Cellular immunotherapy for the treatment of cancer

Abstract

Embodiments of the present disclosure encompass compositions comprising immune effector cells, such as natural killer (NK) cells, wherein the cells comprise one or more exogenously provided interleukins (ILs), and optionally, the cells comprise one or more engineered receptors. In certain embodiments, the ILs are not IL-15, but are IL-12, IL-21, or both. The NK cells can be used to treat any type of cancer, including at least glioblastoma.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 001,275, filed March 28, 2020, which is incorporated herein by reference in its entirety.

[0002] (Technical field) Embodiments of the present disclosure relate to at least the fields of cell biology, molecular biology, immunology, and medicine. [Background technology]

[0003] Glioblastoma (GBM) is an aggressive malignant tumor with a poor prognosis. There is no standard treatment after recurrence, and survival is less than 9 months. Recently, results from three first-in-human chimeric antigen receptor (CAR) T-cell trials targeting IL13Rα2 (Brown et al., 2016), Her2 / CMV (Ahmed et al., 2010), and EGFRvIII (O'Rourke et al., 2017) were reported, with disappointing clinical outcomes.

[0004] Currently, there is a critical need to advance cell therapies for GBM, as well as other solid tumors, particularly with the significant responses demonstrated in leukemia and lymphoma. The present disclosure provides a solution to this long-felt need to advance cell therapies for cancer, including at least glioblastoma. Summary of the Invention

[0005] Embodiments of the present disclosure include methods and compositions related to cell therapy for medical conditions. Cell therapy involves immune effector cells or other cells for administration to an individual in need thereof. In certain embodiments, the cells contain one or more exogenously provided interleukins (ILs) and optionally one or more other heterologous gene products, e.g., one or more engineered receptors, thereby enhancing activity compared to other cell therapies. The cells may be exposed to one or more cytokines exogenously (e.g., during culture) and / or transfected to express heterologous cytokines (as opposed to endogenous cytokines expressed from the cell's genome) from one or more vectors. The present disclosure encompasses immunotherapy with ex vivo expanded and activated natural killer (NK) cells in combination with exogenous cytokines (e.g., IL-2, IL-12, IL-21, IL-18, IL-15, IL-7) or NK cells secreting or surface-expressing membrane-bound or tethered cytokines (e.g., IL-2, IL-12, IL-21, IL-18, IL-15, IL-7) for the treatment of glioblastoma (GBM) and other cancers. Prior to delivery, the cells may be exposed to an effective amount of IL-2, IL-12, IL-21, IL-18, IL-15, and / or IL-7 in culture under appropriate conditions. In certain embodiments, the cells comprise one or more exogenously provided interleukins other than IL-15, while in alternative embodiments, the cells comprise exogenously provided IL-15.

[0006] In certain embodiments, the present disclosure encompasses methods of treating glioblastoma using adoptive cell therapy involving NK cells. In certain aspects, NK cells are particularly effective against glioblastoma stem cells, and the present disclosure provides methods and compositions for enhancing NK cells against any type of glioblastoma cell, including glioblastoma cells. The present disclosure encompasses methods of killing glioblastoma stem cells in an individual with glioblastoma, comprising administering to the individual an effective amount of NK cells engineered to express one or more exogenously provided ILs and / or cultured in the presence of one or more ILs. In some embodiments, glioblastoma stem cells are killed by NK cells expressing one or more engineered receptors, which may or may not target one or more antigens expressed on the glioblastoma stem cells. In some cases, glioblastoma stem cells are killed by NK cells engineered to express one or more engineered receptors, engineered to express one or more exogenous cytokines, and / or cultured in the presence of one or more ILs.

[0007] Embodiments of the present disclosure include compositions containing any type of immune effector cell, including natural killer (NK) cells, wherein the cells comprise one or more exogenously provided ILs, optionally where the IL is not IL-15, and the cells comprise one or more engineered receptors. In certain embodiments, the IL is selected from the group consisting of IL-12, IL-21, IL-2, IL-15, IL-18, IL-7, and combinations thereof. In certain cases, the IL is IL-12, IL-21, or both. In any embodiment of the present disclosure, the IL can be secreted, tethered, or membrane-bound in the cell. As used herein, "exogenously provided" may be further defined as expressed from a vector in the cell and / or the cell being externally exposed to one or more ILs.

[0008] In certain embodiments, the engineered receptor utilized in the cells of the present disclosure is an engineered antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR). The antigen may be a cancer antigen, including a solid tumor antigen, or an antigen associated with a pathogen. Specific examples of when the antigen is a cancer antigen include 5T4, 8H9, α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal Ac The engineered receptors include antibodies selected from the group consisting of hR, FR, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11R, IL-13R2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEMs, HMW-MAA, VEGFR2, and combinations thereof. In some embodiments, the engineered receptor is a cytokine receptor, a chemokine receptor, or a homing receptor, or the cells may have a combination thereof.

[0009] In certain embodiments, the cells of the present disclosure are cells, including NK cells, that contain a suicide gene. In some cases, the cells have reduced or inhibited expression of one or more endogenous genes selected from the group consisting of TDAG8, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and combinations thereof.

[0010] Embodiments of the present disclosure include any population of cells encompassed herein.

[0011] In one embodiment, there is a method of treating cancer in an individual, comprising administering a therapeutically effective amount of any of the compositions encompassed herein. In some cases, cancer cells in the individual have increased expression of NK ligands, such as MICA / B, ULBP1, ULBP2 / 5, ULBP3, B7-H6, CD112, CD155, HLA-ABC, HLA-DR, or HLA-3, or a combination thereof. Engineered NK cells may express one or more engineered antigen receptors that target one or more of these NK ligands or that may target other targets. The composition may be provided to the individual intracranially, by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, topically, by perfusion, within the tumor microenvironment, or a combination thereof.

[0012] For cancer treatment, cancer can be solid tumor or not.Cancer can be lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testicle, endometrium, prostate, rectum, anus, cervical cancer, or blood cancer.In certain cases, cancer is glioblastoma.

[0013] In embodiments of the therapeutic methods of the present disclosure, the individual may be a mammal, such as a human, dog, cat, horse, cow, sheep, pig, or rodent. The individual may be administered one or more additional cancer therapies, including surgery, radiation, chemotherapy, hormone therapy, immunotherapy, or a combination thereof. In some embodiments, any method of the present disclosure further comprises diagnosing cancer in the individual. In some embodiments, any method of the present disclosure further comprises generating cells. Any cells utilized herein may be autologous or allogenic with respect to the individual.

[0014] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features believed characteristic of the designs disclosed herein, both as to organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in connection with the accompanying drawings. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. [Brief explanation of the drawings]

[0015] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0016] [Figure 1]Figures 1A and 1B. Glioblastoma stem cells (GSCs), but not astrocytes, are highly susceptible to NK-mediated lysis. (Figure 1A) NK cells were cocultured with Cr51-labeled GSCs at different ratios for 4 hours (n=4), and Cr51 release was measured. (Figure 1B) Expression of NK ligands on GSCs and astrocytes.

[0017] [Figure 2] Schematic diagram of an example of a retroviral vector incorporating a cytokine gene: hIL-12p40linkerp35 is human IL-12 in which the p35 and p40 subunits are artificially linked together with a linker.

[0018] [Figure 3] Figures 3A and 3B. Spheroid culture of patient-derived glioblastoma stem cell lines (GSCs) as a 3D tumor culture model (Figure 3A) and the cytotoxicity of cytokine-transduced CB-NK cells against GSC targets (Figure 3B). IL-12, IL-21, or IL-21-transduced NK cells (red line (top), green line (third from the top), and blue line (second from the top)) exert superior cytotoxicity against GSCs compared to untransduced CB-NK cells (black line (bottom)), as demonstrated by Incucyte® live imaging. Apoptotic cells are measured by caspase 3 / 7 green signal.

[0019] [Figure 4] Figures 4A-4C. Comparison of non-transduced (NT) versus IL-12 and IL-21 transduced NK cells in a patient-derived xenograft (PDX) model of FFluc-transduced GSCs. A single injection of IL-12 or IL-21 transduced NK cells at a dose of 1 x 10 cells eradicated the tumor as shown by bioluminescence imaging (Figures 4A-4B) and resulted in long-term cure of the animals (Figure 4C). DETAILED DESCRIPTION OF THE INVENTION

[0020] [I. Definition example] In accordance with long-standing patent law convention, the terms "a" and "an," when used herein in conjunction with terms including "one or more," including the claims, mean that some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0021] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to refer to the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of," and thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory. "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or function specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are not optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0022] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0023] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination with or exclusive of each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0024] Throughout this application, the term "about" is used according to its plain and ordinary meaning within the field of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0025] As used herein, the term "engineered" refers to an entity produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered entity is synthetic and includes elements that do not occur in nature or are not constructed in the manner utilized in this disclosure.

[0026] As used herein, the term "exogenous" refers to a polynucleotide (e.g., encoding a gene product or a portion of a gene product) that is not endogenously present in a mammalian cell, such as an immune cell, or that is produced synthetically outside the mammalian cell, such as by recombinant techniques. In certain cases, a particular gene product can be provided exogenously to a cell, which may or may not express the corresponding endogenous gene product in the cell.

[0027] As used herein, "prevent" and similar terms such as "prevented," "preventing," etc., refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition, for example, cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0028] As used herein, the terms "subject" and "individual" are used interchangeably and generally refer to an individual in need of treatment for a medical condition. In certain cases, the individual has cancer or is suspected of having cancer. A subject can be any living organism or animal subject that is the subject of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject can be a patient, e.g., a patient who has or is suspected of having a disease (which may be referred to as a medical condition), such as a benign or malignant neoplasm or cancer. A subject may be undergoing or have undergone treatment. A subject may be asymptomatic. A subject may be a healthy individual but desires cancer prevention. A "subject" or "individual" as used herein may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the Internet. An individual may include humans or non-human animals of any age, and thus includes both adults and adolescents (i.e., children) and infants, including individuals in utero. Thus, the term does not imply the need for medical treatment, and individuals may voluntarily or involuntarily participate in experiments, whether clinical or supporting basic scientific research. In alternative cases, the subject or individual requires pathogen treatment.

[0029] As used herein, "treatment" or "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment may optionally include either a reduction or amelioration of one or more symptoms of the disease or condition, or a delay in the onset or progression of the disease or condition. Treatment does not necessarily indicate a complete eradication or cure of the disease or condition or its associated symptoms. Treatment may include reducing the severity of one or more symptoms of a medical condition.

[0030] Embodiments of the present disclosure utilize immune effector cells, such as NK cells, for immunotherapy of any type of cancer, including glioblastoma. In certain embodiments, the immune effector cells are NK cells. Unlike T and B lymphocytes, NK cells do not possess rearranged V(D)J receptors and are not limited by major histocompatibility complex (MHC)-bound antigen presentation, making them suitable as therapeutic effectors against highly heterogeneous tumors, such as GBM. Instead, their effector function is determined by the integration of signals received through germline-encoded receptors, which can recognize multiple ligands on cancer targets without the need for specific antigen specificity or costimulation.

[0031] NK cells comprise one of the most abundant lymphoid subsets infiltrating glioblastoma (GBM), supporting their role in immune surveillance of this disease. Furthermore, GBM stem cells (GSCs), but not normal astrocytes, express many of the ligands recognized by NK activating receptors, such as MHC chain-related antigen (MICA / B) and NKG2D (recognized by DNAM) and UL16-binding protein (ULBP) (recognized by CD155). GSCs are shown herein to be highly susceptible to lysis by allogeneic healthy NK cells in vitro (Figure 1A). Thus, a major advantage of NK cell immunotherapy over CAR T cells for GBM lies in their inherent ability to target multiple antigens on GSCs via germline-encoded receptors, without the need to target specific antigens using CARs. This property of NK cells may overcome challenges associated with antigen escape and tumor heterogeneity observed with CAR T cell therapy, including GBM. The present disclosure allows for further improvement of NK cell activity against GBM by exposing the NK cells to either one or more exogenous cytokines, such as IL-2, IL-12, IL-21, IL-7, or IL-18, and / or by engineering them to express one or more cytokine genes to aid in their effector function, persistence, and trafficking. NK cells may also be further modified to specifically target glioblastoma cells, including by utilizing engineered antigen receptors that target antigens on glioblastoma cells.

[0032] Certain embodiments of the present disclosure encompass allogeneic NK cells in combination with one or more exogenous cytokines and / or NK cells genetically engineered to secrete or express one or more cytokines on their surface. Such cells can be utilized as an off-the-shelf treatment, reducing costs and expanding this therapy to many patients.

[0033] [II. Cytokines] Embodiments of the present disclosure include immune effector cells that have been exposed exogenously (including, for example, in culture) to one or more cytokines and / or that have been transfected with vectors that express one or more cytokines. The cytokine can be of any type, although in certain embodiments the cytokine is IL-2, IL-12, IL-15, IL-21, IL-7, or IL-18. However, in certain embodiments, the cytokine is not IL-15.

[0034] In certain embodiments, one or more exogenously provided cytokines expressed in the cells from the vectors are membrane-bound. The cytokines can be heterologously linked to any type of membrane-bound molecule, e.g., the transmembrane domain of B7-1, CD40, CD28, CD8, the GMCSF receptor, IgG1, or IgG4, and thus any cytokine can be membrane-bound and expressed on the surface of the NK cells.

[0035] In some cases, one or more cytokines are present on the same vector molecule as another gene product, such as an engineered receptor, while in other cases, they are on separate molecules. In certain embodiments, one or more cytokines are co-expressed from the same vector as one or more engineered receptors. One or more cytokines may be produced as separate polypeptides from the antigen-specific receptor. In certain cases, cytokines induce NK cell development and cell proliferation, promote eradication of established tumors by relieving functional suppression of tumor-resident cells, and / or inhibit activation-induced cell death. Other cytokines are also contemplated, including, but not limited to, chemokines and other molecules that contribute to the activation and proliferation of cells used in human applications.

[0036] In certain embodiments, NK cells express one or more exogenously provided cytokines. Cytokines can be exogenously provided to cells to be expressed from an expression vector in feeder cells or added directly to NK cell cultures. In other cases, endogenous cytokines in cells are upregulated upon manipulation of the regulation of endogenous cytokine expression, such as genetic modification of the cytokine's promoter site. When a cytokine is provided to cells on an expression construct, the cytokine can be encoded from the same vector that expresses another gene product, such as a suicide gene. The cytokine can be expressed as a polypeptide molecule separate from the suicide gene and as a polypeptide separate from the cell's engineered receptor. In some embodiments, the present disclosure relates to the co-use of a cytokine other than IL-15 with a CAR and / or TCR vector.

[0037] [III. Immune Effector Cells] The present disclosure encompasses immune effector cells of any type having one or more exogenously provided interleukins, where the interleukin is not IL-15, and optionally, the cells include one or more engineered receptors and / or other heterologous gene products. In certain embodiments, the exogenously provided ILs to the cells are the direct or indirect result of deliberate human manipulation of the cells. This is true whether the cells are exogenously exposed to one or more cytokines (e.g., in culture) and / or whether the cells are transfected to express one or more ILs from a vector (which may or may not be integrated into the cellular genome). Manipulation of immune effector cells in such a manner can be by any mechanism. Immune effector cells may be NK cells, T cells, T regulatory cells, iNKT cells, B cells, MSCs, etc.

[0038] When immune effector cells have one or more exogenously provided interleukins, the cells may be externally exposed to one or more cytokines by culturing them in a medium containing one or more cytokines. The concentration range of the cytokine in the medium may be, for example, 0.1 ng to 1000 ng, 0.1 ng to 750 ng, 0.1 ng to 500 ng, 0.1 to 250 ng, 0.1 to 100 ng, 0.1 to 75 ng, 0.1 to 50 ng, 0.1 to 25 ng, or 0.1 to 10 ng. The concentration range of cytokines in the medium can be, for example, 1 unit to 5000 units, 1 unit to 4000 units, 1 unit to 3000 units, 1 unit to 2000 units, 1 unit to 1000 units, 1 unit to 750 units, 1 unit to 500 units, 1 unit to 250 units, 1 unit to 100 units, 1 unit to 75 units, 1 unit to 50 units, 1 unit to 25 units, etc. In some cases, the cytokine is present in the medium throughout the time the cells are cultured, while in other cases, the cytokine is added to the culture at a later time point in the culture. The cytokine can be present in the culture medium at the first, second, third, fourth, or subsequent passage, or a combination thereof.

[0039] The present disclosure encompasses any type of immune effector cell, including conventional T cells, γδ T cells, NK cells, NK T cells, invariant NK T cells, regulatory T cells, macrophages, B cells, dendritic cells, tumor-infiltrating lymphocytes, or mixtures thereof. The cells can be allogeneic, autologous, or xenogeneic to an individual, including an individual in need of the cells, such as an individual with cancer.

[0040] In certain embodiments, immune effector cells are artificially modified to express or otherwise produce one or more cytokines that are not endogenous to the cells, such as recombinant cytokines. Immune effector cells can be further modified, such as by expressing additional heterologous proteins, such as engineered receptors, suicide genes, or combinations thereof. Cells can also be modified to reduce or silence expression of one or more endogenous genes.

[0041] When immune effector cells are modified in more than one way, the order in which the immune effector cells are modified can be any type. For example, immune effector cells that express exogenously provided cytokines (and / or have been externally exposed to one or more cytokines, such as in culture) can be transfected with one or more engineered receptors. In other cases, immune effector cells that express one or more engineered receptors can be transfected with exogenously provided cytokines and / or exposed to one or more cytokines in culture.

[0042] In certain embodiments, immune effector cells containing one or more exogenously provided ILs other than IL-15 are the same cells modified to express an engineered receptor, such as an antigen receptor. Any immune effector cell encompassed by the present disclosure expresses an antigen receptor, which may be of any type, including a receptor for an antigen that is a cancer antigen, which may also be a solid tumor antigen. In certain embodiments, the receptor is, for example, a chimeric antigen receptor or a T cell receptor. Immune effector cells may be specifically engineered to contain one or more exogenously provided ILs, or they may be specifically engineered to express an antigen receptor that targets an antigen on an individual's cancer cells. That is, the cells may be engineered to contain one or more antigen receptors that target an antigen known to be present on an individual's cancer cells.

[0043] In certain embodiments, the cells of the present disclosure are produced for use as pre-made cells. For example, cells containing one or more exogenously provided ILs may be present, for example, in a repository, and they may be obtained from the repository and engineered to have additional modifications other than expressing the exogenously provided cytokines. In other cases, cells with modifications other than expressing the exogenously provided cytokines are obtained from the repository and engineered to express one or more exogenously provided cytokines. Following such modifications to the cells after obtaining them from the repository, the cells may be stored, or an effective amount of the cells may be provided to an individual in need thereof.

[0044] Any immune effector cells can be obtained from any form of repository, such as cryogenic storage, and further modified. Cells may be stored with an expression construct expressing one or more cytokines, and then obtained and modified to express one or more specific engineered antigen receptors of interest, such as receptors engineered to target antigens tailored to a specific cancer in an individual in need. Cells may be stored with an expression construct expressing one or more specific engineered antigen receptors of interest, such as receptors targeting antigens for a specific cancer, and then modified to express one or more exogenous cytokines, as needed. Before or after storage, the cells can be modified to contain a suicide gene, optionally expressed from the same vector as the respective cytokine or engineered antigen receptor.

[0045] In certain embodiments, immune effector cells contain one or more exogenously provided ILs and also express one or more engineered antigen-targeting receptors and / or express at least one suicide gene. For cells containing one or more exogenously provided ILs, in some cases, different vectors encode the antigen-targeting receptors, whereas the suicide gene and / or exogenous cytokines. In other cases, they (or a subset) are on the same vector. Immune effector cells, including NK cells, can be derived from any suitable source, such as umbilical cord blood, peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), bone marrow, or a mixture thereof. NK cells can be derived from cell lines, such as, but not limited to, NK-92 cells. NK cells can be cord blood mononuclear cells, e.g., CD56+ NK cells.

[0046] In some cases, immune effector cells (including NK cells) containing one or more exogenously provided ILs are expanded in the presence of an effective amount of universal antigen-presenting cells (UAPCs) in any suitable ratio, e.g., 10:1 to 1:10; 9:1 to 1:9; 8:1 to 1:8; 7:1 to 1:7; 6:1 to 1:6; 5:1 to 1:5; 4:1 to 1:4; 3:1 to 1:3; 2:1 to 1:2; or 1:1 ratio, including, for example, a 1:2 ratio, may be co-cultured with UAPCs. In some cases, the NK cells may have proliferated in the presence of IL-2, for example, in the presence of IL-2 at a concentration of 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 500, 200 to 400, 200 to 300, 300 to 500, 300 to 400, or 400 to 500 U / mL.

[0047] Following genetic modification with any vector, immune effector cells containing one or more exogenously provided ILs may be immediately delivered to an individual or stored (or a portion of the cells may be delivered to an individual and the remainder of the cells may be stored). In certain embodiments, after genetic modification, the cells may be expanded ex vivo as a bulk population for days, weeks, or months within about 1, 2, 3, 4, 5, or more days after gene transfer into the cells. In further embodiments, transfectants are cloned, and clones that indeed contain a single integrated or episomally maintained expression cassette or plasmid are expanded ex vivo. Clones selected for expansion exhibit expression of one or more exogenously provided cytokines. Recombinant immune cells may be expanded by stimulation with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, etc.). Recombinant immune cells may be expanded by stimulation with artificial antigen-presenting cells. In further embodiments, any genetically modified cells may be cryopreserved.

[0048] Embodiments of the present disclosure encompass immune effector cells comprising one or more engineered receptors, including one or more exogenously provided ILs and one or more antigen receptors. The one or more engineered antigen receptors are generated by the hand of man, e.g., using recombinant technology, and are not native to the immune effector cell. While the engineered receptors can be of any type, in certain embodiments, the receptors are chimeric antigen receptors, T cell receptors, homing receptors, clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9-mediated genetic alterations, decoy receptors, cytokine receptors, chimeric cytokine receptors, combinations thereof, and the like.

[0049] Embodiments of the present disclosure encompass cells containing one or more exogenously provided ILs and one or more suicide genes. Immune effector cells may contain one or more exogenously provided ILs and may contain recombinant nucleic acids encoding any type of suicide gene. Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see PCT / US2019 / 062009, incorporated herein by reference in its entirety), which can be affected by delivery of antibodies that bind to the TNF-α mutants. Examples of suicide gene / prodrug combinations that can be used include herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. One can also use the so-called suicide gene, Escherichia coli purine nucleoside phosphorylase, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine. Other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0050] The cells may be obtained directly from the individual or may be obtained from a depository or other repository. Therapeutic cells may be autologous or allogeneic with respect to the individual to whom they are provided therapeutically.

[0051] The cells may be from an individual in need of treatment for a medical condition, and after being engineered (e.g., using standard techniques for transduction and expansion for adoptive cell therapy) to contain one or more exogenously provided ILs, optional suicide genes, optional cytokines, and optional receptors, they may be returned to the individual from whom they were originally sourced. In some cases, the cells are stored for later use for the individual or another individual.

[0052] The immune cells may be included in a population of cells, which may have a majority that includes one or more exogenously provided ILs and / or one or more suicide genes and / or one or more cytokines. The cell population may include 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the immune cells include one or more exogenously provided ILs and / or one or more suicide genes, and / or one or more engineered receptors; each of these genes may or may not be produced as a separate polypeptide.

[0053] Immune cells can be produced to contain one or more exogenously provided ILs and / or one or more suicide genes and / or one or more cytokines, with the aim of being modular with respect to a particular purpose. For example, cells can be produced, including for commercial distribution, containing one or more exogenously provided ILs and / or one or more suicide genes (or distributed with nucleic acids encoding suicide genes for subsequent transduction), and users can modify them to express one or more other genes of interest (including therapeutic genes) as desired. For example, an individual interested in treating cancer cells can obtain or generate suicide gene-expressing cells (or heterologous cytokine-expressing cells) and modify them to contain one or more exogenously provided ILs, or vice versa.

[0054] In certain embodiments, NK cells are used, and the genome of the NK cells can be modified to include one or more exogenously provided ILs and / or one or more suicide genes and / or one or more engineered receptors. The genome can be modified in any manner, but in certain embodiments, the genome is modified by, for example, CRISPR gene editing. The genome of the cell can be modified to enhance the effectiveness of the cell for any purpose. In certain cases, the cell is further modified by inhibiting the expression of one or more genes. In some cases, the edited gene allows the cell to function more effectively in the tumor microenvironment. In certain cases, the gene is one or more of TDAG8, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7. In certain embodiments, one or more of these genes are knocked out or knocked down in the cell.

[0055] When a cell is genetically edited to have the expression of one or more genes knocked out or knocked down, such genetic editing can be performed in any suitable manner. In some embodiments, any genetic editing in a cell is performed using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonucleases (RGENs). For example, modification can be performed using CRISPR and CRISPR-associated (Cas) proteins, and in some embodiments, CpF1 is utilized instead of Cas9. Generally, a "CRISPR system" includes transcripts and other elements involved in the expression of CRISPR-associated ("Cas") genes, such as Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats in the context of endogenous CRISPR systems processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from a CRISPR locus.

[0056] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) that has nuclease functionality (e.g., two nuclease domains). One or more components of the CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system obtained, for example, from a particular organism that contains an endogenous CRISPR system (e.g., Streptococcus pyogenes).

[0057] In some embodiments, a Cas nuclease and a gRNA (comprising a fusion of a crRNA specific for the target sequence and an immobilized tracrRNA) are introduced into cells. Generally, a target site at the 5' end of the gRNA targets the Cas nuclease to the target site, e.g., a gene, using complementary base pairing. The target site may be selected based on its position immediately 5' to a protospacer adjacent motif (PAM) sequence (typically NGG or NAG, etc.). In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR systems are characterized by components that promote the formation of a CRISPR complex at the site of the target sequence. Typically, the term "target sequence" generally refers to a sequence to which a guide sequence is designed to be complementary, and which promotes the formation of a CRISPR complex by hybridization between the target sequence and the guide sequence. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex.

[0058] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by disruption or modification as discussed herein. In other embodiments, a Cas9 variant considered a "nickase" is used to create a single-strand nick at the target site. Paired nickases, each directed by a pair of different gRNAs, can be used to improve specificity, for example, by targeting sequences such that a 5' overhang is introduced during simultaneous introduction of a nick. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain (e.g., a transcriptional repressor or transcriptional activator) to affect gene expression.

[0059] The target sequence may comprise any polynucleotide, such as, for example, a DNA polynucleotide or an RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within an organelle of a cell. Generally, a sequence or template that can be used for recombination into a targeted locus that comprises a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0060] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). The tracr sequence may comprise all or a portion of the wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence), and the tracr sequence may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to the guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0061] One or more vectors expressing one or more components of the CRISPR system can be introduced into a cell so that expression of the components of the CRISPR system leads to the formation of CRISPR complexes at one or more target sites. Components can also be delivered to a cell as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements in separate vectors. Alternatively, two or more components expressed from the same or different regulatory elements can be combined in a single vector, in which case one or more additional vectors provide any components of the CRISPR system not included in the first vector. A vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements in one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

[0062] The vector may include regulatory elements operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Cs m2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0063] The CRISPR enzyme can be Cas9 (e.g., obtained from S. pyogenes or S. pneumonia). In some cases, CpF1 can be used as an endonuclease instead of Cas9. CRISPR enzymes can effect direct cleavage of one or both strands at the location of a target sequence, e.g., within the target sequence and / or within the complement of the target sequence. A vector can encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (a single-strand cleavage occurs). In some embodiments, a Cas9 nickase can be used in combination with guide sequence(s), e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination creates a nick on both strands, allowing both strands to be used to induce NHEJ or HDR.

[0064] In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be a cell of a particular organism, such as, but not limited to, a mammal, including a human, mouse, rat, rabbit, dog, or non-human primate, or a cell derived therefrom. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a target host cell by replacing at least one codon of the native sequence with a codon that is more or most frequently used in the host cell's genes while maintaining the native amino acid sequence. Different biological species exhibit specific biases for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) often correlates with, among other things, the properties of the codon being translated and the translation efficiency of messenger RNA (mRNA), which may consequently depend on the availability of specific transfer RNA (tRNA) molecules. The predominance of a selected tRNA in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0065] Generally, a guide sequence includes any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50% or more, about 60% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, about 99% or more, or more.

[0066] Optimal alignment may be determined by using any suitable algorithm for aligning sequences, non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0067] CRISPR enzymes can be part of fusion proteins containing one or more heterologous protein domains. CRISPR enzyme fusion proteins can contain any additional protein sequence, and can also contain a linker sequence between any two domains if necessary. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). CRISPR enzymes may be fused to genetic sequences encoding proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, fusions with maltose binding protein (MBP), S-tag, the DNA binding domain (DBD) of Lex A, the DNA binding domain of GAL4A, and the herpes simplex virus (HSV) BP16 protein. Additional domains that can form part of fusion proteins comprising CRISPR enzymes are described in US Patent Application Publication No. 20110059502, which is incorporated herein by reference.

[0068] [IV. Treatment method] Embodiments of the present disclosure include, for example, methods of treatment related to cancer immunotherapy, anti-pathogen immunotherapy, autoimmunity, or alloimmunity. In certain cases, the cancer immunotherapy and anti-pathogen immunotherapy at least include a composition comprising immune effector cells comprising one or more exogenously provided interleukins. The methods include providing to an individual with cancer and / or a pathogen an effective amount of immune effector cells comprising one or more exogenously provided interleukins.

[0069] In certain cases, an individual is provided with an effective amount of cells containing one or more exogenously provided interleukins. In certain cases, the cells also express one or more engineered antigen receptors. In certain cases, the cells are also knocked out using CRISPR / Cas9. Genetically engineered immune effector cells are used in various cell therapies to enhance their effectiveness against solid tumors, and these cell therapies are provided to individuals.

[0070] As an example, NK cells containing one or more exogenously provided interleukins are engineered to express one or more CARs and to delete one or more endogenous genes in order to enhance their efficacy in the acidic TME of solid tumors, resulting in the extension of this therapy to solid tumors in certain embodiments. Furthermore, this genetic engineering strategy is used in various other forms of cell therapy, such as CAR-NK cells, T cell receptor (TCR)-T cells, tumor-infiltrating lymphocytes (TILs), etc., to enhance their efficacy against various types of solid tumors.

[0071] In certain embodiments, the cells of the present disclosure are provided to an individual for the purpose of ameliorating a medical condition, such as any type of cancer and / or any type of pathogen infection. Uses of the cells contemplated herein (including pharmaceutical compositions comprising same) are used for the prevention, treatment, or amelioration of cancerous diseases, such as neoplastic diseases, or pathogen infections. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful for the prevention, amelioration, and / or treatment of cancer, including, for example, cancers that may or may not be solid tumors.

[0072] In certain embodiments, the present disclosure contemplates the use of the cells encompassed herein, which may be administered, in part, alone or in any combination with one or more other therapies, and in at least some aspects together with a pharmaceutically acceptable carrier or excipient. In certain embodiments, any nucleic acid molecule or vector may be stably integrated into the genome of the cell prior to delivery of the cell to a subject.

[0073] Any of the cells of the present disclosure may be administered to an individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, topically, by perfusion, into the tumor microenvironment, or a combination thereof.

[0074] Furthermore, the present disclosure relates to methods for the prevention, treatment, or amelioration of neoplastic disease, comprising administering to a subject in need thereof an effective amount of any of the cells comprising one or more exogenously provided interleukins contemplated herein.

[0075] Possible indications for administration of the cellular compositions are cancerous diseases, including, for example, glioblastoma, B-cell malignancies, multiple myeloma, and neoplastic diseases of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, or cervix. Exemplary indications for administration of the cellular compositions are cancerous diseases, including any malignant tumor that expresses one or more specific antigens associated with cancer in an individual. Administration of the compositions of the present disclosure is useful for all stages and types of cancer, including, for example, minimal residual disease, early-stage cancer, advanced cancer, and / or metastatic cancer and / or refractory cancer.

[0076] The present disclosure also encompasses co-administration protocols with other compounds that act via immune cells, such as bispecific antibody constructs, targeted toxins, or other compounds. Clinical regimens for co-administration of the compounds of the present invention may include co-administration simultaneously with, prior to, or after the administration of other components. Specific combination therapies include chemotherapy, radiation therapy, surgery, hormone therapy, or other types of immunotherapy.

[0077] Embodiments of the present disclosure include methods for targeting glioblastoma stem cells with cellular immunotherapy, while excluding astrocytes from the same cellular immunotherapy. The cellular immunotherapy may include any type of immune effector cell, including at least NK cells. The cellular immunotherapy may include immune effector cells containing one or more exogenously provided cytokines.

[0078] Embodiments relate to kits comprising constructs for producing cells, nucleic acid sequences as defined herein, vectors as defined herein, and / or host cells (such as immune effector cells) as defined herein. Kits of the present disclosure are also contemplated to include pharmaceutical compositions described herein, alone or in combination with additional agents to be administered to an individual in need of medical treatment or intervention.

[0079] [V. Genetically Engineered Receptors] The immune cells of the present disclosure, comprising one or more exogenously provided interleukins, can be further modified to express one or more non-endogenous gene products. The gene products may or may not be engineered receptors. The receptors may be of any type, including, for example, receptors for antigens, chemokines, or cytokines. When the receptor is for an antigen, the antigen may be a cancer antigen, including a solid tumor antigen.

[0080] Immune effector cells containing one or more exogenously provided interleukins may be genetically engineered to express antigen receptors that target specific antigens, and such cells may be specifically designed to target one or more antigens present on an individual's cancer cells.

[0081] In certain embodiments, immune effector cells comprising one or more exogenously provided interleukins may comprise an engineered antigen receptor, such as an engineered TCR or CAR. For example, the immune cells may be NK cells modified to express one or more CARs and / or TCRs with antigen specificity for one or more particular antigens. In some aspects, the immune cells are engineered to express an antigen-specific CAR or antigen-specific TCR, for example, by knocking in the CAR or TCR using CRISPR.

[0082] Suitable methods of modification are known in the art. See, e.g., Sambrook and Ausubel, supra. For example, cells can be transduced to express a TCR with antigen specificity for a cancer antigen using the transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009.

[0083] In some embodiments, the cells comprise one or more nucleic acids encoding one or more antigen receptors introduced by genetic engineering, and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or a sample obtained from the cell, e.g., obtained from another organism or cell, e.g., not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring (e.g., a nucleic acid not found in nature (e.g., a chimeric)).

[0084] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing these receptors into cells are described, for example, in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US201301493 37, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416, and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, the genetically engineered antigen receptor includes a CAR such as that described in U.S. Pat. No. 7,446,190 and that described in International Patent Application Publication No. WO / 2014055668 A1.

[0085] When the engineered receptor is an antigen receptor, the antigen may be 5T4, 8H9, α vβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, F BP, fetal AchR, FR, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11R, IL-13R2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEMs, HMW-MAA, and VEGFR2.

[0086] (A. Chimeric Antigen Receptor) In some embodiments, the antigen-specific CAR comprises a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising an antigen-binding region that targets (including specifically binds to) a desired antigen.

[0087] In some embodiments, the engineered receptor comprises a CAR, including activating or stimulatory CARs, costimulatory CARs (see WO 2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). These CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some embodiments via a linker and / or transmembrane domain. Such molecules typically mimic or approximate signaling through natural antigen receptors, signaling through such receptors in conjunction with costimulatory receptors, and / or signaling through costimulatory receptors alone.

[0088] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides (including humanized CARs (hCARs) to reduce immunogenicity) that comprise at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain containing one or more signaling motifs. In certain embodiments, the antigen-specific CAR may recognize an epitope that includes a space shared between one or more antigens. In certain embodiments, the binding region may comprise a complementarity-determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide that binds to a receptor (e.g., a cytokine).

[0089] It is contemplated that the human antigen CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In a specific embodiment, the present disclosure includes a full-length cDNA or coding region of an antigen-specific CAR. The antigen-binding region or domain may include a VH and VL chain fragment of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody (e.g., as described in U.S. Patent No. 7,109,304, incorporated herein by reference). The fragment may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0090] The configuration can be a multimer (e.g., a diabody or multimer). The multimer is most likely formed by cross-pairing of the variable portions of the light and heavy chains into a diabody. The hinge portion of the construct can have several options, ranging from a complete deletion, to maintaining the first cysteine, to a proline substitution rather than a serine substitution, to truncation up to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerizing protein can serve this purpose. Only one of the Fc domains can be used, for example, the CH2 or CH3 domain of a human immunoglobulin. The hinge, CH2, and CH3 regions of a human immunoglobulin modified to improve dimerization can also be used. Only the hinge portion of an immunoglobulin can also be used. A portion of CD8α can also be used.

[0091] In some embodiments, the CAR nucleic acid comprises a sequence encoding another costimulatory receptor, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to the primary signal elicited by CD3ζ, additional signals provided by the human costimulatory receptor inserted into the human CAR are important for full activation of NK cells and may help improve in vivo persistence and therapeutic success of adoptive immunotherapy.

[0092] In some embodiments, antigen-specific CARs are constructed to have specificity for an antigen, for example, an antigen expressed on a normal cell type or a non-diseased or diseased cell type. Thus, the CAR typically comprises one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, domains, or portions thereof) or one or more antibody variable domains, and / or antibody molecules in its extracellular portion. In some embodiments, the antigen-specific CAR comprises an antigen-binding portion of an antibody molecule (e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb)).

[0093] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been found to stabilize mRNA. It may also be beneficial to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0094] It is contemplated that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid expression vector in the appropriate orientation for expression.

[0095] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells. A vector suitable for use in accordance with the methods of the present disclosure is a vector that is non-replicative in immune cells. Many viral vectors are known that maintain a sufficiently low copy number of the virus in the cell to maintain the viability of the cell, including, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0096] In some embodiments, the antigen-specific binding component or antigen-specific recognition component is linked to one or more transmembrane domains and an intracellular signaling domain. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding to the transmembrane domain of the same or a different surface membrane protein to minimize interaction with other members of the receptor complex.

[0097] In some embodiments, the transmembrane domain is derived from natural or synthetic sources. If the origin is natural, the domain is derived from any membrane-bound or transmembrane protein in some aspects. The transmembrane region includes a transmembrane region derived from (i.e., at least the transmembrane region of) the α, β, or ζ chain of the T cell receptor, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain is a synthetic transmembrane domain in some embodiments. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0098] In certain embodiments, the platform technologies disclosed herein for genetically modifying immune cells, e.g., NK cells, include: (i) non-viral gene transfer using an electroporation device (e.g., nucleofector); (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations); (iii) CARs with an extracellular domain of variable length that connects the CD70 recognition domain to the cell surface; and, in some cases, (iv) CARs + and artificial antigen-presenting cells (aAPCs) derived from K562, which can robustly and numerically expand immune cells ( Singh et al., 2008 ; Singh et al., 2011 ).

[0099] (BT cell receptor (TCR)) In some embodiments, the engineered antigen receptor comprises a recombinant TCR and / or TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that comprises variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively) and is capable of specifically binding to an antigenic peptide bound to a major histocompatibility complex (MHC) receptor. In some embodiments, the TCR is of the αβ type.

[0100] TCRs, typically found in αβ and γδ types, are generally structurally similar, although the T cells that express them may differ in anatomical location or function. TCRs can be found on the surface of cells or in soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they generally play a role in recognizing antigens bound to MHC molecules. In some embodiments, TCRs can also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., 1997). For example, in some aspects, each chain of a TCR can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a C-terminal short cytoplasmic tail. In some embodiments, TCRs associate with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs of the αβ or γδ types.

[0101] Thus, for purposes of this specification, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR, that binds to a specific antigenic peptide bound in an MHC molecule, i.e., an MHC-peptide complex. An "antigen-binding portion" or "antigen-binding fragment" of a TCR, which may be used interchangeably, refers to a molecule that includes part of the structural domains of the TCR but binds to the antigen (e.g., an MHC-peptide complex) that a full-length TCR binds. In some cases, the antigen-binding portion includes the variable domains of the TCR, e.g., the variable α chain and variable β chain of the TCR, which are sufficient to form a binding site for binding to a specific MHC-peptide complex, e.g., each chain generally includes three complementarity-determining regions.

[0102] In some embodiments, the variable domains of TCR chains associate to form loops, or immunoglobulin-like complementarity-determining regions (CDRs), which form the binding site of the TCR molecule and thereby confer antigen recognition and determine peptide specificity. Typically, like immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the primary CDR involved in recognizing processed antigens, although CDR1 of the α chain has also been shown to interact with the N-terminal portion of antigenic peptides, while CDR1 of the β chain interacts with the C-terminal portion of peptides. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β chain can include an additional hypervariable (HV4) region.

[0103] In some embodiments, a TCR chain comprises a constant domain. For example, like an immunoglobulin, the extracellular portion of a TCR chain (e.g., a chain, β chain) contains two immunoglobulin domains: a variable domain (e.g., V) at the N-terminus; aor Vp; typically, see Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th and one constant domain (e.g., an a-chain constant domain or C chain constant domain, typically amino acids 117-259 according to Kabat numbering, based on the Kabat numbering system) that is adjacent to the cell membrane. a The TCR may also include a β-chain constant domain or Cp, typically amino acids 117-295 according to Kabat. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing the CDRs. The constant domain of the TCR domain includes a short linking sequence in which cysteine ​​residues form disulfide bonds, forming a link between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.

[0104] In some embodiments, the TCR chain can comprise a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules, such as CD3. For example, a TCR that comprises a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling machinery or complex.

[0105] Generally, CD3 is a multiprotein complex that can have three distinct chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex can contain a homodimer of the CD3γ chain, the CD3δ chain, two CD3ε chains, and the CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to associate with positively charged T cell receptor chains. The cytoplasmic tails of the CD3γ, CD3δ, and CD3ε chains each contain one conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), with three present in each CD3ζ chain. Generally, ITAMs are involved in the signaling function of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR to the cell. The CD3 chain and the ζ chain together with the TCR form what is known as the T cell receptor complex.

[0106] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) linked by a disulfide bond or disulfide bond, etc. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, nucleic acid encoding the TCR can be obtained from various sources, such as polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some aspects, the TCR is obtained from a biological source, such as a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from an isolated cell in vivo. In some embodiments, a high-affinity T cell clone can be isolated from a patient and the TCR isolated. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, TCR clones for target antigens are generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, for example, tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against target antigens (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, TCRs or antigen-binding portions thereof can be synthetically generated with knowledge of the sequence of the TCR.

[0107] [IV. Vector] When immune effector cells contain a non-endogenous engineered or exogenously provided gene product, such as one or more exogenously provided interleukins, the gene product can be delivered to the recipient's immune effector cells by any suitable vector, including viral or nonviral vectors. Examples of viral vectors include at least retroviral, lentiviral, adenoviral, or adeno-associated viral vectors. Examples of nonviral vectors include at least plasmids, transposons, lipids, nanoparticles, etc.

[0108] In cases where immune cells are transduced with a vector encoding an antigen-targeting receptor and require the transduction of another gene or genes, such as a suicide gene and / or cytokine and / or any therapeutic gene product, into the cells, the antigen-targeting receptor, suicide gene, cytokine, and any therapeutic gene may or may not be included on the same vector or with the same vector. In some cases, the antigen-targeting CAR, suicide gene, cytokine, and any therapeutic gene are expressed from the same vector molecule, such as the same viral vector molecule. In such cases, the expression of the cytokine, any antigen-targeting receptor, any suicide gene, and any therapeutic gene may or may not be regulated by the same regulatory element. When the cytokine, any antigen-targeting CAR, any suicide gene, and any therapeutic gene are present on the same vector, they may or may not be expressed as separate polypeptides. If they are expressed as separate polypeptides, they may be separated on the vector by, for example, a 2A element or an IRES element (or both may be used once or more on the same vector).

[0109] A. General Embodiments One of skill in the art would be fully able to construct vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) for expression of the antigen receptors of the present disclosure.

[0110] (1. Regulatory Elements) The expression cassette contained in the vector useful in the present disclosure includes, inter alia (5' to 3' direction), a eukaryotic transcriptional promoter operably linked to the protein-coding sequence, a splice signal including intervening sequences, and a transcription termination / polyadenylation sequence. Promoters and enhancers that control the transcription of protein-encoding genes in eukaryotic cells can be composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information carried by each element, allowing different genes to be differentiated, often resulting in complex patterns of transcriptional regulation. Promoters used in the context of the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is utilized for the production of cancer therapeutics, the promoter may be effective under conditions of hypoxia.

[0111] (2. Promoter / Enhancer) The expression constructs provided herein include promoters for driving the expression of antigen receptor and other cistron gene products. Promoters generally contain sequences that function to position the start site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, separate elements overlapping the start site itself serve to define the start site. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region upstream of the start site, but many promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA and promotes expression of the encoded RNA.

[0112] The spacing between promoter elements is often flexible, so that promoter function is preserved even when elements are inverted or moved relative to one another. In the tk promoter, for example, the spacing between promoter elements can be increased to 50 bp apart without activity beginning to decline. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0113] A promoter may be one naturally associated with a nucleic acid sequence, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter may be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, where a heterologous promoter refers to a promoter not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes and promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are "non-naturally occurring," i.e., contain different elements of different transcriptional regulatory regions and / or expression-altering mutations. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences can be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR™, in connection with the compositions disclosed herein. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria and chloroplasts, can be used as well.

[0114] Of course, it is important to use a promoter and / or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (e.g., Sambrook et al. 1989, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0115] Additionally, any promoter / enhancer combination (e.g., from the Eukaryotic Promoter Data Base EPDB, World Wide Web at epd.isb-sib.ch / ) can be used to drive expression. Use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct.

[0116] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate-early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as the beta-actin promoter, the GADPH promoter, and the metallothionein promoter; and tethered response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the minimal TATA box-proximal response element promoter (tre). Human growth hormone promoter sequences (e.g., the human growth hormone minimal promoter described in GenBank® accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from the American College of Cancer, catalog number ATCC 45007) can also be used. In specific embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, MHC class I, or MHC class II promoter, although any other promoter useful for driving expression of therapeutic genes is applicable to the practice of the present disclosure.

[0117] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase the activity of a promoter and have the potential to act in cis and regardless of their orientation, even over relatively long distances (up to several kilobases from the target promoter). However, enhancer function is not necessarily limited to such long distances and may function in close proximity to a given promoter.

[0118] (3. Initiation signal and linked expression) Specific initiation signals may also be used in the expression constructs provided herein for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art would be readily able to determine this and provide the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression can be enhanced by including appropriate transcriptional enhancer elements.

[0119] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to generate multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at endogenous sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) as well as IRESs from mammalian messages have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, resulting in polycistronic messages. IRES elements allow each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0120] As detailed elsewhere herein, specific 2A sequence elements can be used to link or co-express genes in the constructs provided herein. For example, cleavage sequences can be used to link open reading frames to form a single cistron, thereby co-expressing genes. Exemplary cleavage sequences are equine rhinitis A virus (E2A) or F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) or porcine teschovirus-1 (P2A). In certain embodiments, multiple 2A sequences in a single vector are non-identical, while in alternative embodiments, the same vector utilizes two or more identical 2A sequences. Examples of 2A sequences are provided in U.S. Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety.

[0121] (4.Replication origin) To propagate a vector in a host cell, it may contain one or more origins of replication (often called "ori"), which are specific nucleic acid sequences at which replication is initiated, such as a nucleic acid sequence corresponding to the EBV oriP described above, or a genetically engineered oriP with a similar or enhanced function in programming. Alternatively, origins of replication or autonomously replicating sequences (ARS) of other extrachromosomally replicating viruses, as described above, can be used.

[0122] 5. Selectable and Screenable Markers In some embodiments, NK cells containing a CD70-targeting receptor construct of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers an identifiable change to the cell, allowing cells containing the expression vector to be easily identified. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one whose presence allows for its selection, while a negative selectable marker is one whose presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[0123] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of a condition, other types of markers are contemplated, including screenable markers such as GFP based on colorimetric analysis. Alternatively, screenable enzymes may be utilized as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, perhaps in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0124] B. Multicistronic Vectors In certain embodiments, the cytokine, any antigen-targeting receptor, any suicide gene, and / or any therapeutic gene are expressed from a multicistronic vector (as used herein, the term "cistron" refers to a nucleic acid sequence from which a gene product can be produced). In certain embodiments, the multicistronic vector encodes at least one cytokine, a suicide gene, and / or an engineered receptor, e.g., a T cell receptor and / or an additional non-antigen-targeting CAR. In some cases, the multicistronic vector encodes at least one antigen-targeting CAR, at least one suicide gene, and at least one cytokine. The cytokine may be a specific type of cytokine, such as human or mouse or any species. In certain cases, the cytokine is IL-7, IL-12, IL-2, IL-18, and / or IL-21.

[0125] In certain embodiments, the present disclosure provides a versatile modular system (as used herein, the term "modular" refers to a cistron or cistron components that allows for the exchange of entire cistrons or cistron components, respectively, such as by removing and replacing the entire cistron or cistron components) that utilizes polycistronic vectors capable of expressing multiple cistrons at substantially identical levels. This system can be used for cell engineering that allows for the combinatorial expression (including overexpression) of multiple genes. In certain embodiments, the one or more genes expressed by the vector include one, two, or more antigen receptors. The multiple genes can include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector can further include (1) one or more reporters, such as fluorescent or enzymatic reporters for cellular assays and animal imaging; (2) one or more cytokines or other signaling molecules; and / or (3) a suicide gene.

[0126] In certain cases, the vector may contain at least four cistrons separated by any type of cleavage site, such as a 2A cleavage site. The vector may or may not be based on Moloney murine leukemia virus (MoMLV or MMLV), which contains 3' and 5' LTRs with psi packaging sequences in a pUC19 backbone. The vector may contain four or more cistrons with three or more 2A cleavage sites and multiple ORFs for gene swapping. The system allows for combinatorial overexpression of multiple genes (seven or more) flanked by restriction sites for rapid integration via subcloning, and in some embodiments, the system also contains at least three 2A self-cleavage sites. Thus, the system allows for the expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors, and / or homing receptors. This system may also be applied to other viral and non-viral vectors, including, but not limited to, lentiviruses, adenoviruses (AAVs), and non-viral plasmids.

[0127] The modular nature of the system also allows for efficient subcloning of genes into each of the four cistrons in the polycistronic expression vector and swapping of genes for rapid testing, etc. Strategically placed restriction sites in the polycistronic expression vector allow for efficient gene swapping.

[0128] Embodiments of the present disclosure encompass systems that utilize polycistronic vectors, where at least a portion of the vector is modular, for example, by allowing for the removal and replacement of one or more cistrons (or components of one or more cistrons), for example, by utilizing one or more restriction enzyme sites whose identities and positions are specifically selected to facilitate modular use of the vector. The vector also has embodiments in which multiple cistrons are translated into a single polypeptide and processed into separate polypeptides, thereby offering the advantage that the vector expresses separate gene products at substantially equimolar concentrations.

[0129] The vectors of the present disclosure are modular in design to allow for variation of one or more cistrons of the vector and / or variation of one or more components of one or more specific cistrons. Vectors can be designed to take advantage of unique restriction enzyme sites adjacent to the ends of one or more cistrons and / or adjacent to the ends of one or more components of a specific cistron.

[0130] Embodiments of the present disclosure include polycistronic vectors containing at least two, at least three, or at least four cistrons, each flanked by one or more restriction enzyme sites, with at least one cistron encoding at least one antigen receptor. In some cases, two, three, four, or more of the cistrons are translated into a single polypeptide and cleaved into separate polypeptides, while in other cases, multiple of the cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on a vector may be separated by a self-cleaving site, such as a 2A self-cleaving site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on a vector are separated by an IRES element.

[0131] In certain embodiments, the present disclosure provides a system for cell engineering that enables combinatorial expression, including overexpression of multiple cistrons, which may include, for example, one, two, or more antigen receptors. In certain embodiments, the use of polycistronic vectors described herein allows the vector to produce equimolar levels of multiple gene products from the same mRNA. The multiple genes may include, but are not limited to, cytokines, CARs, TCRs, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector may further include one or more fluorescent or enzymatic reporters, such as for cellular assays and animal imaging. The vector may also include a suicide gene product to shut down cells harboring the vector when they are no longer needed or when the vector becomes harmful to the host in which it is provided.

[0132] In certain embodiments of the present disclosure, at least one cistron on a vector contains two or more modular components, each of which is flanked by one or more restriction enzyme sites. A cistron may contain, for example, three, four, or five modular components. In at least some cases, a cistron encodes an antigen receptor, with different portions of the receptor encoded by corresponding modular components. The first modular component of the cistron may encode the antigen-binding domain of the receptor. Furthermore, the second modular component of the cistron may encode the hinge region of the receptor. Furthermore, the third modular component of the cistron may encode the transmembrane domain of the receptor. Additionally, the fourth modular component of the cistron may encode a first costimulatory domain. Furthermore, the fifth modular component of the cistron may encode a second costimulatory domain. Furthermore, the sixth modular component of the cistron may encode a signaling domain.

[0133] In certain embodiments of the present disclosure, two different cistrons on a vector each encode a non-identical antigen receptor. Both antigen receptors may be encoded by a cistron containing two or more modular components, including separate cistrons containing two or more modular components. The antigen receptors may be, for example, chimeric antigen receptors (CARs) and / or T cell receptors (TCRs).

[0134] In certain embodiments, the vector is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector) or a non-viral vector. The vector may include a Moloney murine leukemia virus (MMLV) 5' LTR, a 3' LTR, and / or a psi packaging element. In certain cases, the psi packaging is integrated between the 5' LTR and the antigen receptor coding sequence. The vector may or may not include a pUC19 sequence. In some aspects of the vector, at least one cistron encodes a cytokine (e.g., interleukin-15 (IL-15), IL-7, IL-21, or IL-2), a chemokine receptor, a cytokine receptor, and / or a homing receptor.

[0135] When 2A cleavage sites are utilized in the vector, the 2A cleavage sites may include P2A, T2A, E2A and / or F2A sites.

[0136] In addition to one cistron encoding the CD70-targeted CAR, any cistron of the vector may contain a suicide gene. Any cistron of the vector may encode a reporter gene. In certain embodiments, a first cistron encodes a suicide gene, a second cistron encodes the CD70-targeted CAR, a third cistron encodes a reporter gene, and a fourth cistron encodes a cytokine. In certain embodiments, a first cistron encodes a suicide gene, a second cistron encodes the CD70-targeted CAR, a third cistron encodes a second CAR or another antigen receptor, and a fourth cistron encodes a cytokine. In certain embodiments, different portions of the CD70-targeted CAR and / or another receptor are encoded by corresponding modular components, with the first component of the second cistron encoding the antigen-binding domain, the second component encoding the hinge and / or transmembrane domain, the third component encoding the costimulatory domain, and the fourth component encoding the signaling domain.

[0137] In certain embodiments, at least one of the cistrons encodes a suicide gene. In some embodiments, at least one of the cistrons encodes a cytokine. In certain embodiments, at least one cistron encodes an antigen-targeting CAR. The cistron may or may not encode a reporter gene. In certain embodiments, at least two cistrons encode two different antigen receptors (e.g., CARs and / or TCRs). The cistron may or may not encode a reporter gene.

[0138] In certain configurations of genetic cargo of interest, a single vector may contain a cistron encoding an antigen-targeting CAR and a cistron encoding a second antigen receptor that is not identical to the antigen-targeting receptor. In certain embodiments, the first antigen receptor encodes the antigen-targeting CAR, and the second antigen receptor encodes a TCR, or vice versa. In certain embodiments, a vector containing separate cistrons encoding the antigen-targeting CAR and the second antigen receptor, respectively, also contains a third cistron encoding a cytokine or chemokine and a fourth cistron encoding a suicide gene. However, the suicide gene and / or cytokine (or chemokine) may not be present on the vector.

[0139] In certain embodiments, at least one cistron contains multiple components that are modular. For example, a cistron can encode a multicomponent gene product, such as an antigen receptor, having multiple portions; in certain cases, the antigen receptor is encoded from a single cistron, thereby ultimately producing a single polypeptide. Cists encoding multiple components may have the multiple components separated by one, two, three, four, five, or more restriction enzyme digestion sites, which may be unique in the vector containing the cistron. In certain embodiments, a cistron with multiple components encodes an antigen receptor with multiple corresponding portions, each attributing a unique function to the receptor. In certain embodiments, each or most of the components of a multicomponent cistron are separated by one or more restriction enzyme digestion sites that are unique to the vector, allowing for interchangeability of the separate components, if desired.

[0140] In certain embodiments, each component of a multicomponent cistron corresponds to a different portion of an encoded antigen receptor, such as an antigen-targeted CAR. In exemplary embodiments, component 1 may encode the antigen-binding domain of the receptor; component 2 may encode the hinge domain of the receptor; component 3 may encode the transmembrane domain of the receptor; component 4 may encode the costimulatory domain of the receptor, and component 5 may encode the signaling domain of the receptor. In certain embodiments, an antigen-targeted CAR may contain one or more costimulatory domains, each separated by a unique restriction enzyme digestion site, due to the interchangeability of costimulatory domains within a receptor.

[0141] In certain embodiments, there is a polycistronic vector with four separate cistrons, with adjacent cistrons separated by 2A cleavage sites; however, in certain embodiments, instead of a 2A cleavage site, there is an element (such as an IRES sequence) that allows for the direct or indirect production of separate polypeptides from the cistron. For example, the four separate cistrons may be separated by three 2A peptide cleavage sites, with each cistron having a restriction site (X1, X2, etc.) flanking each end of the cistron, allowing for the interchangeability of a particular cistron with another cistron or other types of sequences using standard recombinant techniques. In certain embodiments, the restriction enzyme sites flanking each of the cistrons are unique in the vector to facilitate recombination; however, in alternative embodiments, the restriction enzyme sites are not unique in the vector.

[0142] In certain embodiments, vectors provide a unique second level of modularity by allowing interchangeability within a particular cistron, including within multiple components of that particular cistron. Multiple components of a particular cistron may be separated by one or more restriction enzyme sites, including one unique to the vector, to allow for interchangeability of one or more components within the cistron. As an example, cistron 2 may contain five separate components, although there may be two, three, four, five, six, or more components per cistron. For example, a vector may contain five separate components, each with unique restriction enzyme sites X9, X 10 , X 11 , X 12 , X 13 , and X 14 A cistron may include cistron 2 having five components separated by , allowing standard recombination to exchange different components 1, 2, 3, 4, and / or 5. In some cases, there may be multiple restriction enzyme sites between different components (which may be unique, or alternatively, one or more may not be unique), and there may be sequences between the multiple restriction enzyme sites (but alternatively, there may not be). In certain embodiments, all components encoded by the cistrons are designed to be interchangeable. In certain cases, one or more components of the cistron may be designed to be interchangeable, while one or more other components of the cistron may not be designed to be interchangeable.

[0143] In certain embodiments, a cistron encodes an antigen-targeted CAR molecule having multiple components. For example, cistron 2 can be composed of sequences encoding an antigen-targeted CAR molecule, with its separate components designated component 1, component 2, component 3, etc. A CAR molecule can comprise two, three, four, five, six, seven, eight, or more interchangeable components. Specifically, component 1 encodes an scFv, component 2 encodes a hinge, component 3 encodes a transmembrane domain, component 4 encodes a costimulatory domain (although component 4' encoding a second or more costimulatory domains flanked by restriction sites for exchange may also be present), and component 5 encodes a signaling domain. In a particular example, component 1 encodes an scFv; component 2 encodes an IgG1 hinge and / or transmembrane domain; component 3 encodes CD28; and component 4 encodes CD3ζ.

[0144] Those skilled in the art will recognize that in designing a vector, the various cistrons and components must be constructed so that they are maintained in frame when necessary.

[0145] In a particular example, cistron 1 encodes a suicide gene; cistron 2 encodes an antigen-targeting CAR; cistron 3 encodes a reporter gene; cistron 4 encodes a cytokine; component 1 of cistron 2 encodes an scFv; component 2 of cistron 2 encodes an IgG1 hinge; component 3 of cistron 2 encodes CD28; and component 4 encodes CD3ζ.

[0146] The restriction enzyme site may be of any type and may contain any number of bases in its recognition site, e.g., 4 to 8 bases; the number of bases in the recognition site may be at least 4, 5, 6, 7, 8, or more. Upon cleavage, the site may generate blunt or sticky ends. The restriction enzyme may be, for example, type I, type II, type III, or type IV. Restriction enzyme sites may be obtained from available databases such as the Integrated Relational Enzyme Database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0147] An exemplary vector may be circular, with position 1 (the 12 o'clock position at the apex of the circle, with the rest of the sequence in a clockwise direction) by convention set at the start of the 5'LTR.

[0148] In embodiments utilizing a self-cleaving 2A peptide, the 2A peptide may be a viral oligopeptide 18-22 amino acids (aa) long that mediates "cleavage" of a polypeptide during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome, and different viral 2As are generally named after the viruses from which they originate. The first 2A discovered was F2A (foot-and-mouth disease virus), followed by E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A). The mechanism of "self-cleavage" by 2A was discovered to involve the ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A.

[0149] In certain cases, the vector may be a gamma-retroviral transfer vector. Retroviral transfer vectors can include a plasmid-based backbone, such as the pUC19 gene (2.63 kb) between HindIII and EcoRI restriction enzyme sites. The backbone can carry viral components derived from Moloney murine leukemia virus (MoMLV), including a 5' LTR, a psi packaging sequence, and a 3' LTR. LTRs are long terminal repeats found on either side of the retroviral provirus, and in the case of transfer vectors, they enclose the genetic cargo of interest (e.g., an antigen-targeting CAR and related components). The psi packaging sequence, which is the target site for packaging by the nucleocapsid, is also integrated in cis between the 5' LTR and the CAR-encoding sequence. Thus, the basic structure of an example transfer vector can be constructed as follows: pUC19 sequence-5' LTR-psi packaging sequence-genetic cargo of interest-3' LTR-pUC19 sequence. This system can also be applied to other viral and non-viral vectors, including but not limited to lentivirus, adenovirus (AAV), and non-viral plasmids.

[0150] [VII. Pharmaceutical Compositions] Provided herein are pharmaceutical compositions and formulations comprising immune effector cells encompassed herein and a pharmaceutically acceptable carrier. The cells may be in a medium suitable for transfer into an individual, or may be in a medium suitable for transfer into an individual and / or medium suitable for storage, such as cryopreservation, prior to transfer into an individual.

[0151] The pharmaceutical compositions and formulations described herein comprise an active ingredient (such as cells) having a desired degree of purity, in the form of a lyophilized formulation or an aqueous solution, in one or more pharmaceutically acceptable carriers (see Remington's Pharmaceutical Sciences 22 ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately 1 0 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In some embodiments, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0152] [VIII. Combination Therapy] In certain embodiments, the compositions and methods of the present embodiments involve immune cell populations (including NK cell populations) combined with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, hormone therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy.

[0153] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor(s) or anti-metastatic agent(s). In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an antiemetic). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent(s). The additional therapy may be one or more chemotherapeutic agents known in the art.

[0154] Immune cell therapy may be administered prior to, during, after, or in various combinations with an additional cancer therapy (e.g., immune checkpoint therapy, etc.). Administration may occur at intervals ranging from simultaneously, to minutes, to days, to weeks. In embodiments in which immune cell therapy is administered to a patient separately from an additional therapeutic agent, generally, no significant period of time will elapse between the respective delivery times, thereby ensuring that the two compounds still exert their desired combined effect on the patient. In such cases, it is contemplated that the patient may receive antibody therapy and anticancer therapy within about 12 to 24 or 72 hours of each other, more specifically within about 6 to 12 hours of each other. In some situations, it may be desirable to significantly extend the period of treatment, where days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between each administration.

[0155] Various combinations can be used. In the following examples, immune cell therapy is "A" and anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / A A / B / B / BB / A / B / BB / B / B / AB / B / A / BA / A / B / B A / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / B A / A / A / BB / A / A / AA / B / A / AA / A / B / A.

[0156] Administration of any compound or cell therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity that may result from the combination therapy.

[0157] (A.Chemotherapy) A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of action within cells, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on whether they can directly crosslink DNA, cause intercalation into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0158] Examples of chemotherapeutic agents include: alkylating agents (such as thiotepa and cyclophosphamide); alkylsulfonates (such as busulfan, improsulfan, and piposulfan); aziridines (such as benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylamelamines (such as altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide, and trimethylolmelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (e.g., the synthetic analogue topotecan); bryostatin; kallistatin; CC-1065 (e.g., its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duplexes Ocarmycins (e.g., synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyins; spongistatins; nitrogen mustards (e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenestrone, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dynemicins (e.g., dynemicin A); bisphosphonates, such as clodronate; esperamicins;Also included are neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5 -oxo-L-norleucine, doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfilomycin mycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (such as denopterin, pteropterin, and trimetrexate); purine analogs (such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguam). pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine); androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone); anti-adrenal agents (e.g., mitotane and trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;Defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilones; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids (e.g., maytansine and ansamitocins); mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet ( phenamet); pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronchial Tol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids (e.g., paclitaxel and docetaxel; Gemcitabine); 6-Thioguanine; Mercaptopurine; Platinum coordination complexes (e.g., cisplatin, oxaliplatin, and carboplatin); Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin cin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids (e.g., retinoic acid, etc.); capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0159] (B. Radiation Therapy) Other widely used agents that cause DNA damage include what is commonly known as gamma radiation, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of damage to DNA, its precursors, DNA replication and repair, and chromosome assembly and maintenance. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens over prolonged periods (3 to 4 weeks) to single doses of 2,000 to 6,000 roentgens. Dosage ranges for radioisotopes vary widely, depending on the half-life of the radioisotope, the strength and type of radiation emitted, and uptake by tumor cells.

[0160] (C. Immunotherapy) Those skilled in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of this embodiment. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may serve as the therapeutic effector alone, or the antibody may recruit other cells to actually affect cell killing. Antibodies may also be conjugated to drugs or toxins (such as chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as targeting agents. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts either directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0161] Antibody-drug conjugates have emerged as a groundbreaking approach to the development of cancer therapeutics. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. In this approach, the high specificity of the MAb for its antigen target is combined with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers its payload (drug) to tumor cells bearing high levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, thereby reducing toxicity and improving the therapeutic index. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has proven this approach effective. Currently, over 30 ADC drug candidates are in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly relies on the identification and validation of new targets amenable to this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high expression levels in tumor cells and robust internalization.

[0162] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., some marker that is not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. Immune stimulatory molecules also exist, including cytokines (e.g., IL-2, IL-4, IL-12, GM-CSF, γ-IFN, etc.), chemokines (e.g., MIP-1, MCP-1, IL-8, etc.), and growth factors (e.g., FLT3 ligand, etc.).

[0163] Examples of immunotherapies currently under consideration or in use include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); 98); gene therapies, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0164] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints can either enhance or attenuate signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B-lymphocyte and T-lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte-activation gene-3 (LAG3), programmed cell death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0165] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or may be antibodies, particularly human antibodies (e.g., International Patent Publication No. 2015 / 016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human antibodies. As those skilled in the art will recognize, alternative and / or equivalent names may be used for specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0166] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0167] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some aspects, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1-binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0168] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has GenBank® accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch upon binding to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28; both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0169] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0170] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art, or art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used, for example, humanized CTLA-4 antibodies described in International Patent Publication Nos. WO 2001 / 014424, WO 2000 / 037504, and U.S. Patent No. 8,017,114 (all incorporated herein by reference).

[0171] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., International Patent Publication No. WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-described antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-described antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0172] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796 and International Patent Application Nos. WO 1995 / 001994 and WO 1998 / 042752 (all incorporated herein by reference), and immunoadhesins such as those described in U.S. Pat. No. 8,329,867 (incorporated herein by reference).

[0173] (D.Surgery) Approximately 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in combination with other therapies, such as the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0174] When part or all of the cancer cells, tissue, or tumor is removed, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection, or local application to the site with additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be of various dosages.

[0175] (E. Other drugs) It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will enhance the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (e.g., antibody c225) could be used in combination with certain aspects of the present embodiments to improve the efficacy of treatment.

[0176] IX. Kits of the Present Disclosure Any of the compositions described herein can be included in a kit. In a non-limiting example, cells containing one or more exogenously provided interleukins, reagents for producing the cells, vectors, and reagents for producing the vectors and / or their components can be included in a kit. In certain embodiments, NK cells can be included in the kit, which may or may not be modified in any manner. Such kits may or may not include one or more reagents for manipulating the cells. Such reagents include, for example, cytokines, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. A vector expressing one or more cytokines and / or one or more engineered antigen receptors, or reagents for producing either, can be included in the kit. Nucleotides encoding one or more cytokines, nucleotides encoding CRISPR reagents for knockout of one or more specific genes, suicide gene products, receptors, etc. can be included in the kit. Proteins such as cytokines or antibodies, including monoclonal antibodies, can be included in the kit. Nucleotides encoding components of an engineered CAR receptor or TCR receptor, including reagents for producing them, can be included in the kit.

[0177] In certain aspects, the kit also includes an NK cell therapy of the present disclosure and another cancer therapy. In some cases, the kit also includes a second cancer therapy, such as, for example, chemotherapy, hormone therapy, and / or immunotherapy, in addition to the cell therapy embodiment. The kit can be tailored to an individual's particular cancer and can include the individual's respective second cancer therapy.

[0178] The kit may contain appropriately dispensed compositions of the present disclosure. The components of the kit may be packaged in aqueous media or in lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, preferably into suitable aliquots. When two or more components are present in the kit, the kit will usually also include second, third, or other additional containers into which the additional components may be placed separately. However, various combinations of components may be contained in a single vial. The kits of the present invention also typically include a means for containing the composition, as well as any other reagent containers, in close confinement for commercial sale. Such containers may include injection-molded or blow-molded plastic containers for holding the desired vials. [Example]

[0179] The following examples are included to demonstrate certain non-limiting aspects of the present disclosure. Those skilled in the art will appreciate that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the disclosed subject matter. However, those skilled in the art will, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the disclosed subject matter.

[0180] Example 1 NK Cell Immunotherapy for the Treatment of Glioblastoma NK cells were tested for their ability to treat glioblastoma. NK cells were able to kill patient-derived glioblastoma stem cell lines (GCS), but not normal astrocytes (Figure 1). Figure 1B shows the difference in expression of selective NK ligands between GCS and astrocytes.

[0181] NK cells can be engineered to express specific desired genes, such as one or more cytokine genes (e.g., IL-15, IL-12, IL-21). Examples of specific constructs are shown in Figure 2, where the 2A element separates IgG1 production from cytokine production, although both are on the same expression construct. In these specific examples, human IL-15, human IL-21, or human IL-12 are engineered such that the p35 and p40 subunits are artificially linked by a linker. Such expression constructs can be present in any type of vector within the NK cells.

[0182] The activity of cytokine-engineered NK cells was tested using a 3D tumor spheroid culture model of GSCs (Figure 3A). The tumor spheroid model simulates a solid tumor mass. Killing assays were performed in an Incucyte® device using live-cell imaging of tumor cell proliferation and NK cell killing. The indicated cord blood-derived cytokine-engineered NK cells (red, blue, and green lines) demonstrated superior killing of a patient-derived glioblastoma stem cell line compared to untransduced NK cells (black line) (Figure 3B).

[0183] We characterized the in vivo activity of NK cells against GBM. ffLuc+ patient-derived glioblastoma stem cell lines (5 × 10 5 ) was stereotactically implanted into the right forebrain of NSG mice. After 7 days, tumor formation was confirmed by BLI imaging, and the mice were then implanted with 1.0 × 10 5 NK cells were administered intracranially. Animals treated with IL-12- or IL-21-transduced (secretable) cord blood NK cells showed significant tumor regression (tumors no longer detectable by BLI imaging) accompanied by a significant improvement in survival compared to animals treated with non-transduced (NT) NK cells (Figures 4A, 4B, and 4C).

[0184] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of this application is not limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods, and steps described herein. Those skilled in the art will readily recognize from this disclosure other now-existing or later-developed processes, machines, manufacture, compositions of matter, means, methods, or steps that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein may be utilized in accordance with the present disclosure. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0185] (References) All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. <References> Ahmed et al. Clin Cancer Res 16(2): 474-485 (2010). Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.Brown et al., N. Engl J. Med. 375(26): 256-269. (2016). Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998. Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206) Cohen et al., J Immunol. 175:5799-5808, 2005. Chothia et al., 1988 Christodoulides et al., Microbiology, 144(Pt 11):3027-3037, 1998. Davidson et al., J. Immunother., 21(5):389-398, 1998. Davila et al. PLoS ONE 8(4): e61338, 2013. Heemskerk et al. Um Gene Ther. 19:496-510, 2008. Hellstrand et al., Acta Oncologica, 37(4):347-353, 1998. Hollander, Front. Immun., 3:3, 2012. Hanibuchi et al., Int. J. Cancer, 78(4):480-485, 1998. Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998. Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071 Johnson et al. Blood 114:535-46, 2009. Jores et al., PNAS USA 87:9138, 1990. Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed. Leal, M., Ann N Y Acad Sci 1321, 41-54, 2014. Lefranc et al., Dev. Comp. Immunol. 27:55, 2003. Li, Nat Biotechnol. 23:349-354, 2005. Mokyr et al. (1998) Cancer Res 58:5301-5304 O’Rourke et al. Sci Transl Med 9(399). Parkhurst et al., Clin Cancer Res. 15: 169-180, 2009. Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012 Qin et al., Proc. Natl. Acad. Sci. USA, 95(24):14411-14416, 1998. Sadelain et al., Nat.Rev.Cancer 2003;3:35-45. Sadelain et al., Cancer Discov. 3(4): 388-398, 2013. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001. Singh et al., Cancer Research, 68:2961-2971, 2008. Singh et al., Cancer Research, 71:3516-3527, 2011. Turtle et al., Curr. Opin. Immunol., 24(5): 633-39, 2012. Varela-Rohena et al. Nat Med. 14: 1390-1395, 2008. Wu et al., Cancer, 18(2): 160-75, 2012. <Patents and Patent Applications> European patent application number EP2537416 U.S. Patent Publication No. 2005 / 0260186 U.S. Patent Publication No. 2006 / 0104968 U.S. Patent Publication No. 2002131960 U.S. Patent Publication No. 2013287748 U.S. Patent Publication No. 20130149337 U.S. Patent Application No. 2014 / 0294898 U.S. Patent Application No. 2014 / 022021 U.S. Patent Application No. 2011 / 0008369 U.S. Patent No. 5,739,169 U.S. Patent No. 5,801,005 U.S. Patent No. 5,824,311 U.S. Patent Nos. 5,830,880 U.S. Patent No. 5,846,945 U.S. Patent No. 5,844,905 U.S. Patent No. 5,885,796 U.S. Patent No. 6,207,156 U.S. Patent No. 6,410,319 U.S. Patent No. 6,451,995 U.S. Patent No. 7,070,995 U.S. Patent No. 7,109,304 U.S. Patent No. 7,265,209 U.S. Patent No. 7,354,762 U.S. Patent No. 7,446,179 U.S. Patent No. 7,446,190 U.S. Patent No. 7,446,191 U.S. Patent No. 8,008,449 U.S. Patent No. 8,017,114 U.S. Patent No. 8,119,129 U.S. Patent No. 8,252,592 U.S. Patent No. 8,324,353 U.S. Patent No. 8,329,867 U.S. Patent No. 8,339,645 U.S. Patent No. 8,354,509 U.S. Patent No. 8,398,282 U.S. Patent No. 8,479,118 U.S. Patent No. 8,735,553 WO 1995 / 001994 WO 1998 / 042752 WO 2000 / 14257 WO 2000 / 37504 WO 2001 / 014424 WO 2009 / 101611 WO 2009 / 114335 WO 2010 / 027827 WO 2011 / 066342 WO 2012 / 129514 WO 2013 / 071154 WO 2013 / 123061 WO 2013 / 126726 WO 2013 / 166321 WO 2014 / 031687 WO 2014 / 055668 WO 2015 / 016718

Claims

1. A composition comprising natural killer (NK) cells, wherein the NK cells comprise one or more exogenously provided interleukins (ILs), the ILs being IL-21 secreted by the cells, and the NK cells comprise one or more engineered receptors.

2. The composition of claim 1 , wherein exogenously provided is further defined as being expressed from a vector in the cell.

3. The composition described in claim 1, wherein the NK cells are cultured in the presence of one or more ILs.

4. The composition of any one of claims 1 to 3, wherein the engineered receptor is an engineered antigen receptor.

5. 5. The composition of claim 4, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

6. The composition of claim 4 or 5, wherein the antigen is a cancer antigen.

7. The composition of any one of claims 4 to 6, wherein the antigen is a solid tumor antigen.

8. The antigen is selected from the group consisting of 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FAP, FBP, fetal AchR, FRα, GD2, GD3, and glypican-3. The composition of any one of claims 4 to 7, wherein the antibody is selected from the group consisting of (GPC3), HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, L1CAM, Kappa, KDR, MCSP, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, survivin, TAG72, TEMs, HMW-MAA, and VEGFR2.

9. The composition of any one of claims 1 to 3, wherein the engineered receptor is a cytokine receptor, a chemokine receptor, or a homing receptor.

10. 10. The composition of any one of claims 1 to 9, wherein the cells have reduced or inhibited expression of one or more endogenous genes selected from the group consisting of TDAG8, NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and combinations thereof, compared to non-transduced NK cells.

11. A population of cells comprising the composition of any one of claims 1 to 10, contained in a suitable medium.

12. A pharmaceutical composition for treating cancer, comprising the population of cells of claim 11.

13. A method of treating cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of the composition of any one of claims 1 to 10, provided that the individual is not a human.

14. The method of claim 13, wherein the cancer cells in the individual have increased expression of an NK ligand compared to astrocytes.

15. The method of claim 13 or 14, wherein the cancer cells in the individual have increased expression of MICA / B, ULBP1, ULBP2 / 5, ULBP3, B7-H6, CD112, CD155, HLA-ABC, HLA-DR, HLA-3, or a combination thereof, compared to astrocytes.

16. 16. The method of any one of claims 13 to 15, wherein the cancer is cancer of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testis, endometrium, prostate, rectum, anus, cervix, or hematological cancer.

17. The method of any one of claims 13 to 16, wherein the cancer is glioblastoma.

18. The method of any one of claims 13 to 17, further comprising generating the cells.

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