "treatment" refers to treating 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 "administering" or "administration" of a monoclonal antibody or natural killer cell to a subject includes any route by which the antibody or cell is introduced or delivered to perform the intended function. Administration may be by any route suitable for delivery of the cell or the monoclonal antibody. Thus, the delivery route may include intravenous, intramuscular, intraperitoneal, or subcutaneous delivery. In some embodiments, the monoclonal antibody and / or NK-92 cells are administered directly to the tumor, for example, by injection into the tumor. Administration includes self-administration and administration by another person.

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

[0052] As used herein, the term “antibody” refers to an immunoglobulin or a fragment thereof. The antibody can be of any type (e.g., IgG, IgA, IgM, IgE, or IgD). Preferably, the antibody is IgG. The antibody can be non-human (e.g., derived from a mouse, goat, or any other animal), fully human, humanized, or chimeric. The antibody can 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 cells that grow and have the ability to proliferate infinitely in culture. Monoclonal antibodies that can be used include naked antibodies that attach to antigens on cancerous cells and block them. In one embodiment, the naked monoclonal antibody is alemtuzumab, which binds to the CD52 antigen on lymphocytes. Also included among the monoclonal antibodies that can be used are conjugated monoclonal antibodies such as tagged, labeled, or loaded antibodies. Specifically, the antibody can be tagged or loaded with a drug or toxin, or radiolabeled. Examples of such antibodies include, but are not limited to, ibritumomab that targets the CD20 antigen, brentuximab that targets the CD30 antigen, and trastuzumab that targets the HER2 protein. Other monoclonal antibodies that can be used are bispecific monoclonal antibodies such as blinatumomab that 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 can be glycosylated. Non-limiting examples of antibody fragments can include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, rIgG fragments, functional antibody fragments, single-chain recombinant forms thereof, and the like. F(ab')2, Fab, Fab', and Fv are antigen-binding fragments that can be made from the variable regions of IgG and IgM. They differ in terms of size, valence, and Fc content. Fragments can be made by any method, including expression of a construct (e.g., a heavy-chain portion and a light-chain portion) by one cell or cell line, or multiple cells or cell lines. Preferably, the antibody fragment contains a portion of the Fc region sufficient to have the ability to recognize an epitope and bind to an Fc receptor.

[0055] As used herein, the term "cancer" refers to all types of cancers, 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, ovary, sarcoma, stomach, uterus, and medulloblastoma. Further examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström's macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, pre-cancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, hypercalcemia of malignancy, endometrial cancer, adrenocortical cancer, pancreatic endocrine and exocrine neoplasms, and prostate cancer.

[0056] A title or subtitle may be used in this specification for the convenience of the reader and is not intended to limit the scope of the disclosure. In addition, some of the terms used in this specification are defined more specifically below.

[0057] NK-92 cells The NK-92 cell line is a unique cell line that has been found to proliferate in the presence of interleukin 2 (IL-2). Gong et al., Leukemia 8:652-658 (1994). These cells have high cytolytic activity against various cancers. The NK-92 cell line is a homogeneous population of cancerous NK cells with broad anti-tumor cytotoxicity and can be obtained in a predictable yield after expansion. Its safety profile has been confirmed in a Phase I clinical trial. NK-92 was discovered in the blood of a subject with non-Hodgkin's lymphoma and was subsequently immortalized ex vivo. NK-92 cells are derived from NK cells but lack the major inhibitory receptors shown by normal NK cells and retain most of the activating receptors. However, NK-92 cells do not attack normal cells and also do not induce unacceptable immune rejection reactions in humans. 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 has been found to exhibit the CD56 bright , CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers. Furthermore, it does not present the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. The growth of NK-92 cells in culture is dependent on the presence of recombinant interleukin 2 (rIL-2), and even a low dose of about 1 IU / mL is sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term growth, nor do the other cytokines tested, including IL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ. NK-92 has high cytotoxicity even at a low effector:target (E:T) ratio of 1:1. Gong, et al., supra. NK-92 cells are deposited with the American Type Culture Collection (ATCC) under the name CRL-2407.

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

[0060] Modified NK-92 cells are known and include, but are not limited to, those described in U.S. Patent Nos. 7,618,817, 8,034,332, and 8,313,943, and U.S. Patent Application Publication No. 2013 / 0040386, which are hereby incorporated by reference in their entirety, such as 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 retain almost all of the activating receptors and cytolytic pathways associated with NK cells, but do not express CD16 on their cell surface. CD16 is an Fc receptor that recognizes and binds to the Fc portion of an antibody and activates NK cells against antibody-dependent cell 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., rituximab and herceptin).

[0062] Studies on endogenous NK cells have shown that IL-2 (1000 IU / mL) is important for NK cell activation during transportation, but it is not necessary to maintain the cells at 37°C and 5% carbon dioxide. Koepsell, et al., Transfusion 53:398-403 (2013). However, mainly due to the difference in the origin of these cells, where NK-92 is a cancer-derived cell line while endogenous NK cells are collected from donors (or patients) and processed for injection into patients, endogenous NK cells are significantly different from NK-92 cells. Endogenous NK cell preparations are heterogeneous cell populations, while NK-92 cells are a homogeneous clonal cell line. NK-92 cells can easily proliferate in culture while maintaining cytotoxicity, while endogenous NK cells cannot. In addition, the endogenous heterogeneous 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 an antibody. Several Fc receptors are known, and they differ by their preferred ligand, affinity, expression, and effects after binding to an antibody.

[0064] (Table 1) Exemplary Fc receptors TIFF2025089426000002.tif255160TIFF2025089426000003.tif88160

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

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

[0067] In some embodiments, the polynucleotide encoding the CD16 polypeptide has at least about 70% polynucleotide sequence identity with a polynucleotide sequence encoding a full-length, naturally-occurring CD16 that includes a signal peptide and has phenylalanine at position 176 (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 polynucleotide sequence encoding a full-length, naturally-occurring CD16 that includes a signal peptide and has valine at position 176 (corresponding to position 158 of the mature protein). In some embodiments, the polynucleotide encoding CD16 has at least 70% identity with SEQ ID NO:5 and includes a codon encoding valine at the position of the polynucleotide encoding position 176 of the full-length CD16 polypeptide that includes a signal peptide. In some embodiments, the polynucleotide encoding CD16 has at least 90% identity with SEQ ID NO:5 and includes a codon encoding valine at position 176 of the full-length CD16. In some embodiments, the polynucleotide encoding CD16 includes SEQ ID NO:5 and 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 to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 70% identity or at least 80% identity to SEQ ID NO:2 and contains valine at position 158 as determined with reference to SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 90% identity to SEQ ID NO:2 and contains valine at position 158 as determined with reference to SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 95% identity to SEQ ID NO:2 and contains valine at position 2 as determined with reference to 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, or 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, an epitope tag peptide, such as FLAG, myc, polyhistidine or V5, etc., is added to the amino-terminal domain of the mature polypeptide, and cell surface detection can be facilitated 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 the present disclosure, although homologous CD16 polynucleotides may be about 150 to about 700, about 750, or about 800 polynucleotides in length.

[0070] The identical polynucleotide sequences include those encoding a polypeptide sequence encoding a variant of CD16. The identical polynucleotide sequences also include naturally occurring allelic variants related to SEQ ID NO:5. Transfection of NK-92 cells with any polynucleotide encoding a polypeptide having the amino acid sequence shown in either SEQ ID NO:1 or SEQ ID NO:2, its naturally occurring variant, or 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 the present disclosure. In some embodiments, the identical polynucleotide sequences encode conservative amino acid substitutions in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, NK-92 cells are transfected with a degenerate identical CD16 polynucleotide sequence that differs from the native polynucleotide sequence but encodes the same polypeptide.

[0071] In other examples, cDNA sequences having polymorphisms that alter the CD16 amino acid sequence are used to modify NK-92 cells, such as allelic variants among individuals presenting genetic polymorphisms in the CD16 gene. In other examples, CD16 genes from other species having polynucleotide sequences different from the sequence of SEQ ID NO:5 are used to modify NK-92 cells.

[0072] In some examples, variant polypeptides are made using methods known in the art such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning, and PCR mutagenesis. Site-specific mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985) or other known techniques can be performed on the 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 that result 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, where one member of a class of amino acids is substituted for another member of the same class, fall within the scope of the disclosed CD16 variants so long as the substitution does not substantially alter the activity of the polypeptide. Conservative substitutions are well known to those of skill 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 bulk of the side chain at the target site, may alter the function or immunological identity of the CD16 polypeptide. Non-conservative substitutions involve exchanging one member of one of these classes for another. Substitutions can be introduced at conservative substitution sites, more preferably at non-conserved sites.

[0075] In some embodiments, the CD16 polypeptide variant is at least 200 amino acids in length 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 in length 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 valine at position 158 as determined with reference to SEQ ID NO:2.

[0076] In some embodiments, the nucleic acid encoding the CD16 polypeptide may encode a CD16 fusion protein. The CD16 fusion polypeptide comprises any portion of CD16 or the entire CD16 fused to a non-CD16 polypeptide. The fusion polypeptide is readily produced 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 coding polynucleotide (e.g., a polynucleotide sequence encoding a signal peptide of a heterologous protein). In some embodiments, a fusion polypeptide may be produced in which a heterologous polypeptide sequence is fused to the C-terminus of CD16 or positioned within CD16. Typically, up to about 30% of the CD16 cytoplasmic domain can be replaced. Such modifications can enhance expression or increase cytotoxicity (e.g., ADCC responsiveness). In other examples, domains from other lymphocyte activation receptors, including but not limited to, chimeric proteins such as Ig-a, Ig-B, CD3-e, CD3-d, DAP-12 and DAP-10, replace a portion of the CD16 cytoplasmic domain.

[0077] Fusion genes can be synthesized by conventional techniques, which include an automated DNA synthesizer and PCR amplification using anchor primers that generate complementary overhangs between two contiguous gene fragments that can then be annealed and re-amplified to generate chimeric gene sequences (Ausubel, 2002). Many vectors are commercially available that facilitate the subcloning of CD16 in-frame into the fusion portion.

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

[0079] (Table 1) Tumor-Specific Antigens and Associated Malignancies TIFF2025089426000004.tif121164TIFF2025089426000005.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 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 to SEQ ID NO:9. In some embodiments, the CD33 CAR polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity to SEQ ID NO:11. Optionally, the CD33 CAR polypeptide has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:11. In some embodiments, the CSPG-4 CAR polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity to 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 to SEQ ID NO:13. In some embodiments, an epitope tag peptide, such as FLAG, myc, polyhistidine, or V5, etc., is added to the amino-terminal domain of the polypeptide, and cell surface detection can be assisted by using anti-epitope tag peptide monoclonal or polyclonal antibodies.

[0081] In some examples, variant polypeptides are made 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 performed on the 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 that result in amino acid substitutions at "non-essential" amino acid residues can be made in 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, where one member of a class of amino acids is substituted for another member of the same class, fall within the scope of the disclosed variants so long as the substitution does not substantially alter the activity of the polypeptide. Conservative substitutions are well known to those of skill in the art. Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, such as the β-sheet or α-helix conformation, (2) the charge, (3) the hydrophobicity, or (4) the bulk of the side chain at the target site, may alter the function or immunological identity of the polypeptide. Non-conservative substitutions involve exchanging one member of one of these classes for another. Substitutions can be introduced at conservative substitution sites, more preferably at non-conserved sites.

[0084] Optionally, the CAR targets an antigen associated with a specific cancer type. Optionally, the cancer is 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's macroglobulinemia, heavy chain disease, and is selected from the group consisting of solid tumors including, but not limited to, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial sarcoma, 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, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0085] The CAR can be engineered, for example, as described in Patent Publication Nos. WO 2014039523; US 20140242701; US 20140274909; US 20130280285; and WO 2014099671, each of which is incorporated herein by reference in its entirety. Optionally, the CAR is a CD19 CAR, a CD33 CAR, or a CSPG-4 CAR.

[0086] Additional Modification - Cytokine The cytotoxicity of NK-92 cells depends on the presence of cytokines (e.g., interleukin-2 (IL-2)). It is quite costly to externally add and use IL-2, which is required to maintain and expand NK-92 cells in commercial-scale culture. Administration of IL-2 to a human subject in an amount sufficient to continue activating NK92 cells may cause harmful 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 a preferred embodiment, the cytokine is IL-2 (SEQ ID NO:6). In a particular embodiment, IL-2 is a variant that targets the endoplasmic reticulum, and the suicide gene is iCas9.

[0088] In one embodiment, IL-2 is expressed with a signal sequence that directs IL-2 to the endoplasmic reticulum. In some embodiments, the polynucleotide encoding IL-2 encodes a polypeptide having the sequence of SEQ ID NO:7. Without being bound by theory, directing IL-2 to the endoplasmic reticulum enables expression of IL-2 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. Continuous 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" enables negative selection of cells. Suicide genes are used as a safety system, and cells expressing the gene can be killed by the introduction of a selection agent. This is desirable when recombinant genes cause mutations leading to uncontrolled cell growth. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella-zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli gpt gene, and Escherichia coli Deo gene (see, for example, Yazawa K, Fisher W E, Brunicardi F C: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9). As used herein, the suicide gene is active in NK-92 cells. Typically, a suicide gene encodes a protein that has no adverse effect on cells but kills cells in the presence of a specific compound. Thus, a suicide gene is typically part of a system.

[0090] In one embodiment, the suicide gene is the thymidine kinase (TK) gene. The TK gene can 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 aspect, 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 aspect, the suicide gene is cytochrome P450, which 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 aspect, 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 the context of adoptive cell therapy.

[0094] In one aspect, the 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 hereby incorporated by reference in its entirety. In one aspect, modified NK-92 cells that have not been engineered to express a suicide gene are irradiated.

[0095] Transgene expression The transgene (e.g., CD19 CAR and CD16) can be engineered into an expression vector by any mechanism known to those of skill in the art. The transgenes can be engineered into the same expression vector or different expression vectors. In a preferred aspect, the transgenes are engineered into the same vector.

[0096] In some aspects, the vector enables integration of the transgene into the genome of the cell. In some aspects, the vector has a positive selection marker. Positive selection markers include any gene that allows cells to grow under conditions that kill cells that do not express that gene. Non-limiting examples include antibiotic resistance, e.g., geneticin (Neo gene 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, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, and poxviral vectors.

[0098] The transgene 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 gun".

[0099] Antibody Optionally, the antibody can be used to target cancerous cells or cells expressing cancer-related markers. Some antibodies are approved for the treatment of cancer alone.

[0100] (Table 2) Examples of FDA-approved therapeutic monoclonal antibodies TIFF2025089426000006.tif141170TIFF2025089426000007.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 an antibody that is bound to a target cell through an Fc receptor such as CD16.

[0102] Thus, in some embodiments, NK-92 cells expressing CD16 and / or CAR are administered to a patient together with an effective amount of at least one monoclonal antibody against a specific cancer-related protein, 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 that binds to cancer cells and also to a cell surface protein present on the surface of NK-92 cells can be used.

[0103] Cancer-specific antibodies bind to specific protein antigens expressed on the surface of cancer cells. NK-92 cells can be modified such that the antibody associates with the NK-92 cell surface. In preferred embodiments, the antibody is specific for cancer. 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 multiple types of human advanced cancers. It is contemplated that antibodies against YKL-40 can be used to inhibit tumor growth, angiogenesis, and / or metastasis. Faibish et al.,(2011) Mol. Cancer Ther. 10(5):742-751.

[0104] The antibody can be administered in combination with the administration of NK-92 cells. An antibody specific for 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 cancer and recovered or were in recovery at the time of sampling. Methods for identifying, screening, and expanding antibodies (e.g., monoclonal antibodies) from these samples are known. For example, a phage display library can be generated 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 the library using a phage display vector. The library can be prepared and screened as described, for example, in Maruyama, et al., which is hereby incorporated by reference in its entirety. Antibodies can be made 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 hereby incorporated by reference in its entirety.

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

[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 cells (etc.) of NK-92, or any range including the endpoints between any two numbers can be administered.

[0109] In other embodiments, the NK-92 cells can be administered to such an individual in terms of the relative number of cells, for example, the individual can be administered from about 1000 cells per kilogram of the individual up to about 10 billion cells, 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 NK-92 cells (etc.), or any range including the endpoints between any two numbers can be administered.

[0110] In other embodiments, the total dose can be calculated by m 2 of the body surface area, and includes about 1×10 2 、1×10 11 、1×10 10 、1×10 9 、1×10 8 、1×10 7 、or any range including the endpoints between any two numbers. The average person is about 1.6 m 2 to about 1.8 m 2 . In a preferred embodiment, about 1 billion to about 3 billion NK-92 cells are administered to the patient. In other embodiments, the amount of NK-92 cells injected per dose can be calculated by m 2 of the body surface area, and includes 1×10 2 、1×10 11 、1×10 10 、1×10 9 、1×10 8 、1×10 7 per 1 m 2 . The average person is 1.6 - 1.8 m

[0111] NK-92 cells, and optionally other anti-cancer agents, can be administered once to a patient having 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 treatment period, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more than that, or once for any range including the endpoints between any two numbers.

[0112] In some embodiments, the NK-92 cells are administered in a composition comprising the 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 from about 1% to about 15% human serum or human serum equivalent. In some embodiments, the medium comprises from about 1% to about 10% human serum or human serum equivalent. In some embodiments, the medium comprises from about 1% to about 5% human serum or human serum equivalent. In a preferred embodiment, the medium comprises about 2.5% human serum or human serum equivalent. In some embodiments, the serum is human AB serum. In some embodiments, a serum substitute acceptable for use in human therapy is used in place of human serum. Such serum substitutes are known in the art or may be developed in the future. Concentrations of human serum above 15% can be used, but concentrations above about 5% are considered too costly. In some embodiments, the NK-92 cells are administered in a composition comprising the NK-92 cells and an isotonic solution that supports cell viability. In some embodiments, the NK-92 cells are administered in a composition reconstituted from a cryopreserved sample.

[0113] Pharmaceutically acceptable compositions can contain various carriers and excipients. Various aqueous carriers, such as buffered physiological saline, can be used. These solutions are sterile and generally free of unwanted substances. Suitable carriers and excipients and 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, i.e., the material is administered to a subject without causing an undesirable biological effect or interacting in a harmful way with other components of the pharmaceutical composition in which it is contained. When administered to a subject, the carrier may be selected to minimize degradation of the active ingredient and minimize harmful 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 agents for buffering and preservation during storage and may contain buffers and carriers for appropriate delivery depending on the route of administration.

[0114] These compositions for use in vivo or in vitro can be sterilized by conventional well-known sterilization techniques. The compositions can contain acceptable auxiliary substances required for an appropriate physiological state, such as pH adjusters and buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of cells and / or other agents in these formulations can vary and are mainly selected based on factors such as the volume of liquid, viscosity, and body weight according to the particular dosage form selected and the needs of the subject.

[0115] In one aspect, NK-92 cells are administered to a patient in combination with one or more other treatments for the cancer being treated. Without being bound by theory, it is thought that the simultaneous treatment of the patient with NK-92 cells and another therapy for cancer may allow the NK-92 cells and the alternative therapy to give such an endogenous immune system the chance to eliminate cancer that has hitherto suppressed its endogenous action. In some aspects, the two or more other treatments for the cancer being treated include, for example, antibodies, radiation, chemotherapy, stem cell transplantation, or hormone therapy.

[0116] In one aspect, the antibody is administered to the patient in combination with NK-92 cells. In one aspect, the NK-92 cells and the antibody can 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 can be administered by any suitable route, such as intravenous or oral administration.

[0117] Without being bound by theory, NK-92 cells expressing a combination of Fc receptor and CAR are thought to be more likely to prevent escape variants from occurring when administered together with a monoclonal antibody, and may also avoid selecting for escape variants. In addition, the patient's own effector cells can participate in ADCC by the monoclonal antibody and target cancer cells. This dual system (both Fc receptor and CAR) may also be tumor-on-target off-tumor, i.e., more selective for cancer cells than non-cancerous cells. There are few tumor-associated antigens that are uniquely expressed on cancer cells, and it is extremely rare for non-cancerous cells to overexpress two types of tumor-associated / specific antigens. For example, lymphocytes usually express both CD19 and CD20, and often when one is upregulated the other is downregulated, and vice versa. NK-92-CD16-CD19 may be effective in treating certain lymphomas in combination with ibritumomab tiuxetan or rituximab.

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

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

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

[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 contain information on how to prepare (dilute and reconstitute in the case of lyophilized proteins) the antibody and the NK-92 cells (e.g., thaw and / or culture). The instructions may further include guidance on dosage and frequency of administration.

[0122] Materials, compositions, and components that can be used in the methods and compositions of the present disclosure, that can be used in combination therewith, that can be used in their preparation, or that are products thereof are disclosed. When 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 recited, but each is understood to be specifically contemplated and described herein. For example, when a method is disclosed and discussed and some modifications that can be made to several molecules including the method are discussed, each and every combination and permutation of the method, as well as possible modifications, are specifically contemplated unless there is a specific indication to the contrary. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure including, but not limited to, steps in methods of using the disclosed compositions. Thus, if there are various additional steps that can be performed, each of these additional steps can be performed by any particular method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is to be understood as being specifically contemplated and disclosed.

Examples

[0123] The following examples are for illustrative purposes only and are not to be construed as limiting. There are various alternative techniques and procedures available to those skilled in the art, and these are equally capable of successfully performing the following examples.

[0124] Example 1: Extension of survival period after treatment with NK-92-Fc-CAR CD19-positive leukemia cells derived from T-cell acute lymphoblastic leukemia (ALL) patients, acute myeloid leukemia (AML) patients, and pre-B-ALL patients are engrafted and expanded in NSG mice by S.C. inoculation. Leukemia cells (first passage) recovered from the leukemia nodules of the mice are used. NSG mice in each group are inoculated I.P. with 5×10 6 leukemia cells from the first passage in 0.2 mL PBS. All human leukemias grow actively in NSG mice. After 24 hours, treatment is with either (a) rituximab, (b) NK-92-CD16-CD19 cells, or (c) rituximab and NK-92-CD16-CD19 cells. Mice are treated weekly for 4 months. Treatment with either NK-92-CD16-CD19 cells or a combination of rituximab and NK-92-CD16-CD19 cells is thought to significantly extend lifespan and prolong the survival period of the 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 Fc receptors and CARs, in vitro cytotoxicity assays were performed on NK-92 cells electroporated with mRNA encoding CD19-CAR 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 FIGS. 1A, 1B, and 1C. FIG. 1A shows the killing of target cell lines by un-electroporated parental NK-92 cells. FIG. 1B shows the killing of target cell lines by parental NK-92 cells expressing CD19-CAR. FIG. 1C shows the killing 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, while NK-resistant CD19-negative SR-91 cells remain resistant. The killing of K562 is not affected by the expression of CD19-CAR.

[0126] Example 3. Electroporation of chimeric antigen receptor (CAR) mRNA into human NK cell lines results in high transfection efficiency and target-specific cytotoxicity Data on mRNA transfection, expression, and cytotoxicity of three different CARs based on first-generation CAR constructs: CD19, CD33, and CSPG-4 are provided. Target cell lines for mRNA transfection were aNK (parental NK-92 cells) and haNK (high-affinity FcR-expressing NK-92). The scFv sequences were custom-ordered by GeneArt (codon-optimized), and mRNA was transfected using MaxCyte GT to generate taNK (target-activated NK cells). Expression was determined by immunofluorescence using the corresponding antibodies, and cytotoxicity was measured using a standard flow cytometry assay.

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

[0128] Both aNK and haNK can be reliably and consistently transfected with mRNAs of various CAR constructs that maintain high viability of transfected NK cells, excellent expression of CAR, and target cell-specific cytotoxicity for at least 48 hours. This technology can be easily scaled up for the clinical-grade production of CAR-expressing NK cell lines. The fact that haNK can be effectively transfected (to become t-haNK) opens up the possibility of non-cross-reactive dual-receptor targeting of malignant lesions (i.e., CD19 CAR with CD20 antibody).

[0129] The examples and aspects described herein are for illustrative purposes only, and various modifications or changes are suggested to those skilled in the art from those perspectives and should be within the spirit and scope of this application and within the scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0130] Exemplary sequences SEQ ID NO:1 Amino acid sequence (mature form) of low-affinity immunoglobulin gamma Fc region receptor III-A. Phenylalanine at position 158 is underlined. TIFF2025089426000008.tif77158 SEQ ID NO:2 Amino acid sequence (mature form) of high-affinity variant F158V immunoglobulin gamma Fc region receptor III-A. Valine at position 158 is underlined. TIFF2025089426000009.tif77158 SEQ ID NO:3 Amino acid sequence (precursor form) of low-affinity immunoglobulin gamma Fc region receptor III-A. Position 176 for the precursor form corresponds to position 158 for the mature form. Phe at position 176 is underlined. TIFF2025089426000010.tif85159 SEQ ID NO:4 Amino acid sequence of the high-affinity variant immunoglobulin gamma Fc region receptor III-A (precursor form). Position 176 for the precursor form corresponds to position 158 for the mature form. Val at position 176 is underlined. TIFF2025089426000011.tif85159 SEQ ID NO:5 Polynucleotide encoding low-affinity immunoglobulin gamma Fc region receptor III-A (precursor) (encoding phenylalanine at position 158) TIFF2025089426000012.tif70154 SEQ ID NO:6 Wild-type IL-2 TIFF2025089426000013.tif55160 SEQ ID NO:7 IL-2-ER TIFF2025089426000014.tif56160 SEQ ID NO:8 CD19-CAR DNA sequence TIFF2025089426000015.tif215153 SEQ ID NO:9 CD19-CAR amino acid sequence TIFF2025089426000016.tif70150 SEQ ID NO:10 CD33-CAR DNA sequence TIFF2025089426000017.tif208154 SEQ ID NO:11 CD33-CAR amino acid sequence TIFF2025089426000018.tif70149 SEQ ID NO:12 CSPG4-CAR DNA sequence TIFF2025089426000019.tif222153 SEQ ID NO:13 CSPG4-CAR amino acid sequence TIFF2025089426000020.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 acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 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 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 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala 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 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala 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 ggcggatcct 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 Lys 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 Methionine Alanine Glutamic acid Alanine Tyrosine Serine Glutamic acid Isoleucine Glycine Methionine Lysine Glycine Glutamic acid Arginine Arginine Arginine 420 425 430 Glycine Lysine Glycine Histidine Aspartic acid Glycine Leucine Tyrosine Glutamine Glycine Leucine Serine Threonine Alanine Threonine Lysine 435 440 445 Aspartic acid Threonine Tyrosine Aspartic acid Alanine Leucine Histidine Methionine Glutamine Alanine Leucine Proline Proline Arginine 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 acagctatcc tctgacgttc ggtggcggca ccaagctgga aatcaaacgg gctgccgcag 420 aaggtggagg cggttcaggt ggcggaggtt ccggcggagg tggctctggc ggtggcggat 480 aaggtggagg cggttcaggt ggcggaggtt ccggcggagg tggctctggc ggtggcggat 480 cggccatggc ccaggtgaag ctgcagcagt caggaggggg cttggtgcaa cctggaggat 540 cggccatggc ccaggtgaag ctgcagcagt caggaggggg cttggtgcaa cctggaggat 540 ccatgaaact ctcctgtgtt gtctctggat tcactttcag taattactgg atgaactggg 600 ccatgaaact ctcctgtgtt gtctctggat tcactttcag taattactgg atgaactggg 600 tccgccagtc tccagagaag gggcttgagt ggattgcaga aattagattg aaatccaata 660 tccgccagtc tccagagaag gggcttgagt ggattgcaga aattagattg aaatccaata 660 attttggaag atattatgcg gagtctgtga aagggaggtt caccatctca agagatgatt 720 attttggaag atattatgcg gagtctgtga aagggaggtt caccatctca agagatgatt 720 ccaaaagtag tgcctacctg caaatgatca acctaagagc tgaagatact ggcatttatt 780 ccaaaagtag tgcctacctg caaatgatca acctaagagc tgaagatact ggcatttatt 780 actgtaccag ttatggtaac tacgttgggc actattttga ccactggggc caagggacca 840 actgtaccag ttatggtaac tacgttgggc actattttga ccactggggc caagggacca 840 cggtcaccgt atcgagtgcc gcggttctag agctcttgag caactccatc atgtacttca 900 cggtcaccgt atcgagtgcc gcggttctag agctcttgag caactccatc atgtacttca 900 gccacttcgt gccggtcttc ctgccagcga agcccaccac gacgccagcg ccgcgaccac 960 gccacttcgt gccggtcttc ctgccagcga agcccaccac gacgccagcg ccgcgaccac 960 caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc 1020 caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc 1020 cagcggcggg gggcgcagtg cacacgaggg ggctggacct gctggatccc aaactctgct 1080 cagcggcggg gggcgcagtg cacacgaggg ggctggacct gctggatccc aaactctgct 1080 acctgctgga tggaatcctc ttcatctatg gtgtcattct cactgccttg ttcctgagag 1140 tgaagttcag caggagcgca gacgcccccg 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. The NK-92 cell has been modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR) such that the NK-92 cell presents the at least one Fc receptor and the at least one CAR on the cell surface of the NK-92 cell.

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

3. The cell of claim 1, wherein the Fc receptor comprises a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 and comprises a valine at position 158.

4. The cell of claim 1, wherein the Fc receptor comprises the amino acid sequence of SEQ ID NO:

2.

5. The cell of any one of claims 1 to 4, wherein the CAR has at least 90% identity to SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:

13.

6. 5. The cell of any one of claims 1 to 4, wherein 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.

7. The cell of any one of claims 1 to 6, further expressing a cytokine.

8. The cell of claim 7, wherein the cytokine is interleukin-2 or a variant thereof.

9. The cell of claim 7 or 8, wherein the cytokine is targeted to the endoplasmic reticulum.

10. The cell of any one of claims 1 to 9, wherein the Fc receptor and the CAR are encoded on different vectors.

11. 1. An NK-92 cell line, wherein cells of the NK-92 cell line have been modified to express at least one Fc receptor and at least one chimeric antigen receptor (CAR), such that said Fc receptor and said CAR are presented on the cell surface of the NK-92 cells.

12. 12. The NK-92 cell line of claim 11, wherein said Fc receptor is FcγRIII-A (CD16) or a CD16 polypeptide having a valine at position 158 of the mature form of said CD16.

13. The method of claim 11, wherein the Fc receptor comprises a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 and comprises a valine at position 158.

14. The method of claim 11, wherein the Fc receptor comprises the amino acid sequence of SEQ ID NO:

2.

15. 15. The NK-92 cell line of any one of claims 11 to 14, wherein the CAR has at least 90% identity to SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:

13.

16. 15. The NK-92 cell line of any one of claims 11 to 14, 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.

17. 17. The NK-92 cell line of any one of claims 11 to 16, further expressing a cytokine.

18. 18. The NK-92 cell line of claim 17, wherein said cytokine is interleukin-2 or a variant thereof.

19. 19. The NK-92 cell line of Claim 17 or 18, wherein said cytokine is targeted to the endoplasmic reticulum.

20. The cell of any one of claims 11 to 19, wherein the Fc receptor and the CAR are encoded on different vectors.

21. 21. The cell line of any one of claims 11 to 20, wherein cells of said cell line undergo less than 10 population doublings.

22. The cell line of any one of claims 11 to 20, wherein the cells are cultured in a medium containing less than 10 U / ml of IL-2.

23. 23. A composition comprising an amount of a cell from any one of claims 1 to 10 or claims 11 to 22.

24. 24. The composition of claim 23, further comprising at least one monoclonal antibody.

25. 25. The composition of claim 24, wherein the at least one monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody.

26. 25. The composition of claim 24, wherein the monoclonal antibody is selected from the group consisting of alemtuzumab, rituxumab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adotranstuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab.

27. 23. A method of treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of a cell according to any one of claims 1-10 or any one of the cell lines according to any one of claims 11-22, thereby treating said cancer.

28. 28. The method of claim 27, wherein the cells are administered to the patient by a route selected from the group consisting of intravenous, intraperitoneal, and subcutaneous.

29. 28. The method of claim 27, further comprising administering to the patient an effective amount of at least one monoclonal antibody.

30. 30. The method of claim 29, wherein the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody.

31. 30. The method of claim 29, wherein the monoclonal antibody is selected from the group consisting of alemtuzumab, rituxumab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adotrastuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab.

32. The method of any one of claims 29 to 31, wherein the monoclonal antibody and the cells are administered simultaneously.

33. The method of any one of claims 29 to 31, wherein the monoclonal antibody and the cells are mixed prior to administration to the patient.

34. The method of any one of claims 29 to 31, wherein the monoclonal antibody and the cells are administered sequentially.

35. 35. The method of any one of claims 27 to 34, wherein the cancer is selected from the group consisting of leukemia, lymphoma, polycythemia vera, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, sarcoma, and carcinoma.

36. The patient's body surface area of ​​1 m 2 Approximately 1 x 10 8 From 1 x 10 pieces 11 The method of any one of claims 27 to 35, wherein said cells are administered to said patient.

37. 1. A kit for treating cancer comprising: (a) NK-92 cells, the cells having been modified to express at least one Fc receptor on the cell surface and at least one chimeric antigen receptor (CAR) on the cell surface; and (b) instructions for use.

38. 38. The kit of claim 37, further comprising (c) at least one monoclonal antibody.

39. 39. The kit of claim 38, wherein the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody.

40. 39. The kit of claim 38, wherein the monoclonal antibody is selected from the group consisting of alemtuzumab, rituxumab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adotrastuzumab, blinatumomab, avelumab, daratumumab, and elotuzumab.

41. A modified NK-92 cell 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 cell has at least two ligand binding elements on its cell surface.

42. 1. A method for enhancing the binding affinity of an NK-92 cell to a cancer cell comprising using a multilocus binding element on a cell surface of an engineered NK-92 cell, the engineered NK-92 cell comprising a multilocus binding element for one or more Fc receptors and one or more CARs, such that the NK-92 cell has at least two ligand binding elements on the cell surface of the NK-92 cell.

Images