Methods for Activating and Proliferating Natural Killer Cells and Combinations with Bispecific Antibodies - Patent application

Abstract

Embodiments of the present invention provide methods and compositions relating to immunotherapy for medical conditions. [0003] Embodiments of the present disclosure relate to methods and compositions related to the preparation and use of combinatorial immunotherapy. In certain embodiments, compositions comprising NK cells prepared by certain methods also comprise certain antibodies. These compositions are utilized in treatments, such as cancer treatment. In certain embodiments, the compositions comprise a complex of NK cells and an antibody, in which the antibody binds to the NK cell and may also bind to another antigen, such as on a cancer cell.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 172,402, filed April 8, 2021, and also claims priority to U.S. Provisional Patent Application Serial No. 63 / 228,991, filed August 3, 2021, both of which are incorporated by reference in their entireties.

[0002] Embodiments of the present disclosure include at least the fields of cell biology, molecular biology, immunology, and medicine, including cancer medicine. [Background technology]

[0003] In the field of natural killer (NK) cells as a therapy, certain barriers hinder their use, including their limited numbers and the need for ex vivo expansion, in addition to their poor persistence that affects their efficacy as an anti-cancer treatment. Cytokine stimulation is a key signal that enhances the functional capacity of NK cells to respond to tumor target cells. Moreover, it has been shown that preactivating NK cells overnight with a combination of IL-18, IL-15, and IL-12 enhances cytokine production upon restimulation and generates long-lived, memory-like NK cells (Leong et al., 2014). However, the use of preactivated NK cells from peripheral blood (PB) requires cumbersome procedures to generate sufficient quantities for clinical use due to the lack of ex vivo expansion. In addition, there are obstacles associated with defective cancer recognition by NK cells and poor activation of NK cells. Thus, there is an unmet need for improved strategies to generate highly functional preactivated NK cells in sufficient numbers for therapeutic applications and that are particularly efficient in cancer antigen targeting. Summary of the Invention

[0004] Embodiments of the present disclosure relate to methods and compositions related to immunotherapy for disease conditions. Immunotherapy may be utilized for any disease condition where targeting of cells in need of destruction is clinically useful and there is a specific antigen on the targeted cells for which the antibody and / or cell may be specific. In certain embodiments, the immunotherapy comprises, consists of, or consists essentially of adoptive cell therapy and an antibody configured to bind to the cells. In certain embodiments, the adoptive cell therapy consists of NK cells, and the antibody may be multispecific, including at least bispecific. Thus, in certain embodiments, a composition that is combinatorial with both NK cells and antibodies is utilized for therapy, and the combination may be configured such that the antibody can bind to a surface antigen on the NK cells, and the complex of the combination is utilized therapeutically.

[0005] In certain embodiments, NK cells for the methods and compositions can be prepared in a specific manner prior to use, including prior to combination (of any kind) with an antibody. In certain embodiments, NK cells are expanded in a specific manner and optionally pre-activated in a specific manner. For example, NK cells can be expanded in the presence of specific antigen-presenting cells under specific culture conditions, and further, NK cells can be optionally exposed to one or more cytokines as a pre-activation step.

[0006] In certain embodiments, the efficacy of a composition comprising a complex of NK cells and an antibody is enhanced compared to when they are used separately, in certain aspects, the efficacy of the NK cells and the antibody is synergistic when used as a complex, while in other aspects, the efficacy of the NK cells and the antibody is additive.

[0007] The embodiments of the present disclosure relate to compositions comprising: (1) one or more umbilical cord blood-derived, stem cell-derived (including iPSC), or peripheral blood-derived natural killer (NK) cells; and (2) one or more antibody molecules, where (a) the antibody is monospecific, and an Fc region of the monospecific antibody binds to the NK cell and an antigen-binding domain of the monospecific antibody binds to a target antigen; or (b) the antibody is multispecific, and one or more antigen-binding domains of the antibody bind to a target antigen and another antigen-binding domain or domains of the antibody bind to an NK cell surface antigen. The NK cell may or may not be expanded. The NK cell may or may not be pre-activated. In some cases, the multispecific antibody is bispecific, trispecific, or multispecific. In a specific aspect, in (a), the composition is further defined as a complex of the NK cell and the monospecific antibody via the binding of the Fc region of the monospecific antibody to the NK cell. In a specific example, in (b), the composition is further defined as a complex of an NK cell and a multispecific antibody via binding of an antigen-binding domain or domains of the multispecific antibody that bind to an NK cell surface antigen or antigen. Target antigens include CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, C The antigen may be a cancer antigen, stem cell antigen, or autoantigen selected from the group consisting of D38, CD56, CD70, CD38, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, HLA-G, TROP2, CD99, and combinations thereof. Or, the NK cell surface antigen may be CD16, CS1, CD56, NKG2D, NKG2C, or a costimulatory molecule such as any c-type lectin, DNAM, 2B4, CD2, NCR, or KIR.For the present composition, the source of cord blood is cord blood from one donor or pooled cord blood from two or more individual cord blood units. The CB may be pooled from 3, 4, 5, 6, 7, or 8 cord blood units. The NK cells (which may be CD56+) may be derived from cord blood mononuclear cells, cord blood hematopoietic stem cells, iPSCs, peripheral blood, or NK cell lines. The present composition may be used fresh or cryopreserved. The source of the NK cells may be a fresh source or a cryopreserved repository. In some cases, when the NK cells are sourced from cryopreservation, the NK cells were cryopreserved in a medium comprising at least one cryoprotectant, at least one serum or a non-serum alternative to serum. In certain embodiments, at least one cytokine and / or at least one growth factor may be added to the cryopreservation medium. The cryoprotectant may be dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethol starch, dextran trehalose, or a combination thereof. The non-serum substitute may consist of platelet lysate and / or blood product lysate, or human or animal serum albumin. In some cases, the at least one cytokine (which may be interleukin (IL)-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, interferon, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or a combination thereof) is a natural protein, a recombinant protein, a synthetic protein, or a mixture thereof. The NK cells may be modified by the hand in one or more ways, such as to include one or more artificial antigen receptors, including chimeric antigen receptors, T cell receptors, CD16, CD32 and / or CD64 receptors. The NK cells may express heterologous cytokines, such as IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 or IL-23. Additionally or alternatively, the NK cells may express a suicide gene. The composition may be comprised of a solution or solid that includes one or more cryoprotectants.The composition may be constituted in a pharma- ceutically acceptable carrier.

[0008] In one embodiment, a method of making any of the compositions encompassed herein is provided, comprising (a) optionally expanding NK cells in a culture comprising an effective amount of: (1) a cytokine selected from the group consisting of IL-2, IL-15, and / or IL-21; and (2) an antigen presenting cell / feeder or NK activation bead; and (b) providing the NK cells with an antibody molecule, whereby, if expanded, the antibody molecule is provided to the NK cells before and / or after expansion. Optionally, the method comprises a preactivation step before and / or after the expansion step, where the NK cells are preactivated in a culture comprising an effective concentration of one or two or more of IL-2, IL-12, IL-15, and IL-18. Optionally, IL-12 is utilized in the culture instead of IL-15. The providing step can be further defined as culturing the NK cells with the antibody molecule for a certain period of time (e.g., from about 5 minutes to about 24 hours or more) or binding the NK cells to the antibody molecule immediately prior to injection. The culture may or may not include Plasma-Lyte A and / or human serum albumin. After culture, the composition may or may not be infused into the recipient subject without first washing. The NK cells may be depleted of CD3+, CD14+ and / or CD19+ cells. The depletion step may be performed before the preactivation step, and / or before expansion with feeder cells and / or NK cell activating beads, and / or before culture with one or more cytokines, and / or before infusion. Optionally, the method further comprises obtaining the NK cells from umbilical cord blood, where the umbilical cord blood does not include umbilical cord tissue.

[0009] The antigen presenting cells may be artificial cells (aAPCs) and may express CD137 ligand. The aAPCs may further express one or more membrane-bound cytokines, such as membrane-bound IL-21 (mIL-21) and / or membrane-bound IL-15 (mIL-15). The aAPCs may not essentially express endogenous HLA class I, II, and / or CD1d molecules. In some cases, the aAPCs express ICAM-1 (CD54) and / or LFA-3 (CD58) and / or CD48. The aAPCs may be further defined as leukemia cell-derived aAPCs, which may be K562 cells engineered to express CD137 ligand and / or mIL-21. In any case, the aAPCs may be engineered by transduction with any kind of vector, including retroviral transduction, including viral or non-viral vectors. The aAPCs may be irradiated or not. The preactivation step may be for 10 to 20 hours, 14 to 18 hours, or 16 hours. The culture for the preactivation step may include IL-18 and / or IL-15 at a concentration of 1 to 1000 ng / mL. The culture for the preactivation step may include IL-12 at a concentration of 0.1 to 1000 ng / mL. The culture for the preactivation step may include IL-12 at a concentration of 1 to 1000 ng / mL, including 10 ng / mL. In some cases, the method further includes washing the preactivated NK cells before and / or after the expansion step, which may be done once or multiple times. If expanded, the expansion may be for 12 to 16 days, for example 5 to 60 days, including 18 to 24 days. In some matters, the preactivated NK cells and aAPCs are present in the expansion culture at a ratio of 3:1 to 1:3, including a ratio of 1:2. The expansion cultures may or may not further comprise IL-2 at a concentration of 10-500 U / mL, including 100-300 U / mL, such as 200 U / mL. In any embodiment of the method, the IL-12, IL-18, IL-15, and / or IL-2 may or may not be recombinant. In some embodiments, IL-2 is replenished in the expansion cultures every 2-3 days.IL-2 or IL-15 or IL-18 or any combination of these cytokines may be added to the expansion culture at least a second time. APCs may be added to the expansion culture at least a second time. Optionally, one or more steps of the method are performed in serum-free medium.

[0010] In one embodiment, a method of treating a disease or disorder in a subject (including a mammal such as a human) comprises administering a therapeutically effective amount of any of the compositions encompassed herein, wherein the NK cells of the composition may be autologous or allogeneic with respect to the subject. The disease or disorder may be cancer (blood cancer or solid tumor), inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency disease, B-cell malignancy, or infectious disease. Hematological cancers include leukemias selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). The disorder may be graft-versus-host disease (GVHD), multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma, and / or Sjogren's syndrome.

[0011] In some cases, the method includes administering to the subject at least a second therapeutic agent, such as a therapeutically effective amount of one or more anti-cancer agents (such as chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy and / or immunotherapy, such as one or more of calcineurin inhibitors, mTOR inhibitors, antibodies, chemotherapeutic agents, radiation, chemokines, interleukins, or inhibitors of chemokines or interleukins), one or more immunomodulatory agents, and / or one or more immunosuppressive agents. Any second agent for any disease may be an antibody, including a monoclonal antibody, a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0012] In any method, the composition (and / or at least the second therapeutic agent) may be administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intrainvasively, percutaneously, subcutaneously, topically, or by direct injection or perfusion or infusion.

[0013] 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 below which form the subject matter of the claims herein. It should be understood by those skilled in the art that the ideas 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 design. It should also be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as defined by the appended claims. The novel features believed characteristic of the design disclosed herein, both as to organization and method of operation, together with further objects and advantages, will be better understood from the following description. It is to be expressly understood, however, that this disclosure is provided for purposes of illustration and description only, and is not intended as a definition of the limits of the present disclosure. [Brief description of the drawings]

[0014] For a more complete understanding of this disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0015] [Figure 1] FIG. 1 is a simulation diagram detailing one embodiment of an experimental design for expanding cord blood-derived NK cells and preloading them with antibody.

[0016] [Diagram 2] Margetuximab binds with higher affinity to P+E NK cells compared to normal proliferating NK cells.

[0017] [Diagram 3] Margetuximab-loaded NK cells exhibit enhanced cytotoxicity against HER2+ tumor cells in a long-term Xcelligence killing assay.

[0018] [Figure 4A-C]Margetuximab-loaded NK cells enhance tumor control in an NSG mouse model of HER2+ ovarian cancer (SKOV3).

[0019] [Diagram 5] Amivamab binds with higher affinity to P+E NK cells compared to normal proliferating NK cells.

[0020] [Figure 6] Amivantamab-loaded NK cells exhibit enhanced cytotoxicity against EGFR+ / cMET+ tumor cells in short-term 51Cr-release killing assays.

[0021] [Figure 7A-C] Amivantamab-loaded NK cells exhibit enhanced cytotoxicity against EGFR+ / cMET+ tumor cells in a long-term Xcelligence killing assay.

[0022] [Figure 8A-C] Amivumab-loaded NK cells result in enhanced tumor control in an NSG mouse model of EGFR+ / c-MET+ ovarian cancer (SKOV3).

[0023] [Figure 9] Imgatuzumab binds to preactivated, expanded (PE) NK cells with higher affinity compared to normally expanded (NE) NK cells.

[0024] [Figure 10] Imgatuzumab-loaded NK cells exhibit enhanced cytotoxicity against EGFR+ tumor cells in short-term 51Cr-release killing assays.

[0025] [Figure 11A-C] Imgatuzumab-loaded NK cells exhibit enhanced cytotoxicity against EGFR+ tumor cells in long-term Xcelligence killing assays.

[0026] [Figure 12A-C] Imgatuzumab-loaded NK cells result in enhanced tumor control in an NSG mouse model of EGFR+ ovarian cancer (SKOV3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Following long-standing patent law practice, the words "a" and "an" when used in conjunction with the word comprising herein, including in the claims, refer to "one or more." 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.

[0028] Throughout this specification, unless otherwise required by context, the words "containing", "including" and "comprises" are understood to mean the inclusion of a recited step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and other elements may not be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0029] Throughout this specification, references to "one embodiment," "one embodiment," "particular embodiment," "related embodiment," "an embodiment," "additional embodiment," or "further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with an embodiment are 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.

[0030] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. 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.

[0031] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0032] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the field of cell and molecular biology to indicate that the standard deviation of error for the device or method being employed to determine the value.

[0033] The term "engineered" as used herein refers to entities produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, engineered entities are synthetic and consist of elements that do not occur in nature or are constructed by the methods utilized in this disclosure. In specific embodiments, vectors are engineered by recombinant nucleic acid techniques and cells are engineered by transfection or transduction of the engineered vector.

[0034] As used herein, the terms "loading," "loaded," or "loading" refer to adoptive cell therapy cells having one or more antibodies bound to the cells at their surface.

[0035] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other adverse reactions when administered to an animal, such as a human, as appropriate. The preparation of pharmaceutical compositions containing an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Furthermore, it will be understood that for administration to animals (e.g., humans), preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0036] As used herein, "pharmaceutical acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutrient replenishers, such materials and combinations thereof will be known to those skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0037] The term "preactivation" or "preactivation" as used herein refers to exposure of NK cells to IL-12, IL-15 or IL-2, and / or IL-18, resulting in increased enrichment of genes associated with signaling pathways associated with NK cell effector function, e.g., IFN-γ response, TNF signaling, IL-2 / STAT5 signaling, IL-6 / JAK / STAT3 signaling, mTOR pathway, and / or inflammatory immune response. In certain cases, expression of TRAIL, NKp44, and / or CD69 is increased.

[0038] As used herein, the terms "preload," "preloading," or "preloading" refer to adoptive cell therapy cells that have one or more antibodies bound to their cell surface prior to using the cells for any reason.

[0039] As used herein, "prevent" and similar terms such as "prevented," "preventing," and the like refer to an approach to prevent, inhibit, or reduce the likelihood of the onset or recurrence of a disease or condition, such as 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, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0040] The term "subject" as used herein generally refers to an individual who has or is suspected of having cancer. A subject may be any organism or animal subject that is the subject of a method or material, including mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), household pets (e.g., dogs, cats, rodents), horses, and transgenic non-human animals. A subject may be, for example, a patient who has or is suspected of having a disease (sometimes referred to as a pathology), such as a benign or malignant neoplasm, or cancer. A subject may be undergoing or having been treated. A subject may be asymptomatic. A subject may be a healthy individual, but may wish to prevent cancer. The terms "individual" are used interchangeably, at least in some cases. 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. Individuals may include humans or non-human animals of any age, and thus include both adults and juveniles (i.e., children) and infants, including individuals in utero. The term does not imply the need for medical treatment, and therefore individuals may participate voluntarily or involuntarily in experiments, whether clinical or in support of basic science research.

[0041] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathological condition 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, such as cancer. Treatment may optionally include either the alleviation or amelioration of one or more symptoms of a disease or condition, or the delay in the progression of a disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition, or symptoms associated therewith. I. Natural Killer (NK) Cells and Methods for Generation

[0042] NK cells have emerged as a stimulating source of cellular immunotherapy for patients with hematological malignancies as well as solid tumors; however, most studies using adoptively transferred NK cells have been limited by poor persistence of the infused cells, poor in vivo proliferation, and disappointing antitumor activity. Thus, a barrier to overcome in the field of NK immunotherapy is the need for a biologically driven approach to enhance the antitumor function of NK cells, such as by manipulating the cells before administering them as a treatment. Thus, in certain embodiments, the present disclosure provides a method for the generation of NK cells with any type of enhanced efficacy compared to NK cells that have not been so manipulated. Some embodiments of the present disclosure relate to the isolation, activation, and expansion of NK cells, including for cancer immunotherapy.

[0043] In certain embodiments, the present disclosure encompasses loaded, preactivated, (optionally) and (optionally) expanded NK cells generated by the present methods that exhibit enhanced anti-tumor function against cancer. The preactivated and expanded NK cells also, in certain embodiments, exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC). A.NK cells, overview

[0044] In certain embodiments, the NK cells utilized in the present methods may be derived from any suitable source, such as umbilical cord blood (CB), e.g., human CB. In certain cases, the NK cells are not derived from umbilical cord tissue (the isolating material surrounding the blood vessels of the umbilical cord (i.e., Wharton's jelly)). In alternative embodiments, the NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and / or bone marrow, or are NK cell lines, such as NK-92, derived from patients, by methods well known in the art. In certain embodiments, the NK cells are isolated from pooled CBs. The CBs may be pooled from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more units. The NK cells may be autologous or allogeneic with respect to the recipient individual. The isolated NK cells may or may not be haplotype-matched to the subject to whom the cell therapy is administered. NK cells may or may not be detected by specific surface markers, such as CD16 and / or CD56 in humans. In some cases, NK cells are depleted for the presence of one or more surface markers, such as depleted for CD3+, CD14+ and / or CD19+ cells. In certain embodiments, NK cells are CD3-CD56+.

[0045] In one embodiment, NK cells are isolated by previously described methods of ex vivo expansion of NK cells (Spanholtz et al., 2011; Shah et al., 2013). In this method, CB mononuclear cells are isolated by Ficoll density gradient centrifugation. The cell culture can be depleted of any cells expressing CD3 and CD56. + / CD3 - Alternatively, umbilical cord blood can be used to characterize the CD34 + NK cells are derived by cell isolation. B. NK cell loading

[0046] In certain embodiments, NK cells are loaded with antibodies before use. NK cells can be loaded in any particular way, including during culture or immediately before injection, for example to generate a complex between NK cells and antibodies. The conditions are sufficiently suitable to allow an effective amount of antibody to bind to the surface of the NK cells. When a monospecific antibody is used, the Fc region of the monospecific antibody binds to the NK cell, while the antigen-binding domain of the monospecific antibody is free to bind to its target antigen. When a multispecific antibody is used, one or more antigen-binding domains of the antibody bind to the surface of the NK cell, such as via an antigen on the surface of the NK cell, while the other antigen-binding domain is free to bind to its target antigen.

[0047] The culture conditions under which the NK cells are loaded may or may not be type specific with one or more specific parameters. In certain embodiments, loading of the NK cells occurs during culture at a specific temperature, such as 37° C., while in alternative embodiments, the temperature is 36° C. or 38° C., or lower or higher. The duration of the loading step may be any suitable amount of time, such as ranging from 1 minute to 24 hours, or more. For example, the range may be in the range of 1 minute to 24 hours, 1 minute to 18 hours, 1 minute to 12 hours, 1 minute to 6 hours, 1 minute to 1 hour, 30 minutes to 24 hours, 30 minutes to 18 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 1 hour, 1 to 24 hours, 1 to 18 hours, 1 to 12 hours, 1 to 6 hours, 6 to 24 hours, 6 to 18 hours, 6 to 12 hours, 12 to 24 hours, 12 to 18 hours, or 18 to 24 hours. In certain embodiments, the cell culture medium is a basal medium or a complex medium. In some cases, the culture includes one or more reagents utilized during the preactivation and / or expansion steps, while in other cases the culture does not. In certain embodiments, the culture includes one or more cytokines, including, for example, one or more of IL-12, IL-15, IL-2, and IL-18. In some embodiments, the culture includes any type of APC.

[0048] The antibodies of the composition are provided in an effective amount to an effective amount of the NK cells of the present disclosure, thereby generating a complex that is "chimeric antigen receptor-like." In particular, the antigen-binding domain of the antibody binds to the NK cell, such as via an antigen that is a cell surface protein. Multiple antibodies may be provided to multiple NK cells, such that multiple cell / antibody complexes are present. The antibodies may be of any one time, including monospecific, bispecific, or multispecific, and in certain cases, the antibody engages both the NK cell and the target antigen via the antigen-binding domain of the antibody (e.g., by an engager in the art that is a fusion protein consisting of two single-chain variable fragments (scFv) of different antibodies). In an example where the antibody is monospecific, the antigen-binding domain of the antibody binds to a target antigen, such as a cancer antigen, and another portion of the antibody, such as the Fc region of the antibody, binds to the NK cell. When the antibody is multispecific, one or more antigen-binding domains of the antibody bind to the NK cell (e.g., via an NK cell surface antigen) and one or more antigen-binding domains of the antibody bind to one or more target antigens. Multispecific antibodies may be, for example, bispecific, trispecific, or tetraspecific. If the antibody is trispecific or tetraspecific, the additional antigen-binding domains may bind to other cells, such as stem cells.

[0049] In certain embodiments, the antibody binds to any NK cell surface antigen (which may or may not be a receptor) on NK cells, such as CD16 (including CD16a or CD16b), CD56, c-type lectins, e.g., NKG2D, NKG2C, costimulatory molecules, e.g., CS1, DNAM, 2B4, CD2, NCR, or KIR, and can redirect NK cells to targets, thus increasing response and specificity against different tumors.

[0050] Formation of the complex can be by any suitable means such that conditions are sufficient for the appropriate region of the antibody to bind to the appropriate surface region of the NK cell. In some cases, any particular medium can be utilized. In certain cases, plasma-Lyte A and / or human serum albumin are utilized, and in other cases they are not. Once the complex is formed in culture, it may or may not be washed prior to administration to a subject, such as via injection. In an alternative embodiment, the NK cells and the antibody are administered separately, and the complex is formed in vivo. C. Preactivation

[0051] In some embodiments, the NK cells are preactivated prior to administration to the recipient individual. The preactivation step may or may not be performed prior to any expansion step. In certain embodiments, the NK cells are preactivated with one or more cytokines, and in certain embodiments, the NK cells are preactivated with one or more, e.g., two, three, or more, of IL-12, IL-15, IL-2, and IL-18. If less than three of IL-12, IL-15, IL-2, and IL-18 are utilized, it may be IL-12 and IL-15, but not IL-18; or IL-12 and IL-18, but not IL-15; or IL-15 and IL-18, but not IL-12. IL-2 may or may not be used in place of IL-15.

[0052] In certain embodiments, the preactivation cytokines may be IL-12, IL-15 and IL-18. One or more additional cytokines may also be used for the preactivation step. Preactivation may be for a short period of time, such as 5 to 72 hours, for example 10 to 50 hours, in particular 10 to 20 hours, for example 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours, in some cases in particular about 16 hours. The preactivation culture may contain IL-18 and / or IL-15 at a concentration of 10 to 100 ng / mL, for example 40 to 60 ng / mL, in particular 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 ng / mL, in particular about 50 ng / mL. In some cases, the pre-activation culture comprises IL-12 at a concentration of 0.1-150 ng / mL, including at a concentration of 1-20 ng / mL, such as at a concentration of 10 ng / mL. In alternative embodiments, the NK cells may be stimulated with IL-2, or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-21, etc.), which may be in addition to or in place of one or more of IL-12, IL-15, and IL-18. In such cases, the pre-activation culture may comprise IL-12 at a concentration of 0.1-150 ng / mL, including at a concentration of 0.5-50 ng / mL, particularly 1-20 ng / mL, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, particularly at a concentration of about 10 ng / mL. D. Proliferation

[0053] In certain embodiments, the NK cells are expanded to increase their amount before administration to an individual in need thereof. The expanded cells may or may not be derived from preactivated NK cells, such that a preactivation step may occur prior to the expansion step. The NK cell expansion step may be any suitable one such that the NK cell population expands, but in certain cases, the expansion step utilizes a particular one or more reagents, such as during culture, to enhance their expansion. In some cases, the NK cells may not be expanded. IL-2 or IL-15 or IL-18 or any combination of cytokines may be added to the expansion culture before or during expansion. The NK cells may be expanded ex vivo in certain embodiments in flasks or in one of several different bioreactor configurations with continuous perfusion of media / additives.

[0054] In certain cases, the NK cells (whether preactivated or not) may be washed 2, 3, 4, or 5 times, particularly 3 times, before and / or after expansion (e.g., with PBS or Plasma Lyte or human serum albumin or culture medium or a combination thereof). In certain embodiments, the NK cells are expanded in the presence of artificial antigen presenting cells (aAPCs). The aAPCs may be engineered to express CD137 ligand and / or membrane-bound cytokines. The membrane-bound cytokines may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, the aAPCs are engineered to express CD137 ligand and mIL-21. The aAPCs may be derived from cancer cells, such as leukemia cells. The aAPCs cannot express endogenous HLA class I, II, or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58) or CD48. In particular, the aAPCs may be K562 cells, e.g., K562 cells engineered to express CD137 ligand and mIL-21. Engineering may be by any method known in the art, such as retroviral transduction, but may utilize any viral or non-viral vector. The aAPCs may or may not be irradiated. Expansion may be for a particular period of time, e.g., about 2-30 days, e.g., 3-20 days, particularly 12-16 days, e.g., 12, 13, 14, 15, 16, 17, 18 or 19 days, particularly about 14 days. The preactivated NK cells and the aAPCs may be in a ratio of about 3:1-1:3, e.g., 2:1, 1:1, 1:2, particularly about 1:2. The expansion culture may further include one or more cytokines to promote proliferation, such as IL-2. IL-2 may be present at a concentration of about 10-500 U / mL, e.g., 100-300 U / mL, particularly about 200 U / mL. IL-2 may be supplemented in the expansion cultures at some frequency, e.g., every 2-3 days. aAPCs may be added to the cultures at least a second time, e.g., at about day 7 of expansion. Any cytokine(s) used in the preactivation and / or expansion steps may be recombinant human cytokines.

[0055] After expansion, the NK cells can be immediately utilized in any manner, such as by complexing with one or more antibodies, or can be preserved, such as by cryopreservation. In certain embodiments, the cells can be expanded ex vivo as a bulk population within about 1, 2, 3, 4, or 5 days, for days, weeks, or months.

[0056] Activated and / or expanded NK cells secrete type I cytokines, such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells, as well as other cytokines and chemokines. Measurement of these cytokines can be used to determine the activation state of NK cells. Additionally, other methods known in the art for determining NK cell activation can be used to characterize the NK cells of the present disclosure.

[0057] Thus, with regard to certain preactivation and expansion aspects of the present disclosure, in certain embodiments, NK cells preactivated with IL-12, IL15 and IL-18 and subsequently expanded with aAPCs, e.g., K562 cells expressing mIL-21 and CD137 ligand, provide highly potent cell products. Thus, methods of using the present NK cells for the treatment of various diseases, such as immunotherapy of patients with cancer, are provided. In an exemplary method, isolated NK cells can be subjected to a short period of preactivation, e.g., about 16 hours, with a combination of cytokines, such as interleukin-12 (IL-12), IL-15 and / or IL-18, and then expanded using artificial antigen presenting cells (aAPCs), e.g., K562 feeder cells expressing membrane-bound IL-21 and CD137 ligand, and / or exogenous IL-2. IL-2 or IL-15 or IL-18 or any combination of cytokines can be added to the expansion culture at least a second time. E. Other Variations

[0058] In some embodiments, NK cells are modified in one or more ways compared to natural NK cells. NK cells can be engineered by hand to express one or more heterologous proteins. Alternatively, or in addition, NK cells can be modified to reduce or inhibit the expression of one or more endogenous genes. In other cases, NK cells are modified to express one or more cytokines or suicide genes. 1. Engineered antigen receptors

[0059] NK cells may be genetically modified to express one or more engineered antigen receptors, including at least one or more chimeric antigen receptors (CARs) and / or one or more TCRs. In certain embodiments, the engineered antigen receptors are directed to target one or more cancer antigens, which may or may not be the same antigens as those directed by the multispecific antibody (e.g., engager).

[0060] The NK cells may be modified to encode at least one CAR, which may be, for example, a first generation, a second generation, or a third or subsequent generation. The CAR may or may not be bispecific for two or more different antigens. The CAR may include one or more costimulatory domains. The NK cells may also be modified to express receptors to enhance their binding to antibodies, such as CD16, CD32, and / or CD64 receptors. Each costimulatory domain may include, for example, any one or more costimulatory domains of members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD27, NKG2D, 2B4M, CD40, or combinations thereof. In certain embodiments, the CAR includes CD3 zeta. In one embodiment, the CAR lacks one or more specific costimulatory domains, for example, the CAR may lack 4-1BB and / or may lack CD28.

[0061] In certain embodiments, the CAR polypeptide in cells comprises an extracellular spacer domain that connects the antigen-binding domain and the transmembrane domain, which may be called a hinge.The extracellular spacer domain may include, but is not limited to, the Fc fragment of an antibody or its fragment or derivative, the hinge region of an antibody or its fragment or derivative, the CH2 region of an antibody, the CH3 region of an antibody, an artificial spacer sequence, or a combination thereof.Examples of extracellular spacer domains include, but are not limited to, CD8-alpha hinge, CD28, artificial spacer made of polypeptides such as Gly3, or the CH1, CH3 domains of IgG (such as human IgG1 or IgG4). In certain cases, the extracellular spacer domain can comprise (i) an IgG4 hinge region, a CH2 region, and a CH3 region, (ii) an IgG4 hinge region, (iii) an IgG4 hinge region and a CH2, (iv) a CD8-alpha or CD4 hinge region, (v) an IgG1 hinge region, a CH2 region, and a CH3 region, (vi) an IgG1 hinge region or (vii) an IgG1 hinge region and a CH2, (viii) a CD28 hinge region, or a combination thereof. In certain embodiments, the hinge is derived from IgG1, and in certain embodiments, the CAR polypeptide comprises a particular IgG1 hinge amino acid sequence or is encoded by a particular IgG1 hinge nucleic acid sequence.

[0062] The transmembrane domain in the CAR can be derived from either natural or synthetic sources. When the source is natural, in some embodiments the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from (i.e., at least including) the transmembrane region(s) of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules such as DAP10 or DAP12. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some embodiments, the synthetic transmembrane domain comprises mainly hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan and valine can be found on either side of the synthetic transmembrane domain.

[0063] In some embodiments, when the amount of tumor-associated antigen is low, the CAR can be co-expressed with one or more cytokines to improve persistence. For example, the CAR can be co-expressed with one or more cytokines, such as IL-7, IL-2, IL-15, IL-12, IL-23, IL-18, IL-21, IL-7, GMCSF, or a combination thereof. In some embodiments, the NK cells expressing the CAR are engineered to express one or more heterologous cytokines and / or to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected with one or more cytokines on the same vector as other genes.

[0064] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be 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 shown to stabilize mRNA. It may also be more advantageous to stabilize mRNA using endogenous or exogenous non-coding regions.

[0065] It is contemplated that chimeric constructs can be introduced into any kind of immune cells as naked DNA or in suitable vector.Methods for stable transfection of 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 the DNA that codes for chimeric receptors that is contained in a plasmid expression vector in suitable orientation for expression.

[0066] Alternatively, the chimeric construct can be introduced into immune cells using a viral vector (e.g., retroviral vector, adenoviral vector, adeno-associated viral vector or lentiviral vector). The vector suitable for use according to the method of the present disclosure does not replicate in immune cells. Many virus-based vectors are known, such as HIV, SV40, EBV, HSV or BPV-based vectors, in which the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell. Non-viral vectors include plasmids, transposons, nanoparticles, liposomes, lipids, metals, or combinations thereof.

[0067] In some embodiments, the engineered antigen receptor comprises a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that comprises variable a and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively) and can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0068] TCRs, which usually exist in αβ and γδ forms, are generally structurally similar, although the T cells expressing them may have different anatomical locations or functions. 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) and are generally involved in the recognition of antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may 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 embodiments, each chain of a TCR may possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with invariant proteins of the CD3 complex, which are 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.

[0069] 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 to an MHC molecule, i.e., an MHC-peptide complex. The "antigen-binding portion" or antigen-binding fragment of a TCR can be used interchangeably and refers to a molecule that includes a portion of the structural domain of a TCR, yet binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion includes sufficient variable domains of the TCR, such as the variable α and β chains of the TCR, to form a binding site for binding to a specific MHC-peptide complex, and generally each chain includes three complementarity determining regions.

[0070] In some embodiments, the variable domains of the TCR chains are involved in the formation of loops or immunoglobulin-like complementarity determining regions (CDRs), which form the binding site of the TCR molecule, thereby conferring antigen recognition and determining peptide specificity. Usually, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR involved in recognition of processed antigens, while CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, and CDR1 of the beta chain interacts with the C-terminal portion of peptides. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain can include an additional hypervariable (HV4) region.

[0071] In some embodiments, the TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., a chain, β chain) comprises two immunoglobulin domains at the N-terminus, a variable domain (e.g., V aor Vp; typically, the Kabat numbering, 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 ed.) and one constant domain adjacent to the cell membrane (e.g., the a-chain constant domain or C a , typically amino acids 117-259 based on Kabat, and a β chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two membrane proximal constant domains, and two membrane distal variable domains that include the CDRs. The constant domain of the TCR domain includes a short connecting sequence in which cysteine ​​residues form disulfide bonds to form a link between the two chains. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α and β chains such that the TCR includes two disulfide bonds in the constant domain.

[0072] In some embodiments, the TCR chain can include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, this structure allows the TCR to bind to other molecules, such as CD3. For example, a TCR that includes a constant domain with a transmembrane region can anchor the protein to the cell membrane and bind to the invariant subunit of the CD3 signaling apparatus or complex.

[0073] In general, CD3 is a multiprotein complex that can have three different chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex can contain a homodimer of CD3γ, CD3δ, two CD3ε, and CD3ζ chains. CD3γ, CD3δ, and CD3ε chains are cell surface proteins that are highly related to the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that allows these chains to bind to the positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, although each CD3ζ chain has three. In general, ITAMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating signals from the TCR into the cell. The CD3 chain and the ζ chain together with the TCR form what is known as the T cell receptor complex.

[0074] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or a single chain TCR construct. In some embodiments, the TCR is a heterodimer, for example, comprising two separate chains (α and β or γ and δ chains) linked by one or more disulfide bonds. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, for example, from cells such as T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, the T cells can be obtained from cells isolated 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 cells can be cultured T cell hybridomas or clones. In some embodiments, TCR clones against target antigens have been generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, e.g., 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 from knowledge of the sequence of the TCR.

[0075] The CAR and TCR of the present disclosure target one or more specific antigens. Some of the antigens targeted by the engineered antigen receptor are expressed in association with the disease, condition, or cell type targeted by adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including lymphomas, leukemias, and / or myelomas, such as B-cell, T-cell, and myeloid leukemias, lymphomas, and multiple myelomas, blood cancers, and cancers of the immune system. In some embodiments, the antigen is selectively expressed or overexpressed on disease or pathological cells, such as tumor cells or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0076] Any suitable antigen can be targeted in this method. The antigen may in some cases be associated with a cancer cell, but not with a non-cancerous cell. Exemplary antigens include, but are not limited to, antigenic molecules from infectious pathogens, self / autoantigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In certain embodiments, antigens include CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD70, CD38, trop2, HLA-G, CD123, CLL1, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epidermal tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A1 ... E-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosine-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK Receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPI / mbcr-abl,BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-kappa B (NF-B), Notch receptors (e.g., Notch1-4), NY ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Exemplary antigen sequences are known in the art and are found, for example, in the GenBank® database: CD19 (Accession No. NG_007275.1);EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number NC_000023.11), EGFRvIII (accession number NG_007726.3), MUC1 (accession number NG_0029383.1), HER2 (accession number NG_007503.1), CA-125 (accession number NG_007503.1), No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage-A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-A10 (Accession No. NC_000023.11), TRAIL / DR4 (Accession No. NC_000003.12) and / or CEA (Accession No. NC_000019.10).

[0077] Tumor-associated antigens can be derived from, for example, prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3 and MAGE4 (or other MAGE antigens such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also called NY ESO1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six transmembrane epithelial antigen of the prostate (STEAP).

[0078] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. In addition, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), short 10 amino acid long peptides, which are useful in the treatment of many cancers.

[0079] Antigens can also include epitope regions or epitope peptides derived from genes mutated in tumor cells or transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutated ras, bcr / abl rearrangements, Her2 / neu, mutated or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens that include epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins that exhibit tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein. 2. The suicide gene

[0080] In certain embodiments, suicide genes are utilized in conjunction with antibody combination therapy to control their use and allow for the termination of cell therapy at a desired event and / or time. Suicide genes are used in transduced cells to induce the death of the transduced cells as needed. Cells of the present disclosure modified to carry vectors encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that results in the transfer of a gene product to a compound that kills the host cell upon administration of a prodrug or other drug. In other embodiments, the suicide gene encodes a gene product that, when desired, is targeted by a drug (such as an antibody) that targets the suicide gene product.

[0081] In some cases, when an individual undergoing and / or having undergone cell therapy is deemed to be at risk of having one or more symptoms, including imminent, of one or more adverse events, such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity (as examples), the cell therapy may be subjected to the use of one or more suicide genes of any kind. The use of suicide genes may be part of a planned protocol for treatment, or may be used only when its use is recognized as necessary. In some cases, the cell therapy is terminated by the use of an agent(s) targeting the suicide gene or a gene product therefrom, such that the cell therapy is no longer required.

[0082] The use of suicide genes can be triggered upon the onset of at least one adverse event to an individual, which adverse event can be recognized by any means, including during routine monitoring, which may or may not be continuous from the beginning of cell therapy. The adverse event(s) can be detected during examination and / or testing. If an individual has cytokine release syndrome (which may also be referred to as cytokine storm), the individual can have, for example, elevated inflammatory cytokine(s) (just for example: interferon-gamma, granulocyte macrophage colony stimulating factor, IL-10, IL-6 and TNF-alpha); fever; fatigue; hypotension; hypoxia, tachycardia; nausea; capillary leakage; cardiac / renal / liver dysfunction; or a combination thereof. If an individual has neurotoxicity, the individual can have confusion, delirium, hypoplasia and / or seizures. In some cases, the individual is tested for markers related to the onset and / or severity of cytokine release syndrome (such as, for example, C-reactive protein, IL-6, TNF-alpha and / or ferritin).

[0083] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT / US19 / 62009, incorporated herein by reference in its entirety), which can be affected by delivery of antibodies that bind to the TNF-alpha mutants. Examples of suicide gene / prodrug combinations that can be used are 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. E. coli purine nucleoside phosphorylase is a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, and can be used. Other suicide genes include, by way of example, CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYPs), carboxypeptidases (CPs), carboxylesterases (CEs), nitroreductases (NTRs), guanine ribosyltransferase (XGRTPs), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0084] In certain embodiments, the vector encoding the CAR or any vector in the NK cell encompassed herein comprises one or more suicide genes.The suicide gene may or may not be on the same vector as the CAR.When the suicide gene is on the same vector as the CAR, the suicide gene and the CAR can be separated, for example, by an IRES or 2A element. 3. Cytokines

[0085] In some embodiments, the NK cell expressing cells are engineered to express one or more heterologous cytokines and / or engineered to upregulate normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected with the one or more cytokines on the same vector as other genes.

[0086] One or more cytokines may be co-expressed from the vector, for example, as separate polypeptides from the engineered antigen receptor and / or suicide gene. For example, interleukin-15 (IL-15) is tissue restricted and is only observed at any level in serum or systemically under pathological conditions. IL-15 possesses several attributes that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that induces natural killer cell development and cell proliferation, promotes eradication of established tumors by relieving functional suppression of tumor-resident cells, and inhibits activation-induced cell death (AICD). In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. NK cells expressing IL-15 are capable of continued supportive cytokine signaling useful for survival after infusion.

[0087] In certain embodiments, the cells express one or more exogenously provided cytokines. As an example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. The cytokines can be exogenously provided to the NK cells as they are expressed from an expression vector in the cells. In alternative cases, endogenous cytokines in the cells are upregulated by manipulating the regulation of expression of the endogenous cytokine, such as genetic modification at the cytokine's promoter(s). When the cytokine is provided to the cells in an expression construct, the cytokine can be encoded by the same vector as the suicide gene and / or the CAR. In some embodiments, the present disclosure relates to the co-use of a CAR with IL-15, and optionally a suicide gene. 4. Knockout or knockdown of exogenous genes

[0088] The disclosed NK cell generation method may include gene editing of NK cells to remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous genes in the NK cells. In some cases, gene editing occurs in NK cells that express one or more xenogeneic antigen receptors, while in other cases, gene editing occurs in NK cells that do not express xenogeneic antigen receptors, but in at least some cases, ultimately express one or more xenogeneic antigen receptors. In certain embodiments, the gene-edited NK cells are expanded NK cells.

[0089] In certain cases, one or more endogenous genes of NK cells are modified, for example, expression is disrupted so that expression is partially or completely reduced.In certain cases, one or more genes are knocked down or knocked out using the method of the present disclosure.In certain cases, multiple genes are knocked down or knocked out in the same step of the method of the present disclosure.The gene that is edited in NK cells can be of any kind, but in certain embodiments, the gene is a gene whose gene product inhibits the activity and / or proliferation of NK cells.In certain cases, the gene that is edited in NK cells allows NK cells to function more effectively in tumor microenvironment. In certain cases, the gene is one or more of 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, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, beta2-microglobulin, HLA, CD73, and CD39. In certain embodiments, the TGFBR2 gene is knocked out or down in NK cells.

[0090] In some embodiments, gene editing is carried out using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonucleases (RGENs). For example, modification can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In general, "CRISPR system" refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci. Methods of utilizing CRISPR systems are well known in the art. F. Cryopreservation

[0091] In certain cases, the NK cells and / or antibodies of the present disclosure are stored in a cryopreservation medium composition comprising at least one cryoprotectant, serum (human or animal serum) or a non-serum serum substitute (not human or animal serum), and at least one cytokine and / or at least one growth factor. In some cases, the cryoprotectant is dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethol starch, or a combination thereof. The non-serum substitute can be of any type, including at least platelet lysate and / or blood product lysate (e.g., human serum albumin). In embodiments of the composition in which one or more (including two or more) cytokines are utilized, the cytokines can be natural or recombinant or synthetic proteins. At least one of the cytokines can be a Food and Drug Administration (FDA) approved cytokine. Examples of cytokines and growth factors include at least IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferons, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or combinations thereof. For serum embodiments, the serum can be human serum (including human AB serum) or animal derived serum such as bovine serum. When DMSO and other cryoprotectants are utilized, they can comprise 4-10%, 4-6%, 4-8%, 5-10%, 5-8%, 6-10%, 6-8%, 8-10%, etc. of the composition.In embodiments in which serum is used, the serum may comprise 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, 10-1 ... 0~60%, 10~55%, 10~50%, 10~45%, 10~40%, 10~35%, 10~30%, 10~25%, 10~20%, 10~15%, 20~99%, 20~95%, 20~90%, 20~85%, 20~80%, 20~75%, 20~70%, 20~65%, 20~60%, 20~55%, 20~50%, 20~45%, 20~40%, 20~35%, 20~30%, 20~25%, 30~99%, 30~95%, 3 0~90%, 30~85%, 30~80%, 30~75%, 30~70%, 30~65%, 30~60%, 30~55%, 30~50%, 30~45%, 30~40%, 30~35%, 40~99%, 40~95%, 40~90%, 40~85%, 40~80%, 40~75%, 40~70%, 40~65%, 40~60%, 40~55%, 40~50%, 40~45%, 50~99%, 50~95%, 50~90%, 50~85%, 5 Constitutes 0-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99%. The composition may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less serum.In certain embodiments, the composition comprises platelet lysate which may be at any concentration in the composition, however in certain embodiments, the platelet lysate is present in an amount of 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85 ...99%, 10-95%, 10-90%, 10-99%, 10-95%, 10-90%, 10-99%, 10-99 %, 10~80%, 10~75%, 10~70%, 10~65%, 10~60%, 10~55%, 10~50%, 10~45%, 10~40%, 10~35%, 10~30%, 10~25%, 10~20%, 10~15%, 20~99%, 20~95%, 20~90%, 20~85%, 20~80%, 20~75%, 20~70%, 20~65%, 20~60%, 20~55%, 20~50%, 20~45%, 20~40%, 20~35%, 20~30%, 2 0~25%, 30~99%, 30~95%, 30~90%, 30~85%, 30~80%, 30~75%, 30~70%, 30~65%, 30~60%, 30~55%, 30~50%, 30~45%, 30~40%, 30~35%, 40~99%, 40~95%, 40~90%, 40~85%, 40~80%, 40~75%, 40~70%, 40~65%, 40~60%, 40~55%, 40~50%, 40~45%, 50~99%, 50~95%, 50~9 constitute 0%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99%. The composition may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less platelet lysate.

[0092] The composition may have components including cytokines and / or growth factors at specific concentrations. In certain cases, any cytokine, including, for example, IL-2, IL-21 and / or IL-15, is present in the composition at a specific concentration. IL-2 may be present, for example, at a concentration of 1-5000, 1-1000, 1-500, 1-100, 100-5000, 100-500, 500-5000, 500-1000, or 1000-5000 U / mL. In certain cases, IL-2 is present in the composition at a concentration of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 U / mL, or less. In certain embodiments, IL-21 is present in the composition at a concentration of 10-3000, 10-2000, 10-1000, 10-500, 10-100, 100-3000, 100-2000, 100-1000, 500-3000, 500-2000, 500-1000, 1000-3000, 1000-2000 or 2000-3000 ng / mL. IL-21 may be present in the composition at a concentration of at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750 or 3000 ng / mL, or less. IL-15 may be present in the composition at a concentration of 1-2000, 1-1000, 1-500, 1-100, 100-2000, 100-1000, 100-500, 500-2000, 500-1000, or 1000-2000 ng / mL. IL-15 may be present in the composition at a concentration of at least 10, 50, 100, 500, 1000, 1500, or 2000 ng / mL, or less.

[0093] The compositions encompassed herein, which include at least one cryoprotectant, serum or non-serum serum substitute, and at least one cytokine and / or at least one growth factor, may each further comprise any type of multiple immune cells and / or stem cells. In certain embodiments, the cells are NK cells, T cells, B cells, NKT cells derived from mature bone marrow or peripheral blood cells, cell lines such as tumor cell lines (e.g., NK92 or other NK lines), hematopoietic stem cells, induced pluripotent stem cells, MSCs (populations of cells alternatively referred to in the literature as "mesenchymal stem cells" and "mesenchymal stromal cells") that may be derived from bone marrow, peripheral blood, skin, adipose tissue, or combinations thereof, or mixtures thereof. In embodiments in which NK cells are utilized, the NK cells may or may not be expanded NK cells. Embodiments of the present disclosure also encompass pharmaceutical compositions comprising any of the compositions of the present disclosure and a suitable pharma- ceutically acceptable carrier.

[0094] In certain embodiments, the cells of the present disclosure at any step are preserved in the following specific formulations for cryopreservation medium: [Table 1]

[0095] Examples of specific formulations at specific concentrations are available as follows: [Table 2] II.How to use

[0096] An embodiment of the present disclosure relates to a method of using a composition comprising NK cells and an antibody provided herein to treat or prevent a medical disease or disorder. The method includes administering a therapeutically effective amount of loaded, optionally preactivated, and optionally expanded NK cells and an antibody to a subject, and thereby treating or preventing a disease in the subject, including reducing the risk of the disease, reducing the severity of the disease, and / or delaying the onset of the disease. In an embodiment of the present disclosure, a cancer or infectious disease is treated by the transfer of a composition comprising an NK cell population and an antibody that induces an immune response. In at least some cases, due to the release of pro-inflammatory cytokines, NK cells can increase the adaptive immune response by reversing the anti-inflammatory tumor microenvironment and promoting the differentiation, activation, and / or recruitment of accessory immune cells to the malignant tumor site.

[0097] The cancers for which the present method of treatment is useful include any malignant cell type, such as those found in solid tumors or blood tumors.Exemplary solid tumors can include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast.Exemplary blood tumors include tumors of bone marrow, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0098] Cancer may be of the following histological types, including, but not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma adenocarcinoma);papillary adenocarcinoma;chromophobe carcinoma;eosinophilic carcinoma;eosinophilic adenocarcinoma;basophilic carcinoma;clear cell adenocarcinoma;granular cell carcinoma;follicular adenocarcinoma;papillary-follicular adenocarcinoma;nonencapsulated sclerosing carcinoma;adrenal cortical carcinoma;endometrioid carcinoma;adnexal carcinoma;apocrine adenocarcinoma;sebaceous gland carcinoma;ceruminous gland carcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease of the breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia metaplasia);(thymoma, malignant);ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;angiosarcoma;malignant melanoma;amelanotic melanoma;superficial proliferative melanoma;lentigo maligna melanoma;acral lentigo melanoma;nodular melanoma;malignant melanoma of giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant Sex;Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Mullerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Mesenchymal cell tumor, malignant;Brenner tumor, malignant;Phyllodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocoma;Mesonephroma, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parosteal osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal malignant;cerebellar sarcoma;ganglioneoblastoma;neuroblastoma;retinoblastoma;olfactory nerve tumor;meningioma, malignant;neurofibrosarcoma;schwannoma, malignant;granular cell tumor, malignant;malignant lymphoma;Hodgkin's disease;Hodgkin's granuloma;malignant lymphoma, small lymphocytic;malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular;mycosis fungoides;other specified non-Hodgkin's lymphoma;B-cell lymphoma;low-grade / follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL;intermediate-grade / follicular NHL;intermediate-grade diffuse NHL;high-grade immunoblastic NHL;high-grade lymphoblastic NHL;high-grade small noncleaved cell NHL;bulk NHL;mantle cell lymphoma;AIDS-related lymphoma;Wanderström's macroglobulinemia;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoglobulinemia Epidemiological proliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroleukemia;Lymphoblastic cell leukemia;Myeloid leukemia;Basophilic leukemia;Eosinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryoblastic leukemia;Myelosarcoma;Hairy cell leukemia;Chronic lymphocytic leukemia (CLL);Acute lymphoblastic leukemia (ALL);Acute myeloid leukemia (AML);andChronic myeloblastic leukemia.;

[0099] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by the abnormal proliferation (production by doubling) of blood cells, usually white blood cells (leukocytes). It is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that covers a wide range of diseases. Leukemia is clinically and pathologically divided into acute and chronic types.

[0100] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding prevents the bone marrow from producing healthy blood cells. Acute forms of leukemia can occur in children and young adults. In fact, in the United States, leukemia is a more common cause of childhood death than any other type of malignant disease. In acute leukemia, immediate treatment is required due to the rapid progression and accumulation of malignant cells, which then spill into the bloodstream and spread to other organs of the body. Although central nervous system (CNS) involvement is rare, the disease can occasionally cause cranial nerve palsies. Chronic leukemia is distinguished by an excessive proliferation of relatively mature but still abnormal blood cells. Typically, it takes months to years to progress, and the cells are produced at a much faster rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia occurs primarily in older adults, but theoretically can occur in any age group. While acute leukemia must be treated immediately, chronic forms may be monitored for some time before treatment to ensure maximum effectiveness of treatment.

[0101] The disease is further classified as lymphocytic or lymphoblastic, which indicates that cancerous changes have occurred in the type of bone marrow cells that normally go on to form lymphocytes, and myeloid or myeloid, which indicates that cancerous changes have occurred in the type of bone marrow cells that normally go on to form red blood cells, some types of white blood cells, and platelets (see lymphoid vs. myeloid cells).

[0102] Acute lymphocytic leukemia (also known as acute lymphoblastic leukemia or ALL) is the most common type of leukemia in children. The disease also affects adults, especially those over 65 years of age. Chronic lymphocytic leukemia (CLL) most often affects adults over 55 years of age. It can occur in younger adults, but rarely affects children. Acute myeloid leukemia (also known as acute myelocytic leukemia or AML) occurs more often in adults than in children. This type of leukemia was previously called "acute nonlymphocytic leukemia." Chronic myeloid leukemia (CML) occurs primarily in adults. A very small number of children also develop the disease.

[0103] Lymphoma is a type of cancer that originates in lymphocytes (a type of white blood cell in the immune system of vertebrates). There are many types of lymphoma. According to the National Institutes of Health, lymphoma accounts for approximately 5% of all cancer cases in the United States, with Hodgkin's lymphoma accounting for less than 1% of all cancer cases in the United States. Because the lymphatic system is part of the body's immune system, patients with weakened immune systems, such as those with HIV infection or certain drugs or medications, are also at increased risk of developing lymphoma.

[0104] In certain embodiments of the present disclosure, a composition comprising NK cells and an antibody is delivered to an individual in need thereof, such as an individual with cancer or an infectious disease. In at least some cases, the cells can strengthen the individual's immune system to attack the respective cancer or pathogenic cells. In some cases, the individual is provided with one or more doses of the composition comprising NK cells and an antibody. If an individual is provided with two or more doses of NK cells / antibodies, the period between administrations should be sufficient to allow time for proliferation in the individual, and in certain embodiments, the period between doses is 1, 2, 3, 4, 5, 6, 7 days or more.

[0105] The source of the preactivated (optional) and expanded NK cells can be of any type, but in certain embodiments, the cells are obtained, for example, from umbilical cord blood, peripheral blood, a bank of human embryonic stem cells or induced pluripotent stem cells. A suitable dose for therapeutic effect is, for example, at least 10 per dose, preferably in a series of administration cycles. 5 Pieces or about 10 5 ~about 10 12 An exemplary dosing regimen includes at least about 10 cells on day 0. 5 Starting with cells, for example, within a few weeks of initiating an intrapatient dose escalation scheme, 12 The treatment consists of four weekly dosing cycles of increasing doses, incrementally increasing to a target dose of cells. Suitable modes of administration include intravenous, subcutaneous, intracavitary (e.g., via a reservoir access device), intraperitoneal, and direct injection into the tumor mass.

[0106] Compositions comprising NK cells and antibodies produced according to the methods of the present invention have many potential uses, including experimental and therapeutic uses. In particular, it is envisioned that such cell populations are useful for suppressing undesirable or inappropriate immune responses. In such methods, a small number of NK cells are removed from a patient, then manipulated and expanded ex vivo, and then reinjected into the patient. Examples of diseases that can be treated in this manner are autoimmune diseases and conditions in which suppression of immune activity is desirable, for example, for allograft tolerance. The method of treatment includes obtaining NK cells from a mammal, expanding the NK cells ex vivo according to the methods of the present methods described herein, exposing the NK cells to an antibody under sufficient conditions, and administering a composition comprising the expanded NK cells / antibody to the mammal to be treated.

[0107] The pharmaceutical compositions of the present disclosure can be used alone or in combination with other well-established agents useful in the treatment of cancer. Whether delivered alone or in combination with other agents, the pharmaceutical compositions of the present disclosure can be delivered to various sites in the mammalian, particularly human, body via various routes to achieve a particular effect. Those skilled in the art will recognize that more than one route can be used for administration, but that certain routes can provide a more rapid and effective response than another route. For example, intradermal delivery can be used more advantageously than inhalation for the treatment of melanoma. Local or systemic delivery can be achieved by administration including application or instillation of the formulation into a body cavity, inhalation or insufflation of an aerosol, or by parenteral introduction, including intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous or intradermal administration.

[0108] In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal glands, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac polydermatitis, and the like. spate-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (minimal change nephrotic syndrome, focal glomerulosclerosis or membranous nephropathy) nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (such as polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis or dermatitis herpetiformis vasculitis), vitiligo and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis.The subject may also have an allergic disorder, such as asthma.

[0109] In yet another embodiment, the subject is a recipient of a transplanted organ or stem cells, and the expanded NK cells / antibodies of the present disclosure are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a possible complication of any transplant that uses or includes stem cells from either related or unrelated donors. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first three months after transplant. Signs of acute GVHD include a reddish skin rash on the hands and feet, which can spread with peeling and blisters of the skin and become more severe. Acute GVHD can also affect the stomach and intestines, where cramps, nausea, and diarrhea are seen. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is graded based on its severity, with stage / grade 1 being mild and stage / grade 4 being severe. Chronic GVHD develops after 3 months post-transplant. Symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD can affect the mucous glands of the eye, the salivary glands of the mouth, and the glands that lubricate the stomach lining and intestines. Any of the populations of NK cells disclosed herein can be utilized. Examples of transplanted organs include solid organ transplants such as kidney, liver, skin, pancreas, lung and / or heart, or cell transplants such as pancreatic islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The transplant can also be a composite transplant, such as facial tissue. NK cells, such as immunosuppressive CD19+ cells, can be administered with antibodies prior to, simultaneously with, and / or after transplantation. In some embodiments, the composition comprising NK cells and an antibody is administered prior to transplantation, e.g., at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplantation. In one specific, non-limiting example, administration of a therapeutically effective amount of the composition comprising NK cells and an antibody occurs 3-5 days prior to transplantation.

[0110] The NK cells / antibodies administered to the patient undergoing transplantation can be sensitized with antigens specific to the transplanted material prior to administration. According to this embodiment, the transplant recipient will have a reduced immune / inflammatory response to the transplanted material, thus minimizing the possibility of rejection of the transplanted tissue. Similarly, for the treatment of graft-versus-host disease, the NK cells can be sensitized with antigens specific to the host. According to this embodiment, the recipient will have a reduced immune / inflammatory response to self-antigens.

[0111] In further embodiments, administering to a subject a therapeutically effective amount of a composition comprising NK cells and an antibody treats or inhibits inflammation in the subject. Thus, the method comprises administering to a subject a therapeutically effective amount of a composition comprising NK cells and an antibody to inhibit an inflammatory process. Examples of inflammatory disorders include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infection. The methods disclosed herein can also be used to treat allergic disorders.

[0112] The administration of the composition comprising NK cells and antibodies can be utilized whenever immunosuppression or inhibition of inflammation is desired, for example, at the first sign or symptom of disease or inflammation. These may be general, such as pain, edema, high fever, or specific signs or symptoms related to dysfunction of affected organ(s). For example, in renal transplant rejection, there may be an increase in serum creatinine level, while in GVHD, there may be a rash, and in asthma, there may be shortness of breath and wheezing.

[0113] Administration of a composition comprising NK cells and an antibody can also be utilized to prevent immune-mediated disease in a subject of interest. For example, a composition comprising NK cells and an antibody can be administered to a subject who will be a transplant recipient prior to transplant. In another example, a composition comprising NK cells and an antibody is administered to a subject undergoing an allogeneic bone marrow transplant without T cell depletion. In a further example, a composition comprising NK cells and an antibody can be administered to a subject with a family history of diabetes. In another example, a composition comprising NK cells and an antibody can be administered to a subject with asthma to prevent asthma attacks. In some embodiments, a therapeutically effective amount of a composition comprising NK cells and an antibody is administered to a subject prior to symptoms. Administration of a composition comprising NK cells and an antibody results in reduced morbidity or severity of a subsequent immunological event or symptom (such as an asthma attack) or improved patient survival compared to patients receiving other treatments that do not include a composition comprising NK cells and an antibody.

[0114] In some embodiments, the composition comprising NK cells and antibodies is administered in combination with a second therapeutic agent. For example, the second therapeutic agent may comprise T cells, immunomodulatory agents, monoclonal antibodies, chemotherapeutic agents, hormone(s), any kind of drug, surgery, radiation, etc. In a non-limiting example, the immunomodulatory agent is lenalidomide, the monoclonal antibody is rituximab, ofatumumab, or lumiliximab, and the chemotherapeutic agent is fludarabine or cyclophosphamide.

[0115] The composition of the present disclosure can be provided in unit dosage form, and each dosage unit, such as injection, contains a predetermined amount of the composition alone or in appropriate combination with other active agents.The term unit dosage form used herein refers to a physically separate unit suitable as a unitary dosage for human and animal subjects, and each unit contains a predetermined amount of the composition of the present disclosure alone or in combination with other active agents, calculated in an amount sufficient to produce desired effect, and optionally together with pharma-ceutically acceptable diluents, carriers or vehicles.The specification of the unit dosage form of the present disclosure depends on the specific pharmacodynamics associated with the pharmaceutical composition in specific subjects.

[0116] Desirably, an effective amount or sufficient number of isolated transduced NK cells are present in the composition and introduced into the subject such that a long-term specific anti-tumor response is established to reduce tumor size or to eliminate tumor growth or regrowth that would otherwise result in the absence of such treatment. Desirably, the amount of NK cells reintroduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% reduction in tumor size when compared to otherwise identical conditions in which the NK cells are not present.

[0117] Thus, the amount of the composition comprising NK cells and antibodies administered should take into account the route of administration and should be such that the composition comprising NK cells and antibodies is introduced in sufficient numbers to achieve the desired therapeutic response. Furthermore, the amount of each active agent included in the compositions described herein (e.g., the amount per each cell contacted or the amount per specific body weight) may vary for different applications. In general, the concentration of NK cells is desirably at least about 1×10 in the subject being treated. 6 ~Approx. 1×10 12 NK cells, and even more preferably about 1×10 7 ~Approx. 5×10 10 The amount should be sufficient to provide 5 x 10 NK cells, but any suitable amount, e.g. up to 5 x 10 8 More than 1 x 10 cells, or below, for example 7 Either single or multiple cells can be utilized. Administration schedules can be based on well-established cell-based therapies (see, e.g., U.S. Pat. No. 4,690,915), or alternative continuous infusion strategies can be employed.

[0118] These values ​​provide a general guideline for the range of compositions comprising NK cells and antibodies that can be utilized by practitioners when optimizing the method of the present disclosure for the implementation of the method.The recitation of such ranges herein in no way excludes the use of greater or lesser amounts of components, which may be warranted in certain applications.For example, the actual dose and schedule may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on individual differences in pharmacokinetics, drug disposition and metabolism.Those skilled in the art can easily make any necessary adjustments according to the exigencies of a particular situation. III. Antibodies

[0119] Aspects of the present disclosure relate to the use of antibodies or functional fragments thereof in compositions that also include certain NK cells. The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to a target antigen, such as via an antigen-binding domain, and includes chimeric, humanized, fully human, monospecific, and multispecific (at least bispecific and trispecific, or higher) antibodies. As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides that contain antibody CDR domains that retain antigen-binding activity. In certain embodiments, the antibody comprises an scFv.

[0120] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent such as an antibody. An antigen can possess one or more epitopes capable of interacting with different antibodies.

[0121] The term "epitope" includes any region or portion of a molecule that can induce an immune response by binding to an immunoglobulin or T-cell receptor. Epitope determinants can include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture.

[0122] The epitope region of a given polypeptide can be identified using many different epitope mapping techniques well known in the art, including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, and protein display arrays.See, for example, Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986), see, for example, Epitope Mapping Protocols, supra. Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydrophobicity plots.

[0123] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but may include naturally occurring antibodies in camelids that may contain fewer chains, such as only heavy chains, in some instances. The antibodies disclosed herein may be derived from only a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or mouse source, while the constant regions are derived from a different animal source, such as human. Antibodies or binding fragments may be produced in hybridomas, by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes its derivatives, variants, fragments and muteins, examples of which are described below (Sela-Culang et al. Front Immunol. 2013;4:302;2013).

[0124] The term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of about 25,000 daltons and contain a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classes of light chains identified as kappa (κ) and lambda (λ). The term "VL fragment" refers to a fragment of the light chain of a monoclonal antibody that contains all or a portion of the light chain variable region, including the CDRs. The VL fragment may further contain a light chain constant region sequence. The variable region domain of the light chain is at the amino terminus of the polypeptide.

[0125] The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of about 50,000 daltons and contains a variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" refers to a fragment of a heavy chain of a monoclonal antibody that contains all or a portion of the heavy chain variable region, including the CDRs. A VH fragment can further contain a heavy chain constant region sequence. The number of heavy chain constant region domains depends on the isotype. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the -COOH terminus. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE, and is defined by the five classifications of heavy chains present: mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, respectively. IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0126] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies having specificity for more than one antigen. They can also be fragments, including hybrid fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.). Immunoglobulins also include natural, synthetic, or genetically engineered proteins that act like antibodies by binding to specific antigens to form complexes. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as:

[0127] The term "monomer" refers to an antibody that contains only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody that contains two Ig units bound to each other via the constant domains (Fc or fragment crystallizable regions) of the antibody heavy chains. The complex may be stabilized by a joining (J) chain protein. The term "multimer" refers to an antibody that contains two or more Ig units bound to each other via the constant domains (Fc regions) of the antibody heavy chains. The complex may be stabilized by a joining (J) chain protein.

[0128] The term "bivalent antibody" refers to an antibody that contains two antigen-binding sites. The two binding sites may have the same antigen specificity or may be bispecific, meaning that the two antigen-binding sites have different antigen specificities.

[0129] Bispecific antibodies are a class of antibodies with two paratopes that have different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be biparatopic, and bispecific antibodies can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed from a pair of different single domain antibodies called "nanobodies." Single domain antibodies are sourced and engineered from cartilaginous fish and camelids. Nanobodies can be joined together by linkers using techniques typical to those skilled in the art. Such methods for the selection and joining of nanobodies are described in PCT Publication Nos. 2015044386A1, 2010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which is specifically incorporated herein by reference in its entirety.

[0130] Bispecific antibodies can be constructed as whole IgG, Fab'2, Fab'PEG, diabodies, or alternatively as scFvs. Diabodies and scFvs can be constructed without Fc regions using only variable domains, potentially reducing the effects of anti-idiotypic reactions. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. For example, see Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which is specifically incorporated by reference in its entirety.

[0131] In some embodiments, the antigen-binding domain may be multispecific or heterospecific by multimerization with VH and VL domain pairs that bind to different antigens. For example, the antibody may bind or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Thus, embodiments may include, but are not limited to, bispecific, trispecific, tetraspecific and other multispecific antibodies, or antigen-binding fragments thereof that are directed to epitopes and other targets, such as Fc receptors on effector cells.

[0132] In some embodiments, multispecific antibodies can be used and directly linked via short flexible polypeptide chains using routine methods known in the art. One such example is a diabody, a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain and utilize a linker that is too short to allow pairing between the domains on the same chain, thereby allowing the domains to pair with complementary domains on another chain to create two antigen binding sites. Linker functionality is applicable to triabodies, tetrabodies and higher order antibody multimer embodiments. (See, for example, Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0133] In contrast to bispecific whole antibodies, bispecific diabodies can also be easily constructed and expressed in E. coli, so that diabodies (and other polypeptides such as antibody fragments) of suitable binding specificity, which may be advantageous, can be easily selected from libraries using phage display (WO 94 / 13804). If one arm of the diabody is held constant, for example with specificity for a protein, the other arm can be varied to create a library from which antibodies of suitable specificity are selected. Bispecific whole antibodies can also be produced by alternative engineering methods such as those described in Ridgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39:167-173, 2017), each of which is incorporated herein by reference in its entirety.

[0134] Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities, see, e.g., U.S. Patent No. 6,010,902, which is incorporated herein by reference in its entirety.

[0135] The portion of the Fv fragment of an antibody molecule that binds with high specificity to an epitope of an antigen is referred to herein as the "paratope". The paratope consists of amino acid residues that contact the epitope of an antigen to facilitate antigen recognition. Each of the two Fv fragments of an antibody is composed of two variable domains, VH and VL, in a dimeric configuration. The primary structure of each variable domain includes three hypervariable loops separated and flanked by framework regions (FR). The hypervariable loops are the regions of highest primary sequence variability in antibody molecules from any mammal. The term hypervariable loops is sometimes used interchangeably with the term "complementarity determining region (CDR)". The length of the hypervariable loops (or CDRs) varies between antibody molecules. The framework regions of all antibody molecules from a given mammal have a high primary sequence similarity / consensus. The consensus of the framework regions can be used by those skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) interspersed between the framework regions. The hypervariable loops are given identifiers that distinguish their position in the polypeptide and the domain in which they reside. The CDRs in the VL domain are identified as L1, L2, and L3, with L1 occurring at the most distal end and L3 occurring closest to the CL domain. The CDRs can also be named CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is generally the region of highest variability of all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain that are linearly arranged in the primary structure and separated from each other by framework regions. The amino-terminal (N-terminal) end of the VL chain is designated FR1. The region identified as FR2 occurs between the L1 and L2 hypervariable loops. FR3 occurs between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which contains three CDRs identified as H1, H2, and H3. The majority of amino acid residues in the variable domain, or Fv fragment (VH and VL) are part of the framework regions (about 85%).The three dimensional or tertiary structure of an antibody molecule is such that the majority of the structure presents the CDRs on the external surface of the molecule, with framework regions being more internal to the molecule.

[0136] Several methods have been developed and can be used by those skilled in the art to identify the exact amino acids that make up each of these regions. This can be done using any of several sequence alignment methods and algorithms to identify the conserved amino acid residues that make up the framework regions, and thus identify the CDRs that may vary in length but are located between the framework regions. Three commonly used methods have been developed to identify the CDRs of antibodies: Kabat (described in TTWu and EAKabat, “AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY,” J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (described in C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (described in M.-P.Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). Each of these methods includes a unique numbering system for identifying the amino acid residues that make up the variable region. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen are present in the CDRs, but in some cases residues in the framework regions contribute to antigen binding.

[0137] Those skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include: 1) computer prediction of the tertiary structure of the antibody / epitope binding interaction based on the chemical nature of the amino acid sequence of the antibody variable region and the composition of the epitope; 2) hydrogen-deuterium exchange and mass spectrometry; 3) a polypeptide fragmentation and peptide mapping approach, which generates multiple overlapping peptide fragments from the full length of the polypeptide and evaluates the binding affinity of these peptides to the epitope; 4) antibody phage display library analysis, in which mammalian genes encoding antibody Fab fragments are expressed by bacteriophage so that they are incorporated into the coat of the phage. This population of Fab-expressing phages is then allowed to interact with the antigen, which may be immobilized or expressed by a different exogenous expression system. The non-binding Fab fragments are washed away, thereby leaving only the specific binding Fab fragments bound to the antigen. The binding Fab fragments can be easily isolated and the genes encoding them can be determined. This approach can also be used for smaller regions of the Fab fragments, including Fv fragments or specific VH and VL domains as appropriate.

[0138] In some embodiments, affinity matured antibody is enhanced by one or more modifications in one or more of its CDRs, which results in improved affinity of the antibody to the target antigen, compared to the parent antibody without those modifications(s). Some affinity matured antibodies have nanomolar or picomolar affinity to the target antigen. Affinity matured antibodies are produced by procedures known in the art, for example, Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, and random mutagenesis of CDR and / or framework residues used for phage display is described by Rajpal et al., PNAS.24:8466-8471 (2005) and Thie et al., Methods Mol Biol.525:309-22 (2009), together with computational methods such as those demonstrated in Tiller et al., Front.Immunol.8:986 (2017).

[0139] Chimeric immunoglobulins are the product of fusion genes derived from different species; "humanized" chimeras generally have framework regions (FR) derived from a human immunoglobulin and one or more CDRs are derived from a non-human source.

[0140] In some embodiments, a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence from another specific species or belonging to a specific antibody class or subclass, and the remainder of the chain(s) is identical or homologous to a corresponding sequence of an antibody from another species or belonging to another antibody class or subclass, as well as a fragment of such an antibody, so long as it exhibits the desired biological activity. U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods related to chimeric antibodies, see, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985). Each of these is specifically incorporated herein by reference in its entirety. CDR grafting is described, for example, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, all of which are incorporated by reference herein for all purposes.

[0141] In some embodiments, minimizing antibody polypeptide sequences from non-human species optimizes the function of chimeric antibodies and reduces immunogenicity. Certain amino acid residues from non-antigen recognition regions of non-human antibodies are modified to be homologous to corresponding residues in human antibodies or isotypes. One example is a "CDR-grafted" antibody, where the antibody contains one or more CDRs from a particular species or belonging to a particular antibody class or subclass, and the remainder of the antibody chain(s) is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass. For use in humans, V regions consisting of CDR1, CDR2 and partial CDR3 for the dispersed regions of both light and heavy chains from a non-human immunoglobulin are grafted into human antibody framework regions, replacing the naturally occurring antigen receptor of the human antibody with the non-human CDRs. In some examples, the corresponding non-human residues replace the framework region residues of the human immunoglobulin. Furthermore, humanized antibodies may contain residues not found in the recipient antibody or donor antibody to further improve performance. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23:1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0142] Intrabodies are intracellularly-located immunoglobulins that bind intracellular antigens, as opposed to secreted antibodies, which bind antigens in the extracellular space.

[0143] Polyclonal antibody preparations typically contain different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host (e.g., rabbit or goat) is immunized with an antigen or an antigen fragment, typically with an adjuvant and optionally bound to a carrier. Antibodies against the antigen are then recovered from the host's serum. Polyclonal antibodies can be affinity purified against the antigen, which confers a single specificity to the antibody.

[0144] Monoclonal antibody or "mAb" refers to an antibody obtained from a population of homogeneous antibodies from exclusive parent cells, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.

[0145] Functional antibody fragments and antigen-binding fragments may be utilized. Certain aspects relate to antibody fragments, such as antibody fragments that bind and / or neutralize inflammatory mediators. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to an antigen. These fragments are composed of various sequences of heavy chain variable regions (VH) and / or light chain variable regions (VL), and in some embodiments, contain heavy chain constant region 1 (CH1) and light chain constant region (CL). In some embodiments, they lack the Fc region, which is composed of heavy chain 2 (CH2) and 3 (CH3) domains. Embodiments of antigen-binding fragments and modifications thereof may include: (i) Fab fragment type composed of VL, VH, CL and CH1 domains; (ii) Fd fragment type composed of VH and CH1 domains; (iii) Fv fragment type composed of VH and VL domains; (iv) single domain fragment type dAb composed of a single VH or VL domain (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003); (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and Day, ED, Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, NY (1990); Antibodies, 4:259-277 (2015).All citations in this paragraph are incorporated by reference.

[0146] An antigen-binding fragment also includes fragments of an antibody that retain at least or exactly one, two, or three complementarity determining regions (CDRs) from the light chain variable region. Fusions of CDR-containing sequences to an Fc region (or its CH2 or CH3 regions) are included within this definition, e.g., scFvs fused directly or indirectly to an Fc region are included herein.

[0147] The term Fab fragment refers to a monovalent antigen-binding fragment of an antibody that contains the VL, VH, CL and CH1 domains. The term Fab' fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than the Fab fragment. For example, a Fab' fragment contains the VL, VH, CL and CH1 domains and all or part of the hinge region. The term F(ab')2 fragment refers to a bivalent antigen-binding fragment of a monoclonal antibody that contains two Fab' fragments linked by a disulfide bridge at the hinge region. The F(ab')2 fragment, for example, contains all or part of two VH and VL domains and may further contain all or part of two CL and CH1 domains.

[0148] The term "Fd fragment" refers to a fragment of a heavy chain of a monoclonal antibody that contains all or a portion of the VH, including the CDRs. The Fd fragment may further contain a CH1 region sequence.

[0149] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody, comprising all or part of VL and VH, and lacking CL and CH1 domains. VL and VH, for example, comprise CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions are linked by a flexible linker to form a single polypeptide chain that forms the antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Publication No. WO88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a bivalent or bispecific sFv polypeptide chain, comprising an oligomerization domain at the C-terminus, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain comprises a self-associating a-helix, e.g., a leucine zipper, which may be further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as "miniantibodies" or "minibodies."

[0150] Single domain antibody is an antigen-binding fragment that contains only VH or VL domain. In some cases, two or more VH regions are covalently linked with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same antigen or different antigens.

[0151] In some cases, fragment crystallizable region Fc is utilized. The Fc region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain. The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Included are truncated forms of such polypeptides that include the hinge region that promotes dimerization. In some embodiments, antibodies incorporating cytokines (including those mentioned herein) can be utilized (e.g., with TRIKE). IV. Pharmaceutical Compositions

[0152] The pharmaceutical compositions of the present disclosure include compositions comprising an effective amount of NK cells and antibodies dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce side effects, allergic reactions, or other adverse reactions when administered to an animal, such as a human, as appropriate. Preparation of pharmaceutical compositions, including the present compositions, will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, which is incorporated herein by reference. In addition, it will be understood that for animal (e.g., human) administration, preparations must meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0153] As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like materials and combinations thereof, which are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Except in the case that any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated.

[0154] Pharmaceutical compositions may contain different types of carriers depending on whether they should be administered in solid, liquid or aerosol form and whether they need to be sterile for administration routes such as injection.The compositions of the present disclosure can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, by local perfusion bath target cells directly, via catheter, via lavage solution, in cream, in lipid composition (e.g., liposome), or by other methods known to those skilled in the art or any combination of the above (e.g., see Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference).

[0155] The composition comprising NK cells and antibodies can be formulated into a composition in free base, neutral or salt form. Pharmaceutically acceptable salts include, where appropriate, acid addition salts, for example, those formed with the free amino groups of the proteinaceous composition, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, a solution is administered in a manner compatible with the administration formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, such as those formulated for parenteral administration, such as solutions for injection, or those formulated for gastrointestinal administration, such as aerosols for delivery to the lungs, or drug-releasing capsules.

[0156] Further according to the present disclosure, the compositions of the present disclosure suitable for administration are provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier must be assimilable and includes liquid, semi-solid, i.e., paste, or solid carriers. Except where any conventional vehicle, agent, diluent or carrier is deleterious to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for use in carrying out the method of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, bulking agents, and the like, or combinations thereof. The compositions may also include various antioxidants to retard oxidation of one or more components. In addition, prevention of the action of microorganisms can be brought about by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0157] In accordance with the present disclosure, the compositions are combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsion, mixing, encapsulation, absorption, etc. Such procedures are routine to those of ordinary skill in the art.

[0158] In certain embodiments of the present disclosure, the composition is thoroughly combined or mixed with semi-solid or solid carrier.Mixing can be carried out by any convenient method, such as grinding.Stabilizers can also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0159] In further embodiments, the disclosure relates to the use of pharmaceutical lipid vehicle compositions, including compositions comprising NK cells and antibodies, and optionally an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide range of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., designed or manufactured by man). However, lipids are typically biological materials. Biological lipids are well known in the art, and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether and ester-linked fatty acids, and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that would be understood by those of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

[0160] Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in nanoparticles or lipid vehicles.For example, the composition comprising NK cells and antibodies can be dispersed in a solution containing lipids, dissolved in lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained in lipids as a suspension, contained or complexed in micelles or liposomes, or otherwise associated with lipids or lipid structures by any means known to those skilled in the art.Dispersion may or may not result in the formation of liposomes.

[0161] The actual dosage of the composition of the present disclosure administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, type of disease to be treated, previous or concurrent therapeutic interventions, idiopathic disease of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of administrations of an effective amount can vary according to the subject's response. The physician responsible for administration will in any case determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0162] In some embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise, for example, about 2% to about 75% of the weight of the unit, or, for example, about 25% to about 60%, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared so that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0163] In other non-limiting examples, dosages can also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numbers recited herein include ranges based on the above numbers, such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc.

[0164] Therapeutic compositions comprising NK cells and antibodies of the present disclosure can be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. In some embodiments, antibiotics are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the clinical condition of the individual, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0165] The treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, as well as the particular route and formulation, are within the skill of those of ordinary skill in the clinical arts to determine. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some embodiments, a unit dose includes a single administrable dose.

[0166] The amount to be administered depends on the desired therapeutic effect, both depending on the number of treatments and the unit dose. It is understood that an effective dose refers to the amount required to achieve a particular effect. In practice, it is contemplated that in some embodiments, a dose ranging from 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, doses are contemplated to include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such doses can be administered multiple times over the course of a single day and / or multiple days, weeks, or months.

[0167] In one embodiment, an effective dose of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In another embodiment, an effective dose provides a blood level of about 4 μM to 100 μM, or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dose can provide the following blood levels of drug resulting from the therapeutic agent being administered to the subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 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, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114 4, 45, 46, 47, 48, 49, 50, 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, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In some embodiments, the therapeutic agent administered to the subject is metabolized in the body to become a metabolized therapeutic agent, in which case blood levels may refer to the amount of the agent. Alternatively, blood levels discussed herein may refer to a non-metabolized therapeutic agent, so long as the therapeutic agent is not metabolized by the subject. A. Gastrointestinal Compositions and Formulations

[0168] In certain embodiments of the present disclosure, the composition comprising NK cells and antibodies is formulated to be administered via the gastrointestinal route. The gastrointestinal route includes all possible routes of administration in which the composition is in direct contact with the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein can be administered orally, bucally, rectally, or sublingually. Thus, these compositions can be formulated with an inert diluent or an assimilable edible carrier, or can be enclosed in a hard or soft shell gelatin capsule, or can be compressed into a tablet, or can be directly incorporated into dietary food.

[0169] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each of which is specifically incorporated herein by reference in its entirety). Tablets, troches, pills, capsules, etc. may also contain: binders, such as gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients, such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, such as corn starch, potato starch, alginic acid, or combinations thereof; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose, saccharin, or combinations thereof; flavorings, such as peppermint, wintergreen oil, cherry flavoring, orange flavoring, etc. When the dosage unit is a capsule, it may contain a liquid carrier in addition to the above types of materials. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain carriers such as liquid carriers in addition to the above-mentioned types of materials. Gelatin capsules, tablets or pills may be enterically coated. Enteric coatings prevent the composition from denaturing in the stomach or upper intestine, where the pH is acidic. For example, see U.S. Pat. No. 5,629,001. Upon reaching the small intestine, the basic pH therein dissolves the coating, and the composition is released and can be absorbed by specialized cells, such as epithelial intestinal cells and Peyer's patch M cells. Syrup of elixir may contain the active compound sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavor.Of course, any material used in preparing any dosage unit should be pharma- ceutically pure and substantially non-toxic in the amounts used.Furthermore, the active compounds can be incorporated into sustained-release preparations and formulations.

[0170] For oral administration, the composition of the present disclosure may alternatively be incorporated into the form of mouthwash, dentifrices, buccal tablets, oral sprays, or sublingual oral administration preparations with one or more excipients. For example, mouthwashes can be prepared by incorporating the required amount of active ingredient in a suitable solvent, such as sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into oral solutions, such as those containing sodium borate, glycerin, and potassium bicarbonate, or dispersed in dentifrices, or added in a therapeutically effective amount to compositions that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the composition may be formed into a tablet or solution form that can be placed under the tongue or otherwise dissolved in the mouth.

[0171] Further formulations suitable for other modes of gastrointestinal administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, the suppository softens, melts, or dissolves in the cavity fluid. In general, for suppositories, traditional carriers can include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In some embodiments, suppositories can be formed from mixtures containing, for example, about 0.5% to about 10%, preferably about 1% to about 2% of active ingredient. B. Parenteral Compositions and Formulations

[0172] In further embodiments, the composition can be administered via parenteral route.As used herein, the term "parenteral" includes the route that bypasses the digestive tract.Specifically, the pharmaceutical composition disclosed herein can be administered, for example, but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intravenously, intrathecally, intraventricularly, intratumorally, subcutaneously, or intraperitoneally.See U.S. Patent Nos. 6,613,308; 5,466,468; 5,543,158; 5,641,515; and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety).

[0173] Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468; specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, for example, aluminum monostearate and gelatin, in the composition.

[0174] For parenteral administration in aqueous solutions, for example, the solution must be suitably buffered if necessary and the liquid diluent must first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. For example, a dose can be dissolved in an isotonic NaCl solution and added to a subcutaneous infusion or injected at the proposed site of infusion (see, for example, "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0175] Sterile injection solution is prepared by incorporating the required amount of active compound into a suitable solvent together with various other ingredients as listed above as necessary, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above.In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient + any additional desired ingredients from its solution that has been previously sterilized and filtered.Powdered composition is combined with liquid carrier, such as water or physiological saline, with or without stabilizer. C. Miscellaneous Pharmaceutical Compositions and Formulations

[0176] In other specific embodiments of the present disclosure, the active compound compositions comprising NK cells and antibodies may be formulated for administration by a variety of miscellaneous routes, such as topical (i.e., transdermal), mucosal (intranasal, intravaginal, etc.) and / or inhalation.

[0177] Pharmaceutical compositions for topical administration may contain active compounds formulated for pharmaceutical use such as ointments, pastes, creams or powders. Ointments include all oil-based, absorbent, emulsion and water-soluble based compositions for topical application, while creams and lotions are compositions that contain only emulsion bases. Drugs administered topically may contain penetration enhancers to promote the absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum, as well as any other suitable absorbent, emulsion or water-soluble ointment bases. Topical preparations may also include emulsifiers, gelling agents and antimicrobial preservatives as necessary to preserve the active ingredient and provide a homogenous mixture. Transdermal administration of the present invention may also include the use of "patches". For example, a patch may continuously deliver one or more active agents at a predetermined rate over a period of time.

[0178] In some embodiments, pharmaceutical compositions can be delivered by eye drops, intranasal sprays, inhalation, and / or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212. (Each is specifically incorporated herein by reference in its entirety). Similarly, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Patent No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well known in the pharmaceutical field. Similarly, transmucosal drug delivery in the form of polytetrafluoroethylene support matrices is described in U.S. Patent No. 5,780,045, (specifically incorporated herein by reference in its entirety).

[0179] The term aerosol refers to a colloidal system of finely divided solids in liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation consists of a suspension of active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. The appropriate container varies according to the pressure requirements of the propellant. The administration of aerosol varies according to the age, weight, and severity and response of the subject. V. Combination Therapy

[0180] In some embodiments, the compositions and methods of the present invention include cancer treatment in addition to the composition comprising NK cells and antibodies.The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, or a combination of the above.The additional treatment can be in the form of adjuvant therapy or neoadjuvant therapy.

[0181] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor(s) or an 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 the side effects of the therapy, 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.

[0182] Immune cell therapy (as an addition to the composition of the present disclosure) can be administered in conjunction with, before, during, after, or in various combinations with additional cancer treatments such as immune checkpoint therapy. Administration can be at intervals ranging from the same time to minutes to days to weeks. In embodiments where immune cell therapy is provided to a patient separately from the composition(s) of the present disclosure, one must generally ensure that a significant period does not pass between the respective delivery times to allow the two compounds to still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the immunotherapy and the composition of the present disclosure may be provided to the patient within about 12-24 or 72 hours of each other, more specifically within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the treatment period, allowing several days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) to pass between the respective administrations.

[0183] Administration of any compound or treatment of the present embodiments to a patient will follow general protocols for administration of such compounds, taking into account any toxicity of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy. A. Chemotherapy

[0184] A wide variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. The term "chemotherapeutic agent" is used to include the meaning of a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, such as whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to induce chromosomal and mitotic abnormalities by directly crosslinking DNA, intercalating into DNA, or affecting nucleic acid synthesis.

[0185] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelanamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelanin; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryomycins (especially bryomycin and bryomycinone); statins; kallistatins; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatins; sarcodictins; spongistatins; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride (mechlorethamine oxide hydrochloride), nitrogen mustards such as melphalan, nobembitine, phenesterine, prednimustine, trofosfamide and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gamma 1 and calicheamicin omega I1); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyridoxal, cyanomorpholino-doxorubicin ... lorino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; methotrexate and 5-furan antimetabolites such as fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone; mitotane and Antiadrenal drugs such as maytansine and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin;Pentostatin;Phenamet;Pirarubicin;Losoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Rhizoxin;Sizofiran;Spirogermanium;Tenuazonic acid;Triaziquone;2,2',2''-Trichlorotriethylamine;Tricothecenes (especially T-2 toxin, veracrine A, roridin A and anguidin);Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitrol;Mitolactol;Pipobroman;Gacytosine;Arabinoside ("Ara-C");Cyclophosphamide;Taxoids, e.g., paclitaxel and docetaxel gemcitabine;6-Thioguanine;Mercaptopurine;Platinums such as cisplatin, oxaliplatin and carboplatin Coordination complexes; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above. B. Radiation therapy

[0186] Other agents that cause DNA damage and have been widely used include what are commonly known as gamma radiation, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation are also contemplated. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by tumor cells. C. Immunotherapy

[0187] Those skilled in the art will understand that additional immunotherapy (except for the cell therapy of the present disclosure) can be used in combination or in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may function alone as an effector of treatment or may recruit other cells to actually affect cell killing. The antibody can also be conjugated with a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector can be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells other than those with knockdown or knockout of TGF-βR2.

[0188] Antibody-drug conjugates have emerged as a revolutionary approach to the development of cancer therapeutics. Antibody-drug conjugates (ADCs) consist of a monoclonal antibody (MAb) covalently attached to a cell-killing drug. This approach combines the high specificity of the MAb for an antigen target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells with high antigen levels. Targeted delivery of the drug also minimizes exposure to normal tissues, resulting in reduced toxicity and improved therapeutic index. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has validated this approach. Currently, there are more than 30 ADC drug candidates 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 suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high levels of expression in tumor cells and strong internalization.

[0189] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., 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 immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0190] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as any type of interferon, IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, 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 treatments can be used in conjunction with the antibody therapies described herein.

[0191] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either direct signals (e.g., costimulatory molecules) up or direct signals down. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also called CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also called CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed 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. D. Surgery

[0192] Approximately 60% of people with cancer undergo some type of surgery, including preventive, 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 embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Lumpectomy refers to the physical removal of at least a portion of the tumor. In addition to lumpectomy, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0193] Removal of part or all of the cancerous cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment can be accomplished by perfusion, direct injection, or local application of the area with additional anti-cancer therapy. Such treatments can 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 can vary in dosage as well. E. Other drugs

[0194] It is contemplated that other agents can be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the 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 inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. 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, such as the antibody c225, can be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy. VI. Kits of the Disclosure

[0195] In some embodiments, kits are provided that may include, for example, NK cells, optionally one or more media and components for producing NK cells, one or more antibodies or reagent(s) for producing antibodies, and the like. In some embodiments, the formulation may include a cocktail of factors, for example, in a form suitable for combination with NK cells. The reagent system or any kit components may be packaged, as appropriate, in either aqueous medium or lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed, and preferably suitably aliquoted. Where there is more than one component in the kit, the kit will also generally include a second, third or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in a vial. The components of the kit may be provided as a dry powder. Where the reagents and / or components are provided as a dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits will also typically include a means for containing the kit component(s) in close confinement for commercial sale. Such containers can include injection or blow molded plastic containers into which the desired vials are retained. The kit can also include instructions for use, in printed or electronic form, e.g., in digital form.

[0196] In certain embodiments, the kit may include one or more cytokines, including at least IL-12, IL-15, IL18 and / or IL-2, including specific concentrations as described elsewhere herein. The kit may include any type of medium, any components of cryopreservation medium, as described elsewhere herein. The kit may include cord blood (including pooled cord blood), any type of antigen presenting cells, beads for depleting specific NK cells (as described herein), vectors encoding one or more proteins as described herein, NK cells, antibodies or reagents for generating antibodies, etc.

[0197] Individual components may also be provided in concentrated amounts in the kit, and in some embodiments, components are provided individually at the same concentration as they are present in solution with the other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x or 20x or more. EXAMPLES

[0198] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention, and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Example 1 Preparation and Use of the Composition

[0199] A composition comprising specific NK cells and an antibody capable of binding to said NK cells is prepared and, in certain cases, administered in an effective amount to an individual in need of treatment. In certain embodiments, autologous or allogeneic NK cells for the individual are administered in an effective amount to the individual, and the NK cells are particularly umbilical cord blood cells. The NK cells may or may not be sourced from a cryopreservation repository, and in some cases have been cryopreserved in a specific cryopreservation medium. The NK cells are optionally preactivated and optionally expanded under conditions that allow the antibody to bind to the NK cells via the appropriate antigen, and then an effective amount of the NK cells is exposed to the antibody. Following this, an effective amount of the composition is provided to the individual in need thereof. Example 2 Monoclonal antibodies and NK cells

[0200] This example includes the preparation and use of NK cells preloaded with antibodies. Figure 1 shows an example of an experimental procedure for NK cell expansion and antibody loading. NK cells may be obtained from processed blood, and in certain embodiments, specific NK cells are isolated, for example, in certain embodiments, they are CD3-CD56+. Optionally, the NK cells are expanded in the presence of, for example, IL-2 and universal antigen presenting cells or other feeder cells, and the expansion step may be for any suitable period of time, such as up to about 14 days in certain cases. In another optional embodiment, the NK cells are preactivated prior to expansion. Any preactivation step may be utilized, but in certain embodiments, the preactivation step includes exposure to one or more cytokines, such as one or more of IL-12, IL-15, and / or IL-18. The preactivation step may take any suitable period of time, but in certain embodiments, it is up to about 16 hours. Following this step, the NK cells may be optionally expanded, including, for example, in the presence of universal antigen presenting cells and IL-2. The cultured cells may be reactivated with IL2, IL-18 or IL-15 or any combination of these cytokines during proliferation. After proliferation, the NK cells are exposed to an appropriate amount of antibody under sufficient conditions for the antibody to bind to the outside of the NK cells via the specific antigen to which the antibody is directed. In certain cases, the antibody is a monoclonal antibody. The conditions for loading the antibody onto the NK cells may be a specific period of time under a specific temperature, for example, 1 hour at 37°C.

[0201] Figure 2 provides validation of margetuximab (anti-HER2 monoclonal antibody) binding to NK cells. NK cells were derived from umbilical cord blood and were either standardly grown (NE) with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium, or preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and grown with irradiated uAPC and IL-2 (200 U / mL) (PE). NK cells were loaded with margetuximab 1 μg / ml for 1 hour at 37°C in Click / RPMI medium, washed, and then binding of margetuximab to NK cells was validated by flow cytometry. Unloaded NE and PE NK cells were used as negative controls. Margetuximab binding was detected by flow cytometry staining with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody. The graph shows that PE NK cells exhibit higher levels of margetuximab binding compared to NE NK cells. The histogram provides data from three different cord blood NK cell donors.

[0202] Margetuximab-loaded NK cells show enhanced cytotoxicity against HER2+ tumor cells (Figure 3). In it, Xcelligence cytotoxicity assays are provided showing normalized cell index of HER2+ SKOV3 (ovarian cancer) cells cultured alone or at a 2:1 effector-to-target (E:T) ratio with NK cells. NK cells were either expanded normally (NE) or pre-activated and expanded (P+E). NE NK cells were grown with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium, and PE cells were preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and grown with irradiated uAPC and IL-2 (200 U / mL). NK cells were left unloaded or loaded with 1 μg / ml margetuximab in Click / RPMI medium for 1 hour at 37° C. and then washed prior to assay. The data show that margetuximab loading enhances NE and PE NK cell cytotoxicity against tumor cells.

[0203] In a NSG mouse model of HER2+ ovarian cancer (SKOV3), margetuximab-loaded NK cells result in enhanced tumor control (Figure 4). In Figure 4A, an example of an experimental design schema for the SKOV3 mouse model is shown. In Figure 4B, bioluminescence imaging shows tumor growth over time in mice implanted with firefly luciferase (FFluc)-transduced SKOV3 and left untreated or treated with NK cells alone, margetuximab alone, or NK cells preloaded with margetuximab. Figure 4C provides a graph showing the average radiance over time for the four groups of mice described in panel 4A.

[0204] In Figure 5, validation of amivantamab (EGFR-MET bispecific antibody) binding to NK cells is demonstrated. NK cells were derived from umbilical cord blood and were either standardly grown with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium (NE) or preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and grown with irradiated uAPC and IL-2 (200 U / mL) (PE). NK cells were loaded with amivantamab 1 μg / ml for 1 hour at 37°C in Click / RPMI medium, washed, and then validation of amivantamab binding to NK cells was performed by flow cytometry. Unloaded NE and PE NK cells were used as negative controls. Amivantamab binding was detected by flow cytometry staining with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody. The graph shows that PE NK cells exhibit higher levels of amivantamab binding compared to NE NK cells. The histogram shows data from three different cord blood NK cell donors.

[0205] Amivantamab-loaded NK cells show enhanced cytotoxicity against EGFR+ / cMET+ tumor cells (Figure 6). Three different cord blood NK cells (N=3 / assay) were used at various effector-to-target (E:T) ratios (20:1, 10:1, 5:1, 1:1) to provide chromium release assays showing specific lysis of three EGFR+ / c-MET+ cancer cell lines, SKOV3 (ovarian cancer), UMRC3 (renal cell carcinoma), and PATC148 (pancreatic ductal adenocarcinoma). NK cells were either expanded normally (NE) or preactivated and expanded (P+E). NE NK cells were grown with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium, and PE cells were preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 h, then washed and grown with irradiated uAPC and IL-2 (200 U / mL). NK cells were left unloaded or loaded with 1 μg / ml amivantamab in Click / RPMI medium for 1 h at 37° C. and then washed prior to assay. Amivantamab loading enhances NE and PE NK cell cytotoxicity against tumor cells.

[0206] In Figures 7A and 7B, amivantamab-loaded NK cells show enhanced cytotoxicity against EGFR+ / cMET+ tumor cells. Xcelligence cytotoxicity assay showing normalized cell index for two EGFR+ / c-MET+ cancer cell lines, SKOV3 (ovarian cancer; Figure 7A), and PATC148 (pancreatic ductal adenocarcinoma; 7B), using three different cord blood NK cells (N=3 / assay) at an effector to target (E:T) ratio of 2:1. NK cells were either expanded normally (NE) or pre-activated and expanded (P+E). NE NK cells were grown with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium, and PE cells were preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 h, then washed and grown with irradiated uAPC and IL-2 (200 U / mL). NK cells were left unloaded or loaded with 1 μg / ml amivantamab in Click / RPMI medium for 1 h at 37° C. and then washed prior to assay. Amivantamab loading enhances NE and PE NK cell cytotoxicity against tumor cells.

[0207] In the NSG mouse model of EGFR+ / c-MET+ ovarian cancer (SKOV3), amivantamab-loaded NK cells result in enhanced tumor control (Figure 8). In Figure 12A, an example of an experimental design schema for the SKOV3 mouse model is provided. In Figure 8B, bioluminescence imaging shows tumor growth over time in mice implanted with firefly luciferase (FFluc)-transduced SKOV3 and left untreated or treated with NK cells alone, amivantamab alone, or NK cells preloaded with amivantamab. Figure 8C provides a graph showing the average radiance over time for the four groups of mice described in Figure 8A.

[0208] NK cells were loaded with Imgatuzumab (anti-EGFR monoclonal antibody), an example of an antibody (Figure 9). NK cells were derived from umbilical cord blood and were either grown normally with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium (NE) or preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and grown with irradiated uAPC and IL-2 (200 U / mL) (PE). NK cells were loaded with Imgatuzumab 1 μg / ml for 1 hour at 37°C in Click / RPMI medium, washed, and then binding of Imgatuzumab to NK cells was verified by flow cytometry. Unloaded NE and PE NK cells were used as negative controls. Imgatuzumab binding was detected by flow cytometry staining with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody. The graph shows that PE NK cells exhibit higher levels of imgatuzumab binding compared to NE NK cells. The histograms show data from three different cord blood NK cell donors.

[0209] Imgatuzumab-loaded NK cells show enhanced cytotoxicity against tumor cells (Figure 10). Chromium release assay showing specific lysis of three EGFR+ cancer cell lines, (SKOV3 (ovarian cancer), UMRC3 (renal cell carcinoma), PATC148 (pancreatic ductal adenocarcinoma)) using three different cord blood NK cells (N=3 / assay) at various effector to target (E:T) ratios (20:1, 10:1, 5:1, 1:1). NK cells were either expanded normally (NE) or preactivated and expanded (P+E). NE NK cells were grown with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium, and PE cells were preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and grown with irradiated uAPC and IL-2 (200 U / mL). NK cells were left unloaded or loaded with 1 μg / ml imgatuzumab in Click / RPMI medium for 1 hour at 37° C. and then washed prior to assay. The data show that imgatuzumab loading enhances NE and PE NK cell cytotoxicity against tumor cells.

[0210] In Figures 11A-11C, Imgatuzumab-loaded NK cells show enhanced cytotoxicity against EGFR+ tumor cells. These figures show Xcelligence cytotoxicity assays demonstrating normalized cell index for three EGFR+ cancer cell lines, SKOV3 (ovarian cancer; Figure 11A), PATC148 (pancreatic ductal adenocarcinoma; Figure 11B) and UMRC3 (renal cell carcinoma; Figure 11C), using three different cord blood NK cells (N=3 / assay) at an effector to target (E:T) ratio of 2:1. NK cells were either expanded normally (NE) or pre-activated and expanded (P+E). NE NK cells were grown with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK cell ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium, and PE cells were preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and grown with irradiated uAPC and IL-2 (200 U / mL). NK cells were left unloaded or loaded with 1 μg / ml imgatuzumab in Click / RPMI medium for 1 hour at 37° C. and then washed prior to assay. The data show that imgatuzumab loading enhances NE and PE NK cell cytotoxicity against tumor cells.

[0211] In an NSG mouse model of EGFR+ ovarian cancer (SKOV3), imgatuzumab-loaded NK cells result in enhanced tumor control (Figure 12). In Figure 12A, there is an example of an experimental design schema for the SKOV3 mouse model. Figure 12B provides bioluminescence imaging showing tumor growth over time in mice implanted with firefly luciferase (FFluc)-transduced SKOV3 and left untreated or treated with NK cells alone, imgatuzumab alone, or NK cells preloaded with imgatuzumab. In Figure 12C, there is a graph showing the average radiance over time for the four groups of mice described in Figure 12A.

[0212] 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 herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described herein. As one skilled in the art will readily appreciate from this disclosure, any currently existing or hereafter developed process, machine, manufacture, composition of matter, means, method or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein may be utilized in accordance with the present disclosure. Therefore, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions of matter, means, methods or steps.

Claims

1. (1) one or more umbilical cord blood-derived natural killer (NK) cells; and (2) One or more antibody molecules, (a) the antibody is monospecific, wherein an Fc region of the monospecific antibody binds to the NK cell and an antigen-binding domain of the monospecific antibody is capable of binding to a target antigen, or (b) The antibody is multispecific, wherein one or more antigen-binding domains of the antibody bind to a target antigen and another one or more antigen-binding domains of the antibody are capable of binding to a NK cell surface antigen.

2. 2. The composition of claim 1, further defined in (a) as a complex of said NK cell and said monospecific antibody via binding of an Fc region of said monospecific antibody to said NK cell.

3. The composition of claim 2 , wherein the complex further comprises the antigen-binding domain of the monospecific antibody bound to its target antigen.

4. 2. The composition of claim 1, further defined in (b) as a complex of said NK cell and said multispecific antibody via binding of one or more of said antigen binding domains of said multispecific antibody that bind to one or more antigens on the surface of said NK cell.

5. The composition of claim 1 , wherein the one or more antigen-binding domains of the antibody are bound to its target antigen.

6. The target antigen is a stem cell antigen, an autoantigen, or a combination of CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, epidermal growth factor receptor (EGFR), epithelial tumor antigen, melanoma associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD 5, CD123, CD23, CD30, CD38, CD56, CD70, CD38, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof.

7. 2. The composition of claim 1, wherein the NK cell surface antigen is CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, or KIR.

8. The composition of claim 1 , wherein the NK cells comprise one or more engineered antigen receptors.

9. 9. The composition of claim 8, wherein the engineered antigen receptor is a chimeric antigen receptor, a T cell receptor, or both.

10. The composition of claim 1 , wherein the NK cells express a heterologous cytokine.

11. The composition of claim 11, wherein the heterologous cytokine is IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 or IL-23.

12. The composition of claim 1 , wherein the NK cells express one or more receptors to enhance their binding to an antibody.

13. The composition of claim 12, wherein the receptor is an Fc receptor.

14. The composition of claim 12, wherein the receptor is CD16, CD32, CD64, or a combination thereof.

15. The composition of claim 1 , wherein the NK cells express a suicide gene.

16. 13. A method for producing the composition of claim 1, comprising: (a) optionally expanding NK cells in a culture comprising an effective amount of: (1) a cytokine selected from the group consisting of IL-2, IL-15, IL-18, IL-21, and combinations thereof; and (2) antigen-presenting cells / feeders or NK-activating beads, and (b) providing said antibody molecules to said NK cells and, if expanded, providing said antibody molecules to said NK cells before and / or after expansion.

17. 17. The method of claim 16, comprising a preactivation step before and / or after said expanding step, wherein said NK cells are preactivated in culture containing an effective concentration of one or more of IL-2, IL-12, IL-15 and IL-18.

18. The method of claim 17, wherein the culture comprises effective concentrations of two or more of IL-2, IL-12, IL-15 and IL-18.

19. The method of claim 17, wherein the culture comprises effective concentrations of IL-12, IL-15 and IL-18.

20. 17. The method of claim 16, wherein said providing step is further defined as culturing said NK cells with said antibody molecule for a specified period of time or combining said NK cells with said antibody molecule immediately prior to infusion.

21. 17. The method of claim 16, wherein the NK cells are activated at least twice or more during said expansion step with IL-12, IL-15, IL-18, IL-2 or any combination thereof.

22. The method of claim 16, wherein the growing step is for 5 to 60 days.

23. 1. A composition for treating a disease or disorder in a subject, comprising: (1) one or more umbilical cord blood-derived natural killer (NK) cells; and (2) One or more antibody molecules, (a) the antibody is monospecific, wherein an Fc region of the monospecific antibody binds to the NK cell and an antigen-binding domain of the monospecific antibody is capable of binding to a target antigen, or (b) the antibody is multispecific, such that one or more antigen-binding domains of the antibody bind to a target antigen and another one or more antigen-binding domains of the antibody can bind to a NK cell surface antigen; A composition comprising:

24. 24. The composition of claim 23, wherein the disease or disorder is cancer, inflammation, graft-versus-host disease, transplant rejection, an autoimmune disorder, an immunodeficiency disease, a B-cell malignancy, or an infectious disease.

25. 25. The composition of claim 24, wherein the disorder is graft-versus-host disease (GVHD).

26. 24. The method of claim 23, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma, or Sjogren's syndrome.

Images