Universal antigen presenting cells and uses thereof

Abstract

To provide universal antigen presenting cells.SOLUTION: Also provided herein are methods of expanding immune cells using the UAPCs and methods for the treatment of a disease, such as cancer, using the expanded immune cells.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application is the benefit of U.S. Provisional Patent Application No. 62 / 633,587, filed February 21, 2018. No. 6,399,433, which is incorporated herein by reference in its entirety.

[0002] KB (measured on Microsoft Windows) A file named "UTFCP1355WO_ST25.txt" created on the 1st The sequence listing included in the accompanying application is filed herewith by electronic submission and is hereby incorporated by reference. It is incorporated herein. [Background technology]

[0003] The present invention relates generally to the fields of medicine and immunology. More specifically, the present invention relates to a method for the treatment of antigen presentation. The present invention relates to indicator cells and uses thereof, for example, the expansion of natural killer (NK) cells.

[0004] Natural killer (NK) cells, in contrast to T cells, require antigen prior to activation. This is highly effective in tumor immunosurveillance since it does not require exposure to a tumor. Although activation of N is not required, it is important to prevent unintended cytotoxicity and autoimmunity. The enormous killing power of K cells requires tight control. Generating NK cells that are both effective and function optimally is another obstacle. To design helper cells to improve NK cell-mediated cancer immunotherapy, The molecular mechanism that governs the function of NK cells to distinguish between "resistant" and "sensitive" target cells It is necessary to pay attention. Summary of the Invention

[0005] Thus, certain embodiments of the present disclosure provide for clinical applications in cell therapy and immunotherapy. Methods and methods for the production, expansion, quality control and functional characterization of clinical grade NK cells A composition is provided.

[0006] In a first embodiment, (1) CD48 and / or CS1 (CD319), (2) membrane (3) 41BB ligand ( Universal antigen-presenting cells (UAPCs) engineered to express 41BBL are provided. In some embodiments, the UAPCs express CD48. In certain embodiments, the UAPCs express CS1. It expresses S1.

[0007] In some embodiments, the UAPCs are endogenous HLA class I, II or CD1d In certain embodiments, the UAPCs do not essentially express ICAM-1 (CD 54) and LFA-3 (CD58).

[0008] In certain embodiments, UAPCs are further defined as aAPCs derived from leukemia cells. In some embodiments, the leukemia cell-derived UAPCs are further defined as K562 cells. can be.

[0009] In some embodiments, the manipulation is further defined as retroviral transduction. In one embodiment, the retroviral transduction further comprises SEQ ID NO:1 and / or SEQ ID NO: In certain embodiments, the UAPCs are irradiated. can be.

[0010] In a further embodiment, a method for expanding immune cells is provided, the method comprising: The immune cells are administered in the form of an effective amount of the UAPCs of the above embodiments (e.g., (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), etc. and (3) a universal 41BB ligand (41BBL)-expressing In some embodiments, the method further comprises culturing the cells in the presence of undifferentiated antigen-presenting cells (UAPCs). In this case, the ratio of immune cells to UAPCs is 3:1 to 1:3, for example, 3:1, 3:2, 1:1, 1 In certain embodiments, the immune cells and UAPCs are cultured at a ratio of 1:2 or 1:3. They are cultured in a ratio of 1:2.

[0011] In some embodiments, the expansion is in the presence of IL-2. In this case, IL-2 is administered at a concentration of 10 to 500 U / mL (e.g., 10 to 25, 25 to 50, 5 0~75, 75~10, 100~150, 150~200, 200~250, 250~3 0, 300-350, 350-400, or 400-500 U / mL) In certain embodiments, IL-2 is present at a concentration of 100 to 300 U / mL. In certain embodiments, IL-2 is present at a concentration of 200 U / mL. In a specific embodiment, the IL-2 is recombinant human IL-2. Replenish every 2 to 3 days (e.g., every 2 or 3 days).

[0012] In certain embodiments, the UAPC is added at least twice. In certain embodiments, the immune cells are NK cells or T cells. They are NK cells.

[0013] In certain embodiments, the immune cells are derived from cord blood (CB), peripheral blood (PB), stem cells or bone marrow. In a specific embodiment, the stem cells are induced pluripotent stem cells. In embodiments, the immune cells are obtained from CB. In certain embodiments, the CB comprises 2 or more units of In a specific embodiment, the CB is pooled from 3, 4, 5, Pooled from 6, 7 or 8 individual cord blood units.

[0014] In some embodiments, the NK cells are CB mononuclear cells (CBMCs). In embodiments, the NK cells further comprise CD56 + Defined as NK cells.

[0015] In certain embodiments, the method is carried out in serum-free medium.

[0016] In a further embodiment, the immune cells are engineered to express a chimeric antigen receptor (CAR). In some embodiments, the CAR is engineered to target CD19, CD123, mesothelial cells, or IL-1. Lin, CD5, CD47, CLL-1, CD33, CD99, U5snRNP200, C Contains the antigen-binding domain of D200, CS1, BAFF-R, ROR-1 or BCMA In some embodiments, the CAR is a humanized CAR. The CAR comprises IL-15. In certain embodiments, the CAR comprises a suicide gene. In some embodiments, the suicide gene is CD20, CD52, EGFRv3 or an inducible It is caspase 9.

[0017] The above embodiments (e.g., the above immune cells) are administered with an effective amount of the UAPCs of the above embodiments (e.g., (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin -21 (mbIL-21), and (3) 41BB ligand (41BBL). The cells were cultured in the presence of universal antigen-presenting cells (UAPCs) engineered to Further provided herein are populations of immune cells generated and expanded according to the methods described above.

[0018] In another embodiment, the expanded population of immune cells of the above embodiment and a pharma- ceutically acceptable The present invention provides a pharmaceutical composition comprising a carrier capable of administering the compound of the present invention to a subject for treating a disease or disorder. The composition comprising an effective amount of the expanded immune cells of the above embodiment for use in et al. are provided herein.

[0019] Further embodiments provide methods of treating a disease or disorder in a subject, the methods comprising administering The method includes administering to the subject a therapeutically effective amount of the expanded immune cells of the above embodiments. include.

[0020] In some embodiments, the disease or disorder is cancer, inflammation, graft-versus-host disease, graft rejection, an autoimmune disorder, an immunodeficiency, a B-cell malignancy or an infectious disease. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is acute lymphoblastic leukemia. Allogeneic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) or chronic In certain embodiments, the disorder is graft-versus-host disease (GVHD). In some embodiments, the disorder is multiple sclerosis, inflammatory bowel disease, Rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact sensitivity, asthma or Shague Renn's syndrome. In some embodiments, the subject is a human.

[0021] In certain embodiments, the immune cells are allogeneic. The immune cells are autologous. In some embodiments, the immune cells are NK cells or T cells. It is.

[0022] In a further embodiment, the method further comprises administering at least one second therapeutic agent. In some embodiments, the at least one second therapeutic agent is In certain embodiments, the anti-cancer agent is an immunomodulatory agent or an immunosuppressant agent. Treatment options include chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, antiangiogenic therapy, or immunotherapy. It is an epidemic.

[0023] In some embodiments, the immunosuppressant is a calcineurin inhibitor, an mTOR inhibitor, Antibodies, chemotherapy, radiation, chemokines, interleukins, or chemokines or insulins It is an inhibitor of turtleukin.

[0024] In further embodiments, the immune cells and / or the at least one second therapeutic agent are Intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, Administered subcutaneously, regionally, or by direct injection or perfusion. In certain embodiments, the second therapeutic agent is an antibody. In some embodiments, the antibody is a monospecific antibody, a bispecific antibody, or a trispecific antibody. , rituximab.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it will be appreciated that those skilled in the art will appreciate that various changes and modifications may be made without departing from the spirit and scope of the present invention. As will become apparent from the detailed description, the detailed description and specific examples are intended to provide preferred embodiments of the invention. It should be noted that while configurations are shown, they are given by way of example only.

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure, in combination with the detailed description of specific embodiments presented herein, The present invention may be better understood by reference to one or more of these drawings. [Brief description of the drawings]

[0027] [Figure 1A] Zipper model of the interaction between human natural killer cell receptors and target cell ligands. Human NK cells interact with target cells through a number of receptors, including killer immunoglobulin receptors (KIRs), natural cytotoxicity receptors (NCRs), NKG2 family receptors, nectin-binding receptors, and SLAM family receptors. Receptors on NK cells are either activating (KIR2DL4, KIR3DS1, NKp30, NKp44, NKp46, NKp80, CD94-NKG2C, DAP10, NKG2D, DAP10, CRTAM, DNAM, 2B4, NTB-A, CD3 zeta, CD100, CD160) or inhibitory (KIR2DL1 / 2 / 3 / 5A / 5B, KIR3DL1 / 2 / 3, CD94-NKG2A, TIGIT, CD96, CEACAM-1, ILT2 / LILRB1, KLRG1, LAIR1, CD161, Siglec-3 / 7 / 9). [Figure 1B]Zipper model of the interaction between human natural killer cell receptors and target cell ligands. Human NK cells interact with target cells through a number of receptors, including killer immunoglobulin receptors (KIRs), natural cytotoxicity receptors (NCRs), NKG2 family receptors, nectin-binding receptors, and SLAM family receptors. Receptors on NK cells are either activating (KIR2DL4, KIR3DS1, NKp30, NKp44, NKp46, NKp80, CD94-NKG2C, DAP10, NKG2D, DAP10, CRTAM, DNAM, 2B4, NTB-A, CD3 zeta, CD100, CD160) or inhibitory (KIR2DL1 / 2 / 3 / 5A / 5B, KIR3DL1 / 2 / 3, CD94-NKG2A, TIGIT, CD96, CEACAM-1, ILT2 / LILRB1, KLRG1, LAIR1, CD161, Siglec-3 / 7 / 9). [Figure 1C] Zipper model of the interaction between human natural killer cell receptors and target cell ligands. Human NK cells interact with target cells through a number of receptors, including killer immunoglobulin receptors (KIRs), natural cytotoxicity receptors (NCRs), NKG2 family receptors, nectin-binding receptors, and SLAM family receptors. Receptors on NK cells are either activating (KIR2DL4, KIR3DS1, NKp30, NKp44, NKp46, NKp80, CD94-NKG2C, DAP10, NKG2D, DAP10, CRTAM, DNAM, 2B4, NTB-A, CD3 zeta, CD100, CD160) or inhibitory (KIR2DL1 / 2 / 3 / 5A / 5B, KIR3DL1 / 2 / 3, CD94-NKG2A, TIGIT, CD96, CEACAM-1, ILT2 / LILRB1, KLRG1, LAIR1, CD161, Siglec-3 / 7 / 9).

[0028] [Figure 2A] Fold expansion of fresh or frozen NK cells with IL-2 or APC. [Figure 2B]Fold expansion of fresh or frozen NK cells with IL-2 or APC. [Figure 2C] Flow cytometry on days 0, 7 or 14 for CD3 and CD56 expression on NK cells. [Figure 2D] Cell counts of NT-NK cells or SG4-NK cells stimulated with APC. [Figure 2E] Proliferation kinetics of NK cells stimulated with clone 9 APC or UAPC.

[0029] [Figure 3A] Flow cytometry of parental K562 cells for the indicated markers. No expression of mb-IL21, 41BBL, CD48 and SLAMF7 (CS1) was observed. [Figure 3B] Flow cytometry analysis of APCs after clone 46 transduction (mbIL-21, 41BBL). [Figure 3C] Flow cytometric analysis of APCs following transduction of uAPC constructs to express CD48 (nmIL-21, 41BBL and CD48). [Figure 3D] Flow cytometric analysis of APCs following transduction of uAPC2 constructs to express CS1 (mbIL-21, 41BBL and CS1).

[0030] [Figure 4A] Map and annotation of the MMLV-retroviral transfer construct. Retroviral transfer vector for mb-IL21. [Figure 4B] Map and annotation of the MMLV-retroviral transfer construct. Retroviral transfer vector for 41BBL. [Figure 4C] Map and annotation of the MMLV-retroviral transfer construct. Retroviral transfer vector for CD48-Katushka. [Figure 4D]Map and annotation of the MMLV-retroviral transfer construct. Retroviral transfer vector for CS1-EGFP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] In this study, NK cells were initially characterized as distinct from other components of the innate immune compartment. The antitumor and anti-allolytic functions of NK cells were characterized. The myriad immune receptors that govern the immune system are illustrated in the “Shipper” graph in Figure 1. Signal transmitters can be broadly classified into two major groups: activating and inhibitory. Depending on their structure and signaling mode, these categories are further divided into immune receptor associations. The membrane proteome of NK cells has not yet been fully elucidated, Functional redundancy within the NK cell "zipper" allows for a complex and dynamic interaction of complementary and antagonistic pathways. These have been utilized to regulate cytolytic function while maintaining a physiological balance.

[0032] Thus, certain embodiments of the present disclosure provide for clinical applications in cell therapy and immunotherapy. Methods and methods for the production, expansion, quality control and functional characterization of clinical grade NK cells The composition is provided in clinically relevant quantities for injection into patients while meeting time constraints. Expanding and generating NK cells is difficult under the best of circumstances. In the present disclosure, the method and composition provide a technical process for the production of NK cells, an achievable NK cell Details and kinetics of K cell expansion, as well as molecular characterization to verify successful cell formation will be detailed below.

[0033] In a further embodiment, human natural killer (NK) cells are conditioned against tumors. As a universal antigen-presenting cell (UAPC) to A robust platform technology that is optimized for the application is provided herein. Described herein are methods for the optimized expansion of immune cells, such as NK cells. This UAPC is an antigen-presenting cell that acts to overcome inhibitory signals and to stimulate NK cells. To induce optimal and specific killing function of cells, we have combined a unique combination of costimulatory molecules. Large-scale studies have demonstrated that UAPCs are able to enhance the mechanism of NK cells and improve tumor This UAPC has been shown to fine-tune the disappearance of NK cells. It is possible.

[0034] An exemplary APC is the NK cell-sensitive K562 antigen-presenting cell line (APC) (clone 46 Membrane-bound interleukin 21 (mbIL-21) and 4-1B in the IL-1 protein (IL-1 protein) In another embodiment, the gene is generated by forced expression of mB ligand in K562 cells. By forcibly expressing bIL-21, 4-1BB ligand and CD48, UAP In another embodiment, K5 Forced expression of mbIL-21, 4-1BB ligand and CS1 in 62 cells These UAPCs were generated by the following procedure (referred to as UAPC2): L-21, 41BBL and NK cell specific antigens (e.g., SLAM family antigens (Table 1) can be engineered to express

[0035] The UAPC platform is designed to efficiently express T cells (e.g., alpha-beta and gamma-delta It can also be applied to expand other immune effectors, including immunized T cells. These immune cells (e.g., NK cells and T cells) are derived from peripheral blood, umbilical cord blood, bone marrow or stem cells. These immune cells can be derived from human immune cells (including induced pluripotent stem cells). The UAPCs are required for culture to reach meaningful numbers for practically relevant applications. They can be subjected to expansion and activation in vivo.

[0036] Thus, the present disclosure provides a method for preparing human immune cells (e.g., NK cells) for cancer immunotherapy. Designed for attaching, adjusting, priming and expanding , provides a series of produced helper cell lines. I. Definition

[0037] As used herein, "essentially free" with respect to a particular component means that Certain components are not intentionally formulated in a composition and / or are contaminants is used herein to mean present only as a trace or in trace amounts Therefore, the total amount of a particular component resulting from any unintentional inclusion of that composition is , well below 0.05%, preferably below 0.01%. Most preferred are compositions in which the amount of is undetectable by standard analytical methods.

[0038] As used herein, "a" or "an" can mean one or more. When used in a paragraph, the words "a" or "an" are used in conjunction with the word "including." When used herein, it can mean one or more than one.

[0039] The use of the term "or" in the claims, unless expressly stated to refer to alternatives only. or is used to mean "and / or" unless the options are mutually exclusive. However, the present disclosure supports the definition and "and / or" as referring to alternatives only. As used herein, "another" means at least one second or more The terms "about," "substantially," and "approximately" generally refer to a range of values. That means plus or minus 5%.

[0040] "Immune disorder," "immune-related disorder," or "immune-mediated disorder" means a disorder in which the immune response contributes to the development of disease. Immune-mediated disorders are disorders that play a significant role in the development or progression of the disease. Autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory and allergic The status is mentioned.

[0041] An "immune response" refers to the activation of cells of the immune system (e.g., B cells or T cells or innate immune cells) in response to a stimulus. In one embodiment, the response is specific to a particular antigen. ("antigen-specific response").

[0042] An "autoimmune disease" is a condition in which the immune system reacts to antigens that are part of the normal host (i.e., self-antigens). ) to induce an immune response (e.g., a B cell response or a T cell response) against the tissue. Autoantigens can be derived from host cells or are usually They come from commensal organisms, such as microorganisms that colonize mucosal surfaces (known as symbionts). obtain.

[0043] "Treating" a disease or condition or treating such a disease or condition includes alleviating the signs or symptoms of the disease. The implementation of a protocol that may include administering one or more drugs to a patient for the purpose of alleviating The desired effects of treatment include slowing the progression of the disease and alleviating the disease state. This includes reducing or alleviating, as well as remission or improved prognosis. It can occur before the signs or symptoms of a disease or condition appear, as well as after they appear. Thus, "treat" or "treatment" includes "preventing" a disease or undesirable condition. In addition, "treating" or "treatment" can include alleviating or alleviating symptoms or or complete relief of symptoms, does not require a cure, and specifically, does not require any limiting effect on the patient. Contains protocols that only bring about results.

[0044] The terms "therapeutic effect" or "therapeutically effective" as used throughout this application refer to this condition. It refers to anything that enhances or improves the health of a subject in relation to medical treatment. includes, but is not limited to, a reduction in the frequency or severity of signs or symptoms of a disease. For example, treatment of cancer can include, for example, a reduction in tumor size, a decrease in the invasiveness of tumors, an increase in the growth of cancer, or the like. Treatment of cancer may include reducing the rate of progression or preventing metastasis of a cancer patient. It can also refer to an extension of.

[0045] "Subject" and "patient" refer to a human or non-human (e.g., primates, mammals, and vertebrate). In certain embodiments, the subject is a human.

[0046] The phrase "pharmaceutical or pharmacologically acceptable" refers to a compound that is suitable for administration to an animal, such as a human, as appropriate. Molecular entities and methods that, when administered to humans, do not cause side effects, allergic reactions or other adverse reactions. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is described in the present disclosure. In light of the disclosure, the method is known to those skilled in the art. The product is required by the FDA Office of Biological Standards. should meet the required standards of sterility, pyrogenicity, general safety and purity; is understood.

[0047] As used herein, "a pharma- ceutically acceptable carrier" includes any of the compounds known to those of skill in the art. Any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions) may be used. solutions, saline solutions, parenteral vehicles, e.g., sodium chloride, Ringer's dextrose, etc. etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and and injectable organic esters, e.g., ethyl oleate), dispersion media, coatings, interfaces Active agents, 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 agents, disintegrants, lubricants, sweeteners, flavorings, colorants, fluid and nutritional supplements, The pH and precise control of the various components in the pharmaceutical composition are included. The concentration is adjusted according to known parameters.

[0048] The term "haplotyping or tissue typing" refers to, for example, the determination of the lymphocyte markers of a particular subject. Determine which HLA locus (or loci) is expressed on the sphere The method used to identify a haplotype or tissue type of a subject by The HLA genes are located on the short arm of chromosome 6, which is the major histocompatibility complex. They are located in the major histocompatibility complex (MHC) and play important roles in cell-cell interactions, immune responses, organ transplantation, cancer development, and There are six loci important in transplantation: HLA-A, These are named HLA-B, HLA-C and HLA-DR, HLA-DP and HLA-DQ. At each locus, any of several different alleles can exist.

[0049] A widely used method for haplotyping is the polymerase chain reaction (PCR). ) to compare the subject's DNA to known segments of genes that code for MHC antigens. Variations in these regions of these genes may be associated with a subject's tissue type or haplotype. Serological methods are also used to detect serologically defined antigens on cell surfaces. HLA-A, -B and -C determinants are measured by known serological techniques. Briefly, lymphocytes (isolated from fresh peripheral blood) from a subject can be cultured using any of the known methods. The cells are then incubated with antisera that recognize all HLA antigens. The cells were spread onto a tray with microwells containing various types of antisera. The cells were incubated for 30 minutes. After incubation, the incubation is further supplemented for 60 minutes. If the lymphocyte has an antigen on its surface that is recognized by an antibody in the serum, the lymphocyte will be lysed. The addition of dyes can indicate changes in cell membrane permeability and cell death. The pattern of cells disrupted by the solution indicates the degree of histological incompatibility. For example, HL Lymphocytes from a person being tested for HLA-A3 express anti-HLA-A3 If it is destroyed in the wells containing the supernatant, the test is positive for this antigen group.

[0050] The term "antigen-presenting cell (APC)" refers to a cell that is recognized by specific effector cells of the immune system. The present invention can present one or more antigens in the form of peptide-MHC complexes that can be used to Thus, a cluster of cells capable of inducing an effective cellular immune response against the presented antigen is produced. The term "APC" refers to an intact whole cell (e.g., a macrophage). , B cells, endothelial cells, activated T cells and dendritic cells) or capable of presenting antigens Naturally occurring or synthetic molecules that are complexed with y2-microglobulin Purified MHC class I molecules. II. Universal antigen-presenting cells

[0051] Some embodiments of the present disclosure include methods for generating universal antigen presenting cells (UAPCs) and Use of UAPCs for the expansion of immune cells (e.g., NK cells and T cells) UAPCs can be used to express membrane-bound IL-21 (mbIL-21) and 41BBL ( The cells can be engineered to express the CD137 ligand.

[0052] UAPCs are adapted to express CD137 ligands and / or membrane-bound cytokines. The membrane-bound cytokine may be mIL-21 or mIL-15. In certain embodiments, the UAPCs express CD137 ligand and mIL-21. The APCs can be derived from cancer cells, such as leukemia cells. They may not express any of the endogenous HLA class I, II or CD1d molecules. C may express ICAM-1 (CD54) and LFA-3 (CD58). PCs were cultured using K562 cells (e.g., expressing CD137 ligand and mIL-21). The APCs may be irradiated. The engineering may be by any method known in the art, for example, by retroviral transduction.

[0053] Cytokines exert a highly potent regulatory role on all classes of immune cells, including NK cells. They exert effects on cell fate, activity and the efficacy of cellular responses. They activate NK cells. The power of cytokine stimulation is that their “natural” effector functions are highly sensitive to environmental intervention. Receptivity and priming can regulate NK cell behavior in vivo This confirms the above.

[0054] To address the problem of obtaining clinically relevant quantities of NK cells, the present method Interleukin-1 as a driver of NK cell expansion in APC platform technology In humans, NK cell proliferation by IL-10 and IL-21 can be used. Cell stimulation induces NKG2D expression in a STAT3-dependent manner. Cytokine receptor Although many immune cells share similar cell signaling components, NK cells Specific signal transduction depends on IL-21 receptor-STAT3 interaction for proliferation Remains.

[0055] Cytokine signaling is important for maintaining lymphocyte survival and proliferation In vivo, IL-2 administration is the only FDA-approved method for expanding NK cells. Another potent NK cell activator, IL-15, is a promising IL-2 inhibitor. It is currently undergoing Phase I clinical trials as an alternative, but has not shown significant toxicity after systemic administration. Besides significant toxicity, these cytokines limit NK cell survival while It also induces T cell proliferation. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors has different and specific effects on various cells. A. Membrane-bound IL-21

[0056] In certain embodiments, the UAPCs are generated to specifically activate NK cells. IL-21 may be used to stimulate the expression of IL-21. The IL-21 receptor (IL21R) is a common Transmits signals by dimerizing with the cytokine receptor gamma chain (gamma(c)) It is closely related to the IL-2 receptor beta chain, which can activate human NK cells. (cells primed for induction). Type I cytokines IL-21, which is a cytotoxic T cell, B cell and NK cell, can regulate the functions of T cells, B cells and NK cells. Only human NK cells are significantly expanded via activation of the IL-21 receptor signaling pathway (i.e., 1000-fold over 21 days). CD8 + Plays an important role in T cell reduction.

[0057] IL21R signaling is primarily mediated by STAT3, a highly potent activator of cell proliferation. The IL21R-STAT3 relationship was confirmed using an anti-phosphotyrosine antibody. GAS and cis-inducibility verified by immunoprecipitation and western blotting Driven by IL-21-induced STAT3 DNA binding to the IL21 element. The molecular basis of IL-21-mediated proliferation is mediated by STAT1 and IL-21-induced Tyrosine 510 (Y510) on IL21R mediates phosphorylation of STAT33 This mechanism can be specifically identified in Stat1 / Stat3 double knockout mice. In mice, this is manifested by a weakened IL-21 response.

[0058] IL21R signaling is important for NK cytotoxicity. Human IL21R deficiency is associated with impaired cytolysis of 51Cr-labeled K562 target cells, but not antibody-dependent Cytotoxicity is unaffected. Monogenic non-embryonic lethal defect (loss-of-function mutation in IL21R) provides an excellent opportunity to clarify the innate NK cell lytic response. Useful for modeling, enhancing and shaping K cell lytic reactions.

[0059] CD56 dim Cell populations and CD56 bright Both cell populations expressed similar numbers of surface IL IL-21 signaling selectively targets a subset of NK cells, even those with 21R. IL-21 induction of STAT1 and STAT3 phosphorylation was mediated by CD56dimN It is higher in CD56bright NK cells versus K cells. In contrast, IL-21 does not affect STAT5 activation, an IL-2-activated pathway that also drives T cell expansion. IL-21 signaling involves STAT3 activation as well as the MAPK and PI3K pathways In NK cells, the innate immune response genes (IFN-gamma, T-bet, IL-1) are involved. Induce the expression of IL-12R beta 2 and IL-18R, which promotes tumor cell killing. Prime NK cells in this way.

[0060] To efficiently utilize IL-21, expression of mbIL-21 (Figure 4A) was used to It may focus and localize trans-interactions with IL21R on K cells. 21 is in close proximity to the membrane and is readily available when needed. Optimal cell proliferation can be maintained without the need for exogenous high amounts and concentrations of IL-21. By co-culture with injected K562-mb15-41BBL, CD56+CD41 cells were isolated from peripheral blood. 3-NK cell expansion was induced by a median of 21.6-fold. This expansion was accompanied by the inclusion of IL-2, IL- 12, from the shared γc family, including IL-15 and IL-21, alone or in combination This is greater than the stimulation with soluble cytokines alone. was able to expand NK cells by at least 1000-fold (3-log) over a period of 14-21 days. Expression of mbIL21 on UAPCs also eliminates the need for exogenous clinical-grade cytokines. It is possible. B. 4-1BBL (4-1BB ligand, CD137 ligand, CD137L, TNF SF9)

[0061] In addition to the idea of ​​priming NK cell function with cytokines, Direct physical interaction with activation molecules on the surface of the CD137 (4 -1BB) is a member of the tumor necrosis receptor (TNF-R) gene family, Mediates cell proliferation, differentiation and programmed cell death (apoptosis). Mouse receptor was first characterized, followed by the characterization of the human homolog, which is identical to the 6 They share 0% identity and show significant conservation of the cytoplasmic / signaling domain. CD1 37 is expressed primarily in activated T cells and activated NK cells, and in thymocytes, myeloid It is expressed at varying levels in cells and is also detectable in endothelial cells at sites of inflammation. CD137 signaling mediates 1) the proliferation and proliferation of pro-survival NF- κB, and 2) the PI3K / ERK1 / 2 pathway, which specifically drives cell cycle progression. It is mediated through.

[0062] In activated NK cells, CD137 is a cytokine-induced costimulatory molecule, and this co- Stimulatory molecules stimulate NK cells by increasing cell proliferation and IFN-γ secretion. CD137L- / - knockout mice have demonstrated that CD137L- / - knockout mice induce anti-tumor responses. Reveals the importance of CD137 / CD137L signaling in the development of antitumor immune cells CD137- / - knockout mice showed reduced tumor progression compared to control mice. The frequency of migration was four times higher.

[0063] CD137 ligand, a 34 kDa glycoprotein member of the TNF superfamily CD137L, the 4-1BB ligand, is primarily expressed on B cells, macrophages and dendritic cells. It is detected on activated antigen-presenting cells (APCs), including IL-1, and at low levels on activated T cells. Transiently expressed. Human CD137L is only 36% homologous to its murine counterpart. Consistent with the antitumor efficacy of agonistic CD137 antibodies, binding of CD137L inhibits CT L and has been shown to induce antitumor activity.

[0064] Therefore, this UAPC is a physiological counter-receptor for CD137. The cells can be engineered to express the 4-1BB ligand for stimulation. C.SLAM / CD48

[0065] In addition to conditioning with cytokines and costimulation with 4-1BBL, NK cells and target Direct physical interaction with target cells also influences the cellular response, i.e., killing of the target cell. A signaling lymphocyte activation molecule ( Homology of the SLAM (formerly known as the CD2 superfamily) family of immunosuppressants Immunoglobulin receptors are of particular importance with respect to cell-cell interactions.

[0066] Thus, the UAPCs of the present disclosure may express one or more SLAM family antigens. Members of the SLAM family include CD2, CD48, CD58 (LFA-3), CD244 (2B4), CD229 (Ly9), CD319 (CS1 (CD2 subset 1); CRACC (CD2-like receptor-activated cytotoxic cell)) and CD352 (N In certain embodiments, the UAPCs include CD 48 and / or CS1. NK cells express the SLAM family (SLAMF) They express at least three members of the IL-1 receptor 2B4, NK cells, T cells and B NTB-A and CD2-like receptor-activated cytotoxic cell (CRAC C) (These are the respective ligands on target cells and possibly on other NK cells. (Recognizing CD48, NTB-A, and CRACC) 6, 7, 8 and 9 are homophilic (self-ligand) receptors, whereas SLAMF2 and SLAMF4 are counter-receptors (heterophilic) for each other. The homophilic affinity of the antibody is broad (dissociation constant Kd ranges from <1 μM to 200 μM). This (3 digits) indicates that the signal transduction mechanisms for SLAM glycoproteins are overlapping but distinct. It suggests a mechanistic basis for becoming.

[0067] Table 1: Binding affinities of SLAM family members. [Table 1]

[0068] In certain embodiments, the UAPCs support cell-cell interactions and enhance NK cell responses. CD48 is expressed by NK cells, T cells, monocytes, and and glycosylphosphatidylinositol-anchored proteins found on the surface of basophils. (GPI-AP) and is involved in adhesion and activation pathways in these cells. Despite lacking an intracellular domain, stimulation of CD48 results in its expression in lipid rafts. Induce signal transduction factor rearrangement, Lck-kinase activity and tyrosine phosphorylation in the As an adhesion costimulatory molecule, CD48 mediates the expression of B and T lymphocytes, NK cells, and mass stimulatory In human NK cells, CD48 induces numerous effects in NK cells and eosinophils. , a counter-receptor for 2B426, an important activator of NK cells. Heterophilic interactions are thought to compete for the interaction of MHC-I with CD244. Therefore, 2B4 / CD48 interaction is thought to mediate activation of human NK cells. In mouse NK cells, it induces a signal, whereas in mouse NK cells it sends an inhibitory signal.

[0069] 2B4-CD48 interactions between cells of the same population, i.e., NK cell-NK cell interactions or The interaction between T cells is mediated by APCs (e.g., K562 myeloid cells, which normally lack CD48). By expressing CD48 on the NK cell lineage, activation of UAPCs is enhanced. It can turn on the powerful 2B4 signaling pathway in trans to cells. D.SLAM / CS1

[0070] In some embodiments, the UAPC switches on the killing power of NK cells. The cells were then adapted to express CS1, another member of the SLAM family, as a costimulatory molecule. Unlike CD48, which interacts with 2B4, the CS1 interaction is homophilic. and can be characterized in the context of cis versus trans interactions. K562 cells, which normally do not contain CS1, are engineered to express CS1. E. Delivery of Nucleic Acid

[0071] mbIL-21, 41BBL and costimulatory antigens can be prepared by any method known in the art. The nucleic acid is usually in the form of an expression vector, such as a viral expression vector. In some embodiments, the expression vector is a retroviral expression vector, Adenovirus expression vector, DNA plasmid expression vector or AAV expression vector In some embodiments, the delivery comprises one or more vectors, one or more transfection vectors, by delivery of the transcript and / or one or more proteins transcribed therefrom. , is delivered to the cells.

[0072] Methods for introducing polynucleotide constructs into animal cells are known, including As a non-limiting example, a polynucleotide construct may be integrated into the genome of a cell. A method for stable transformation in which the polynucleotide construct is not integrated into the genome of the cell. Some embodiments include transient transformation methods and viral-mediated methods. In some embodiments, the polynucleotide may be, for example, a recombinant viral vector (e.g., a retroviral vector). The vector can be introduced into cells by a variety of methods, including the use of viruses, adenoviruses, liposomes, etc. In some embodiments, transient transformation methods include microinjection, electroporation, and the like. In some embodiments, the method includes the steps of: The peptide is expressed in a cell by a vector, more particularly a vector. The nucleic acid may be contained in a nucleic acid sequence of a nucleic acid vector or a virus.

[0073] Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, Microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, poly Cationic or lipid:nucleic acid conjugates, naked DNA, artificial virions, and DNA Agent-enhanced uptake Lipofection is described in detail in US Pat. Nos. 5,049,386, 4,946, 787; and 4,897,355), and are used as lipofection reagents. are commercially available (e.g., Transfectam TM and Lipofectin TM ) Cationic ligands suitable for efficient receptor-recognition lipofection of polynucleotides As for the neutral lipids, see Feigner's lipids, WO91117424; Delivery can include delivery to cells (e.g., by in vitro administration or by injection). The delivery may be to a target tissue (e.g., ex vivo administration) or to a target tissue (e.g., in vivo administration).

[0074] In some embodiments, delivery is by using an RNA virus or D Delivery by use of a NA virus-based system. Viral vectors are used in some embodiments. In vivo, the antibody can be administered directly to the patient or can be delivered using a viral vector. The cells may be treated in vitro or ex vivo and then administered to a patient. In embodiments, viral-based systems include retroviral vectors for gene transfer. vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors and herpes simplex viral vectors. III. Immune cells

[0075] Some embodiments of the present disclosure relate to the use of immune cells (e.g., NK cells or relates to the isolation and expansion of T cells.

[0076] In certain embodiments, immune cells are derived from human peripheral blood by methods well known in the art. Blood mononuclear cells (PBMCs), unstimulated leukocyte transplants (PBSCs), human embryonic stem cells (hESCs), C) Induced pluripotent stem cells (iPSCs) are obtained from bone marrow or umbilical cord blood. The cells may be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow or stem cells. In an embodiment, the immune cells are isolated from pooled CB. The immune cells may be pooled from 5, 6, 7, 8, 10 or more units. The isolated immune cells may be haploidentical to the subject receiving the cell therapy. NK cells are classified into two types: NK cells that express specific surface markers (e.g., CD16 in humans, It can be detected by CD56 and CD8).

[0077] In certain embodiments, the NK cells may be cultured using a method previously described in which the NK cells are expanded ex vivo. According to the reported method (Spanholtz et al., 2011; Shah et al. In this method, CB mononuclear cells are isolated by Ficoll density gradient chromatography (Ferro et al., 2013). The cell culture is then depleted of any cells expressing CD3. CD56 + / CD3 - Measure the percentage of cells or NK cells Alternatively, umbilical CB can be used to characterize CD34 + Thin NK cells are obtained by isolating CD3, CD14 and / or CD5+ cells. This may include depletion of D19 positive cells.

[0078] The isolated immune cells can be expanded in the presence of the UAPCs. ~30 days (e.g., 3 to 20 days, especially 12 to 16 days, e.g., 12, 13, 14, The immune cells and UAPC is about 3:1 to 1:3 (e.g., 2:1, 1:1, 1:2, specifically about 1:2 The expansion cultures may be supplemented with expansion-promoting cytokines (e.g., IL-2) IL-2 may further comprise about 10 to 500 U / mL (e.g., 100 to 300 U / mL). IL-2 may be present in the expansion cultures at a concentration of, for example, about 200 U / mL. , may be replenished every 2-3 days. UAPCs should be replenished at least twice (e.g., approximately 7 days after expansion). ) may be added to the culture.

[0079] Once expanded, the immune cells can be injected immediately or preserved, e.g. by cryopreservation. In certain embodiments, the cells may be cultured within about 1, 2, 3, 4, 5 days, or more. The cells may be expanded as a bulk population ex vivo for periods of time, weeks or months.

[0080] Expanded NK cells express type I cytokines that activate both innate and adaptive immune cells. Interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage cytokines secretes GM-CSF), as well as other cytokines and chemokines. By measuring these cytokines, the activation state of NK cells can be measured. In addition, other methods known in the art for measuring NK cell activation can be used. The methods of the present disclosure may be used for characterization of NK cells. IV. Chimeric Antigen Receptors

[0081] In certain embodiments, the immune cells (e.g., T cells or NK cells) are In some embodiments, the cells are genetically modified to express a CAR. In the present invention, CAR comprises a) an intracellular signaling domain, b) a transmembrane domain, and c) a ) an extracellular domain that contains an antigen-binding region.

[0082] CAR recognizes cell surface tumor-associated antigens that are unrelated to human leukocyte antigens (HLA) and These signaling molecules are used to genetically direct the killing, proliferation and cytokine production. In certain embodiments, the NK cells are activated by the following: ) Non-viral vectors using electroporation devices (e.g., Nucleofector) (ii) transfection with endodomains (e.g., CD28 / CD3-ζ, CD13 (iii) CARs that signal through the CD3+ / CD3-ζ or other combinations; CARs with extracellular domains of various lengths linking the original recognition domain to the cell surface, and In some cases, (iv) CAR + K, which can reliably and numerically expand NK cells Artificial antigen presenting cells (aAPCs) derived from 562 (Singh et al., 2011 ) can be genetically modified by methods including

[0083] Embodiments of the present disclosure include an intracellular signaling domain, a transmembrane domain, and one or more The extracellular domain contains a signaling motif of The present invention also includes nucleic acids encoding antigen-specific CAR polypeptides, including human CARs (hCARs). In certain embodiments, the CAR is a nucleic acid that encodes one or more antigens. In certain embodiments, the present invention can recognize epitopes that include a space shared between the The binding region may be a complementarity determining region of a monoclonal antibody, a variable region of a monoclonal antibody, or a combination thereof. and / or antigen-binding fragments thereof. In another embodiment, the specificity is , derived from peptides (eg, cytokines) that bind to receptors.

[0084] Human CAR nucleic acids are used to improve cellular immunotherapy in human patients. It is contemplated that the gene may be a full-length CA. In a specific embodiment, the method comprises: The antigen-binding region or domain is a specific and human monoclonal antibodies of the type described herein (see, e.g., U.S. Pat. No. 7,333,636, which is incorporated herein by reference). 109,304)) )V H Chain and V L The fragment may include a fragment of a human antigen-specific chain. The antigen-binding domains may be any number of different antigen-binding domains of a target antibody. The fragments are optimized for human codon usage for expression in human cells. It is an antigen-specific scFv encoded by an optimized sequence.

[0085] The configuration may be a multimer, for example a diabody or a multimer. In most cases, the variable portions of the light and heavy chains pair with each other to form diabodies. There are several options for the hinge portion of the construct, including: the complete deletion of the cysteine ​​residue, the retention of the first cysteine ​​residue, and the substitution of a proline instead of a serine residue. The Fc portion is truncated up to the first cysteine. Any protein that is stable and / or dimerizes may be suitable for this purpose. One of the Fc domains, for example, a CH2 or CH3 domain from a human immunoglobulin, may be used. Any of the following may be used: human immunoglobulins modified to improve dimerization The hinge, CH2 and CH3 regions of purine may be used. The hinge portion of globulin or a portion of CD8α may be used.

[0086] In some embodiments, the CAR nucleic acid is capable of binding to other costimulatory receptors (e.g., transmembrane The CD28 polypeptide includes sequences encoding the CD28 polypeptide domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include CD28, CD27, OX-40 (CD134), DAP10 and 4-1BB (CD137), In addition to the primary signal induced by CD3ζ, the human CAR may also include a signal Additional signals provided by the human co-stimulatory receptors required full activation of NK cells. This is important for activation and may improve in vivo persistence and therapeutic success in adoptive immunotherapy. It may help to improve.

[0087] The intracellular signaling domain of the chimeric antigen receptor is It is involved in the activation of at least one of the normal effector functions of the activated immune cells. The effector function of NK cells is a specialized function of differentiated cells such as NK cells. In this study, the intracellular receptor signaling domain in the CAR contains the T cell antigen receptor The intracellular receptor signaling domains of the receptor complex (e.g., the zeta chain of CD3, , FcγRIII costimulatory signaling domain, CD28, CD27, DAP10, CD 137, OX40, CD2 alone or in series with, for example, CD3 zeta). In a specific embodiment, the intracellular domain (which may be referred to as the cytoplasmic domain) is a TCR Zeta chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, Fc εRIγ, ICOS / CD278, IL-2R beta / CD122, IL-2R alpha / Contains part or all of one or more of CD132, DAP10, DAP12, and CD40 Any part of the endogenous T cell receptor complex may be used in the intracellular domain. So-called third generation CARs are, for example, fused together for additive or synergistic effects. Since it has at least two or three signaling domains, one or more The cytoplasmic domain of may also be used.

[0088] In certain embodiments of a CAR, the antigen-specific portion of the receptor (the antigen-binding region) is The extracellular domain (which may be referred to as the extracellular domain containing the tumor-associated antigen or pathogen-specific antigen-binding domain) Antigens include those recognized by pattern recognition receptors such as Dectin-1. Tumor-associated antigens include any antigen that is expressed on the cell surface of tumor cells. The tumor antigen binding domain may be of the following types: CD19, CD20, carcinoembryonic antigen, alfa Fafetoprotein, CA-125, MUC-1, 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 gp4 1, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, G D3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ER It may be, but is not limited to, BB2, EGFRvIII or VEGFR2. The AR may comprise a humanized scFv (e.g., humanized CD19 or CD123).

[0089] In certain embodiments, the CAR improves survivability when the amount of tumor-associated antigen is low. To improve the expression of CAR, it can be co-expressed with cytokines. For example, CAR can be co-expressed with IL-15. It can be done.

[0090] The sequence of the open reading frame encoding the chimeric receptor is The source may be obtained from a cDNA source or may be synthesized (e.g., by PCR) Introns can stabilize mRNA, Therefore, depending on the size of the genomic DNA and the number of introns, It may be desirable to use a combination of A, B, C, D, E, and E, or a combination of these. It may also be advantageous to use endogenous or exogenous non-coding regions to define the obtain.

[0091] The chimeric construct can be introduced into NK cells as naked DNA or in the form of a suitable vector. It is contemplated that naked DNA can be used to stably transfect cells by electroporation. Methods for transfecting a gene into a mammalian cell are known in the art. See, e.g., U.S. Patent No. 6,410, See, e.g., 319. Naked DNA is generally DNA that has been inserted into a plasmid in the proper orientation for expression. It refers to DNA encoding the chimeric receptor that is included in an expression vector.

[0092] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, vectors) The chimeric construct can then be introduced into NK cells. Suitable vectors are non-replicating in NK cells. Many virus-based vectors It is known that the number of copies of the virus maintained in a cell is a function of the number of copies that can maintain the viability of the cell. low enough to be able to detect, for example, HIV, SV40, EBV, HSV or BPV-based He is a fighter.

[0093] CARs are designed to target suicide genes (e.g., CD20, CD52, EGFRv3 or inducible caspases) It can express pase 9).

[0094] The CAR can comprise a tumor antigen-binding domain. The tumor antigen-binding domain can be CD19 , CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigens, melanoma-associated antigens, mutated p53, mutated ras, HER2 / Neu, ERBB2 , folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 E Envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56 , c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain , lambda chain, CSPG4, ERBB2, EGFRvIII or VEGFR2. CARs can be, but are not limited to, humanized scFvs, such as humanized CD19, CD 123, mesothelin, CD5, CD47, CLL-1, CD33, CD99, U5snR May include NP200, CD200, CS1, BAFF-R, ROR-1 or BCMA . V. How to use

[0095] Embodiments of the present disclosure provide a method for treating medical diseases by transferring immune cell populations that induce an immune response. Immune cells, such as N, provided herein for treating or preventing a disease or disorder. Methods for using K or T cells (e.g., those expanded by the present UAPCs) The method comprises administering to the subject a therapeutically effective amount of expanded immune cells. and treating or preventing the disorder in the subject. In these conditions, cancer or infectious diseases are treated by transferring immune cell populations that induce an immune response. Immune cells are placed in the inflammatory bowel due to the release of proinflammatory cytokines, which induce the recruitment of accessory immune cells. Anti-inflammatory properties by promoting differentiation, activation and / or recruitment to sites of malignancy It can reverse the tumor microenvironment and enhance the adaptive immune response.

[0096] Tumors for which the present treatment methods are useful include any malignant cell type, e.g., solid tumors or blood tumors. Exemplary solid tumors include those found in the pancreas, colon, cecum, Stomach, brain, head, neck, ovaries, kidneys, larynx, sarcoma, lungs, bladder, melanoma, prostate and breast The tumors may include, but are not limited to, tumors of organs selected from the group consisting of: Hematological malignancies include tumors of the bone marrow, T- or B-cell malignancies, leukemias, lymphomas, blastomas, myelomas, and myelomas. Further examples of cancers that can be treated using the methods provided herein include erythroblasts, leukemia, and ovarian cancers. Examples include lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma). , peritoneal cancer, gastric cancer or stomach cancer (digestive cancer and digestive (including ductal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, and rectal cancer Bowel cancer, endometrial or uterine cancer, salivary gland cancer, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer These include, but are not limited to, adenocarcinoma, various types of head and neck cancer, and melanoma. .

[0097] The cancer may specifically be, but is not limited to, the following histological types of cancer: neoplasm, malignant Gender;Carcinoma;Carcinoma, undifferentiated;Giant cell and spindle cell carcinoma;Small cell carcinoma;Papillary carcinoma;Squamous Cancer; lymphoid epithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastri Normoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; Adenocarcinoma in adenomatous polyp; adenocarcinoma; familial adenomatous polyposis; solid carcinoma; carcinoid tumor, Malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma ;clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary / follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma ;Endometrioid carcinoma;Skin adnexal carcinoma;Apocrine gland carcinoma;Sebaceous gland carcinoma;Earwax 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, breast; acinic cell carcinoma; adenosquamous carcinoma Skin carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant ;Granulosa cell tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell carcinoma alveolar tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;brown Glomus angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna Melanoma; Acral lentiginous melanoma; Nodular melanoma; Giant intrapigmented nevus malignant melanoma; Epithelioid cell melanoma Chromoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma; leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;Mullerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymal tumor,malignant;Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, Malignant;Ovarian goiter,Malignant;Choriocarcinoma;Mesonephroma,Malignant;Hemangiosarcoma;Hemangioendothelioma,Malignant;Capo hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; cartilage Blastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant; Enamel epithelial odontosarcoma; ameloblastoma, malignant; ameloblastoma, malignant ;chordoma;glioma, malignant;ependymoma;astrocytoma;protoplasmic astrocytoma ;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive nerve Transectomy;cerebellar sarcoma;ganglioneoblastoma;neuroblastoma;retinoblastoma;olfactory neurogenic tumor;spinal Membrane, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hojiki Hodgkin's disease; Hodgkin's lymphoma; lateral granuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell follicular, 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 NH L; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky disease ky disease)NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldemar Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma ;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymph Sarcoma cell leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Fatty Full cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia acute lymphoblastic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and Diffuse myeloblastic leukemia.

[0098] Certain embodiments relate to methods of treating leukemia, which is a cancer of the blood or bone marrow. It is characterized by the abnormal proliferation (production by division) of blood cells, usually white blood cells (leukocytes). Leukemia is part of a broad group of diseases called blood neoplasms. Leukemia is a broad term that encompasses a range of diseases. Clinically and pathologically, leukemia can be classified into acute and chronic forms. It can be divided into types.

[0099] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding causes the bone marrow to The body is no longer able to produce healthy blood cells. Acute forms of leukemia occur in children and young adults. Acute leukemia is characterized by rapid progression and accumulation of malignant cells that then flood the bloodstream. Prompt treatment is required as the virus can spread to other organs of the body. Although involvement of NS is uncommon, the disease can occasionally cause cranial nerve palsies. Chronic leukemia is , distinguished by the excessive accumulation of relatively mature, but still abnormal, blood cells. Over a period of months to years, the cells are produced at a rate much faster than normal cells. This causes many abnormal white blood cells to appear in the blood. Chronic leukemia usually occurs in older people, In theory, it can occur in any age group. Acute leukemia must be treated immediately. In contrast, chronic cases sometimes require pre-treatment to ensure maximum effectiveness of treatment. Will be monitored for a while.

[0100] Furthermore, the above diseases may be characterized as lymphocytic or lymphoblastic (cells that normally continue to form lymphocytes) (indicating the occurrence of cancer in a certain type of bone marrow cell) and myeloid or myeloid ( A type of bone marrow that normally continues to produce red blood cells, some types of white blood cells, and platelets It is classified as a type of cancer that occurs in marrow cells.

[0101] Acute lymphocytic leukemia (also known as acute lymphoblastic leukemia, or ALL) is a It is the most common type of leukemia in young children. The disease is more prevalent in adults, especially those aged 6 It can occur in people over the age of 5. Chronic lymphocytic leukemia (CLL) occurs most often in people over the age of 55. Chronic lymphocytic leukemia (CLL) occurs occasionally in younger adults. Acute myeloid leukemia is a rare disease that occurs in children. ogenous leukemia) (acute myeloid leukemia) Myeloid leukemia (AML), also known as acute myeloid leukemia, usually occurs in adults rather than children. This type of leukemia was previously called acute nonlymphocytic leukemia. Myeloid myeloid leukemia (CML) occurs primarily in adults.

[0102] Lymphoma is a cancer that arises from 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 percent of all cancer cases in the United States, particularly Hodgkin's lymphoma. accounts for less than 1 percent of all cancer cases in the United States. The lymphatic system is the body's immune system. It is part of the immune system, and can be caused by HIV infection or certain drugs or drug therapies. Patients with weakened immune systems also have a higher incidence of lymphoma.

[0103] In certain embodiments of the present disclosure, the immune cells are administered to an individual in need thereof (e.g., The cells are then delivered to a patient (an individual with cancer or an infectious disease) who is then able to stimulate the immune system of that individual. In some cases, the individual The body is provided with immune cells one or more times. If an individual is provided with immune cells more than once, the administration The time between doses should be long enough to allow time for transmission in the individual. and in specific embodiments, the time between administrations is 1, 2, 3, 4, 5, 6, 7 days or Or even more.

[0104] The source of the preactivated and expanded immune cells may be of any type, but specific In embodiments, the cells are derived from, for example, umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells. The appropriate dose for therapeutic effect is preferably determined over a series of doses. In a dosing cycle, for example, at least 10 5 or about 10 5 ~about 10 10 An exemplary dosing regimen includes at least about 10 days on day 0. 5 Starting with cells , for example, within a few weeks of initiating an intrapatient dose escalation scheme, 10 The target dose of cells The treatment consists of four escalating weekly cycles of gradually increasing the dose. Modes of administration include intravenous, subcutaneous, intracavitary (e.g., reservoir access device), and intraperitoneal. and direct injection into the tumor mass.

[0105] In one exemplary method, the NK cells are isolated from a biological sample, such as one or more units of umbilical cord blood. The cord blood unit may be a frozen cord blood unit. The cord blood unit may be thawed and Mononuclear cells can be obtained by subjecting them to a Ficoll gradient. It has been shown that CD3, CD14 and CD19 positive cells are depleted by iMAS and other methods. The negatively selected NK cells are then incubated with APC and IL-2 (e.g., 20 The NK cells can then be cultured in the presence of 50 U / mL of CAR-expressing NK cells. The retroviral supernatant was transduced with γ-irradiated APC and IL-2. Finally, the cells can be cultured to identify positive expression of the CAR (e.g., CD56, CD16, The antibodies may be sorted for CD3, CD19, CD14 or CD45.

[0106] Immune cells produced according to this method have many uses, including experimental and therapeutic applications. In particular, such cell populations may be useful in treating unwanted or inappropriate immune disorders. It is anticipated that such a method will be extremely useful in suppressing the immune response. Immune cells are removed from a patient and then manipulated and expanded ex vivo before being introduced back into the patient. Examples of diseases that can be treated in this way are immune disorders, e.g., due to allograft tolerance. Therapeutic methods include administering to mammals a therapeutically effective amount of the IL-1 receptor agonist, which inhibits the activity of the IL-1 receptor agonist. providing an article; obtaining immune cells from the mammal; and the method described above. and treating the expanded immune cells ex vivo according to the method of claim 1. The method may include administering the compound to a mammal to be treated.

[0107] The pharmaceutical compositions of the present disclosure may be used alone or in combination with other established agents useful in the treatment of cancer. They may be delivered alone or in combination with other agents. Regardless of whether the pharmaceutical composition is administered intravenously or intramuscularly, the pharmaceutical compositions of the present disclosure can be administered via a variety of routes to mammals, particularly humans. By delivering the drug to various parts of the body, specific effects can be achieved. More than one route can be used for administration, although certain routes may be more rapid and effective than others. For example, in the case of melanoma treatment, inhalation therapy may be used to provide an effective response. Local or systemic delivery may also be achieved by application of the formulation to a body cavity. or instillation, inhalation of aerosol or gas injection, or intramuscular, intravenous, intraportal, This can be accomplished by administration, including parenteral introduction, including intrahepatic, peritoneal, subcutaneous or intradermal administration. .

[0108] Certain embodiments of the present disclosure provide methods for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Dyson's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune egg Focal inflammation and autoimmune orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid Acne, cardiomyopathy, celiac spate-dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic Inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, Cicatricial pemphigoid, C REST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulin fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroid Inflammation, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile Arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis , type 1 diabetes or immune-mediated diabetes, myasthenia gravis, nephrotic syndrome (e.g., microvascular change group, focal glomerulosclerosis or membranous nephropathy), pemphigus vulgaris, pernicious anemia, nodular multifocal idiopathic nephropathy Phlebitis, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, Primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon Elephant, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, Stiff body syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveal vasculitis (e.g., polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, etc.) These include dermatitis herpetiformis (or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Some examples of autoimmune diseases that can be treated using the methods disclosed herein include: These include multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, and 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 an organ transplant or a stem cell transplant. and immune cells are used to prevent and / or treat rejection. In the method, the subject is diagnosed as having or at risk of developing graft-versus-host disease. GVHD can be treated with stem cells from related or unrelated donors or GVHD is a complication that can occur in any transplant, including those with a graft. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first 3 months after transplant. Signs of acute GVHD include swelling of the hands and Symptoms include a reddish rash on the legs and feet that can spread and become more severe. Acute GVHD can also affect the stomach and intestines. Symptoms include muscle cramps, nausea and diarrhea. Yellowing of the skin and eyes (jaundice) may occur in acute This indicates that GVHD is affecting the liver. Chronic GVHD is classified into stages based on its severity. Stage / Grade 1 is mild; Stage / Grade 4 is severe Chronic GVHD develops 3 months or more after transplant. Symptoms of chronic GVHD include: Although symptoms are similar to those of acute GVHD, chronic GVHD also affects the mucous glands of the eyes, the salivary glands of the mouth, etc. It may also affect the glands that lubricate the stomach lining and intestines. Any population of immune cells that can be transplanted may be utilized. Examples of organs that can be transplanted include solid organ transplants. Explants (e.g., kidney, liver, skin, pancreas, lung and / or heart) or cell transplants (e.g. , pancreatic islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells). The transplant may be a composite transplant (e.g., facial tissue). The immune cells may be administered to the transplant prior to transplantation. In some embodiments, the immune cells may be administered simultaneously with or after transplantation. Prior to transplantation (e.g., at least 1 hour, at least 12 hours, at least 1 day prior to transplantation) at least 2 days ago, at least 3 days ago, at least 4 days ago, at least 5 days ago, at least At least 6 days in advance, at least 1 week in advance, at least 2 weeks in advance, at least 3 weeks in advance, at least In one non-limiting embodiment, the treatment is administered at least 4 weeks or at least 1 month prior to the treatment. The therapeutically effective amount of immune cells is administered 3 to 5 days prior to transplantation.

[0110] In certain embodiments, the immune cells are administered in combination with a second therapeutic agent. For example, the second therapeutic agent may be a T cell, an immunomodulatory agent, a monoclonal antibody, or a chemotherapy agent. In a non-limiting example, the immunomodulatory agent is lenolidomide, The clonal antibody is rituximab, ofatumab, or lumiliximab, and chemotherapy The agent is fludarabine or cyclophosphamide.

[0111] The compositions of the present disclosure can be provided in unit dosage form, where each dosage unit, e.g., an injection, It contains a predetermined amount of that composition alone or in appropriate combination with other active agents. The term unit dosage form, as used herein, refers to a single dosage form for human and animal subjects. It refers to physically discrete units suitable for individual doses, each of which has a desired effect. A predetermined amount of the composition of the present invention, calculated as an amount sufficient to cause In combination with the active ingredient, optionally a pharma- ceutically acceptable diluent, carrier or The specifications for the novel unit dosage forms of the present invention depend on the specific subject. The extent to which this occurs will depend on the particular pharmacodynamics associated with the pharmaceutical composition.

[0112] Desirably, a long-term specific anti-tumor response is established, resulting in a reduction in tumor size or or to eliminate the growth or regrowth of tumors that may occur in the absence of such treatment; An effective amount or sufficient number of the isolated and introduced immune cells are present in the composition and administered to the subject. Desirably, the amount of immune cells reintroduced into the subject is otherwise the same. Compared to the control (no immune cells present), tumor size was increased by 10%, 20%, 30%, or 40%. , 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% .

[0113] Therefore, the amount of immune cells administered should take into account the route of administration, and sufficient numbers of The amount should be such that immune cells are introduced to achieve the desired therapeutic response. In addition, the amount of each of the active agents (e.g., adjuvant) included in the compositions described herein may be adjusted. The amount of each cell contacted or the amount per specific body weight may vary depending on the application. In general, the concentration of immune cells is desirably at least about 100% in the treated subject. 1×10 6 ~Approx. 1×10 9 Immune cells, even more preferably about 1×10 7 ~Approx. 5×10 8The amount should be sufficient to provide immune cells, but more than 5×10 8 More than or less than 1 x 10 cells 7 Subcellular amounts, Any suitable amount can be utilized. Dosing schedules are based on established cell-based therapies. (e.g., Topalian and Rosenberg, 1987; U.S. Pat. see US Pat. No. 4,690,915), or use an alternative continuous infusion strategy. It is also possible.

[0114] These values ​​are useful for the practitioner in optimizing the methods of the present invention to practice the present invention. This document provides general guidance on the range of immune cells that may be used. In no event shall the use of more or less of any component be permitted than may be warranted in a particular application. For example, the exact dose and schedule may vary depending on whether the composition is combined with other pharmaceutical compositions. Depending on whether or not they are administered in combination, or due to individual differences in pharmacokinetics, drug disposition, and metabolism Those skilled in the art will be able to make any necessary adjustments depending on the exigencies of a particular situation. It can be done easily. VI. Kit

[0115] In some embodiments, e.g., to generate UAPCs and / or immune cells, Kits are provided which may contain one or more media and components for the preparation of such formulations. It may contain a cocktail of factors in a form suitable for combination with APCs and / or immune cells. The reagent system may be packaged, as appropriate, in an aqueous medium or in lyophilized form. The container means of the kit generally includes a container into which the components may be disposed, preferably in a suitably aliquoted form. At least one vial, test tube, flask, bottle, syringe or other container into which the sample can be dispensed. When there is more than one component in the kit, the kit generally comprises may also include a second, third or other further container into which additional components may be separately placed. However, various combinations of components may be included in one vial. The components of the kit may be provided as dry powders. When provided as a dry powder, the powder may be reconstituted by adding a suitable solvent. It is envisioned that the solvent may be provided in another container means. , usually procedures for containing kit components in close confinement for commercial sale. Such containers may include injection molded or otherwise molded containers in which the desired vials are held. The kit may include a blow molded plastic container. The kit may include instructions for use (e.g. For example, it may include a printed format or an electronic format (e.g., a digital format). EXAMPLES

[0116] VII. Examples The following examples are included to demonstrate preferred embodiments of the invention. The techniques disclosed are techniques that the inventors have discovered to work well in the practice of this invention. and therefore, those approaches may be considered to constitute preferred modes for carrying out the invention. However, it should be recognized by those skilled in the art that, in light of this disclosure, In the specific embodiments disclosed, many modifications may be made without departing from the spirit and scope of the invention. It should be recognized that various modifications can be made and still obtain the same or similar results.

[0117] Example 1 - Combination of mbIL21, 4-1BBL and SLAM (CD48 / CS1) Match Membrane-bound type I, together with either CD48-Katushka or CS1-EGFP Retroviral constructs against L-21 and 41BBL (CD137 ligand) were used in Universal antigen-presenting cells (UAPCs) were generated by transducing 62 cells. The maps and annotations of the MMLV-retroviral constructs are shown in Figures 4A-4D. NK cell-sensitive K562 (HLA-A - , HLA-B - ) in APCs (Figure 3A) Forced expression of mbIL-21 and 41BBL generated clone 46 (FIG. 3B). Forced expression of mbIL-1, 41BBL and CD48 in K562 cells resulted in U APCs were generated (Figure 3C). UAPC2 was generated by forced expression of CS1 (Fig. 3D ).

[0118] Numerical expansion of NK cells was determined using activation with clone 46, UAPC, or UAPC2. The proliferation and expansion of NK cells by APC coculture was performed using clone 46, which was previously Compared to the published clone 9 (Figure 2B), non-transduced NK cells (NT-NK ) and CAR-transduced NK cells (SG4-NK). It showed that there is.

[0119] The expansion of NK cells by co-culture with UAPCs was confirmed by the previously published clone 9 (33 h The doubling time of NK cells was reduced to 31.38 hours compared to 1.08 hours. showed that the IL-16-dependent IL-16 expression of UAPCs was associated with a 44% proliferation advantage at 2 weeks ( 1671-fold expansion for clone 1 vs. 1161-fold expansion for clone 9) (Figure 2E). The relationship between time, magnification and starting cell population is simplified by the following formula:

number

[0120] In the formula, N f is the final cell number, N b is the starting cell, e=2.718 28 (a mathematical constant also known as Euler's number), and T is the duration of the culture in hours. range, T D is the doubling time in hours. The doubling time is indicative of the final cell number. This exponentially affects cell proliferation over a given cell culture period, resulting in a greater proliferative advantage. This allows for the collection of clinically relevant numbers of cells in a compressed time frame. Therefore, this method allows for the efficient generation of NK cells by co-culture with UAPCs.

[0121] The platform technology utilizes specific therapeutic combination pathways to deliver cancer immunotherapy. To generate large numbers of clinical grade highly functional and cytotoxic NK cells for the purpose of The expanded cells were 100% NK cells and no T cells were detected. This is a common problem in many immune cell preparations, as it is not possible to extract Side effects in the host are avoided.

[0122] All of the methods disclosed and claimed herein are herein intended to be within the scope of the present disclosure. The compositions and methods of the present invention can be made and executed without undue experimentation. Although various embodiments have been described, they are not intended to depart from the concept, spirit and scope of the present invention. However, variations may be applied to the methods and method steps or to the order of steps described herein. More specifically, certain characteristics that are both chemically and physiologically relevant will be apparent to those skilled in the art. Certain agents may be substituted for the agents described herein, but may not be the same or similar. It will be apparent that similar results can be achieved by using similar methods that are apparent to those skilled in the art. All such substitutions and modifications are within the spirit, scope and spirit of the present invention as defined by the appended claims. It is considered not to go beyond the concept.

[0123] The following documents provide exemplary procedural or other details supplementary to those described herein: To the extent provided, they are specifically incorporated herein by reference. Czerkinsky et al., J. Immunol. Methods 1988;110:29-36. Fast et al., Transfusion 2004;44:282-5. He Y, et al. Journal of immunology research. 2014;2014:7. International Publication No. PCT / US95 / 01570 International Publication No. WO2000 / 06588 International Publication No. WO2005 / 035570 Olsson et al. J. Clin. Invest. 1990;86:981-985. Taitano et al., The Journal of Immunology, 196, 2016. US Patent No. 5,939,281 US Patent No. 6,218,132 US Patent No. 6,264,951 U.S. Patent No. 7,488,490 U.S. Patent Publication No. 2007 / 0078113

Claims

1. (1) universal antigen presenting cells (UAPCs) engineered to express CD48 and / or CS1 (CD319), (2) membrane-bound interleukin 21 (mbIL-21), and (3) 41BB ligand (41BBL).

2. The UAPC described in claim 1, wherein the UAPC expresses CD48.

3. The UAPC described in claim 1, wherein the UAPC expresses CS1.

4. The UAPC described in claim 1, wherein the UAPC expresses CD48 and CS1.

5. The UAPC described in claim 1, wherein the UAPC does not essentially express endogenous HLA class I, II, or CD1d molecules.

6. The UAPC described in claim 1, wherein the UAPC expresses ICAM-1 (CD54) and LFA-3 (CD58).

7. The UAPC described in claim 1, wherein the UAPC is a UAPC derived from a leukemia cell, a macrophage, a B cell, an endothelial cell, an activated T cell, or a dendritic cell.

8. The UAPC described in claim 7, wherein the leukemia cell-derived UAPC is further defined as a K562 cell.

9. The UAPC described in claim 1, wherein the UAPC is stably transformed and engineered by retroviral introduction to express (1) CD48 and / or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL) on the cell surface.

10. The UAPC described in claim 9, wherein the retroviral introduction is further defined as introduction using a viral construct of sequence number 1 and / or sequence number 2 of the sequence listing.

11. The UAPC described in claim 1, wherein the UAPC is irradiated with gamma rays.

12. A composition comprising a UAPC described in any one of claims 1 to 11, and IL-2.

13. The composition of claim 12, wherein IL-2 is present at a concentration of 10 to 500 U / mL.

14. The composition of claim 12, further comprising natural killer (NK) cells.

15. The composition of claim 14, wherein the NK cells are engineered to express a chimeric antigen receptor (CAR).

16. A method for expanding immune cells, comprising culturing immune cells in the presence of an effective amount of UAPC described in any one of claims 1 to 11.

17. The method according to claim 16, wherein the immune cells are NK cells.

18. A population of NK cells activated according to the method of claim 17.

19. A pharmaceutical composition comprising the expanded population of NK cells of claim 18 and a pharma- ceutically acceptable carrier.

20. A composition comprising an effective amount of the expanded NK cells of claim 18 for use in treating a disease or disorder in a subject.

21. The composition of claim 20, wherein the disease or disorder is cancer, inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency, B-cell malignancy, or infectious disease.

22. The composition described in claim 21, wherein the cancer is leukemia.

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